**3. IGF-1 and wound healing**

exhibit a rhythm with a characteristic period of ~24 hours (the so-called circadian rhythms). There is a relationship between feeding, the organs involved in food intake, metabolic networks, and circadian physiology. One of the most important endocrine axes involved in circadian rhythm is the axis Ghrelin-GH-IGF-1. The coordinating role of these hormones lies in regulating appetite, behavior, growth, and cell proliferation, with a clear influence in the metabolic regulation of nutrients and all those processes dependent of them, as it is wound healing. Some hormones have been implied in the regulation of circadian GH production, as cortisol, thyrotropin (TSH), and insulin, in addition to some important neurotransmitters [62]. Although GH is mainly released by the anterior pituitary gland, there is a peripheral GH production in practically all the organism, highly dependent on developmental stages, at the level of tissues as nervous system, or the immune, cardiovascular, gonadal, and musculoskeletal system. This peripheral GH plays an autocrine and paracrine role [63]. In humans, plasma GH shows a circadian pattern of secretion, different according to sex and age; during puberty, the hormone reaches its highest plasma values, but once puberty ends, the secretion of the

126 Wound Healing - Current Perspectives

hormone begins to decline until being practically undetectable in elder people [64].

and obesity has been associated to the suppression of circulating GH [69].

The circadian pattern of GH is affected by nutritional status (caloric intake), age, stress, sex, physical exercise, and lack of sleep. Nutritional status is a key determinant in the regulation of GH secretion; thus, while fasting increases the frequency of GH secretion pulses, while IGF-1 levels decrease, in obesity the opposite occurs, at least during childhood [65]. During fasting, GHR are downregulated [66, 67]. Evidence for a circadian effect on the reduction of human GH gene expression has been demonstrated in response to excess caloric intake [68],

The highest pulse amplitude of GH secretion is observed during the REM phase of the sleep,

Several studies have shown that prolonged sleep deprivation, with the subsequent stress, leads to a reduction in body mass, elevated energy metabolism, changes in circulating hormones, and loss of immune system integrity [72]. Stress mediators act on immune cells to modulate the production of key regulatory cytokines [73, 74]. Thus, circadian rhythm disorders affect the levels of IL-1, IL-2, IL-6, TNFα, natural killer cells, adrenocorticotropic hormone (ACTH), cortisol, GH, and melatonin, all of them playing a key role in wound healing [75–77]. Melatonin has potential effects on the immune system, as inhibition of pineal melatonin synthesis with propranolol or pinealectomy results in immunosuppression and negative effects on wound healing [78]. Yet another study found that melatonin improved wound healing

Notwithstanding all these data, some studies disagree with the concept that sleep exerts a predominant influence on GH release and its effects whatever the conditions be, as it seems to

Thus, GH influences on wound healing progression. Physiologic circadian rhythm, with higher levels of the hormone during the night, will make a faster healing of wounds during the night, and the alteration of this pattern by different factors might exert a deleterious effect on wound healing via GH and others hormones related to it, although compensatory mecha-

while sleep deprivation leads to a strong inhibition of nocturnal GH secretion [70, 71].

when given at night, coinciding with its normal circadian period of secretion [79].

occur compensatory mechanisms promoting GH pulses during wakefulness [80].

nisms have been described in the long-term.

IGF-1 is considered as the main mediator of GH actions, and it has been considered as the "authentic" GH, at least for growing, although GH exerts many actions directly without the participation of IGF-1 [27].

IGF-1 is a polypeptide structural and functionally similar to insulin. It is produced in the liver and practically all extrahepatic tissues, and its production depends not only of GH, but also is strongly influenced by the nutritional status of the organism, at least in the liver. The local production of IGF-1 has been shown to regulate many physiological and pathophysiological states such as fetal development, atherosclerosis, and tissue repair. During tissue repair, IGF-1 is secreted by platelets, macrophages, and fibroblasts of the wound [5].

In wounds, IGF-1 increases protein production and cell proliferation and migration, which are crucial in the healing process [81, 82]. IGF-1 expression is enhanced in subcutaneous [5], and incisional [83] wounds, and in postburn injuries [84]. Some studies have shown that the administration of exogenous IGF-I enhanced protein synthesis in severely burned experimental animals [85].

Moreover, the levels of this growth factor are reduced in the wound environment of diabetic patients. Wound-related parameters as proteins, DNA, hydroxyproline, and macrophages have been shown to be decreased as a consequence of diabetes. After 14 days of treatment with IGF-1 in rats with diabetes produced by streptozotocin, it was observed that the total values of hydroxyproline, DNA, proteins, and macrophages increased by 48, 52, 31, and 40%, respectively [5]. These data support the fact that the suppression of IGF-1 and the macrophage function impairment within the wound environment by the diabetic state are responsible, at least in part, for the delay of wound healing in this disease.

In this context, the relationship between the IGF-1 receptor (IGF-1R) and the estrogen receptor (ER) is of interest. Locally administered IGF-1 promotes wound repair in an estrogen-deprived animal model, the ovariectomized (Ovx) mouse, mainly by dampening the local inflammatory response and promoting re-epithelialization. Using specific IGF-1R and ER antagonists it has been shown how IGF-1-mediated effects on re-epithelialization were directly mediated by IGF-1R [86]. In contrast, the anti-inflammatory effects of IGF-1 were predominantly mediated by ERs, in particular ERa (**Figure 6**). When ERa-null mice were used, IGF-1 could not promote healing and local inflammation increased [86]. These findings illustrate the great complexity of interactions between growth factors at the cutaneous level.

Recent data on the systemic administration of IGF-1 have shown an apparent lack of effect in wound healing. Therefore, perhaps only the IGF-1 produced locally by fibroblasts and macrophages contributes to the regulation of wound healing [46, 47], although it is also possible that the dose used and the type of administration do not have been the most appropriate in this case. If the systemic IGF-1 is ineffective in wound healing, topical administration of IGF-1 could be considered, as other growth factors such as EGF, TGFβ, or the own GH. In addition, IGF-1 systemic administration produces mild complications as hypoglycemia and hypotension. These limit its clinical usefulness.

that fibroblast stimulation by GHRH agonist could be mediated by GH/IGF-1. Some authors have found that using MR-409 and MR-502 GHRH agonists, there was a promotion of wound healing by stimulating the proliferation and survival of dermal fibroblast through phosphorylation of the ERK1/2 and AKT pathways, although neither GH nor IGF-1 was found to be significantly increased in fibroblasts after 4 hours exposure to these agonists. Moreover, none of the agonists showed an effect on the expression levels of either IGF-1 receptor (IGF1-R) or its phosphorylated isoform. Thus, these findings imply direct effects of GHRH and its agonists

Growth Hormone (GH) and Wound Healing http://dx.doi.org/10.5772/intechopen.80978 129

GHRH affects the proliferation of fibroblasts as well as their migration and the expression of smooth muscle actin α (α-SMA) [92], which is organized into stress fibers and exerts contractile forces on the extracellular matrix [96]. Therefore, it seems that GHRH can regulate, simultaneously, both the kinetic profile and the differentiation of fibroblasts in myofibro-

The suppression of growth of fibroblasts in not healing-wound environment is partially due to the decreased sensitivity of resident cells and rapid degradation of growth factors used in different therapies by proteases released from inflammatory cells and bacteria [97, 98]. Therefore, it would be necessary to have a factor that exerted a strong mitogen action on the fibroblasts, while being resistant to proteolytic degradation. In this sense, unlike the natural GHRH [95], the above mentioned MR agonists seem to have an increased resistance to degradation by proteases, because many of the coded amino acids in the peptide chain have been replaced with synthetic non-natural and/or non-coded amino acids which are much less susceptible to such degradation [99]. Consequently, these analogs have demonstrated a greatly prolonged half-life in vivo, making them promising agents for use in wound healing, where an environment rich in proteases is often found. Even more, it was found that MR class

Another factor supporting the use of GHRH agonists has been found in human dermal microvascular endothelial cells (HDMEC), that seems to express both pituitary GHRH-R and its splicing variant 1 (SV1). HDMEC is responsible for angiogenesis, a critical event for granula-

The endogenous GHRH produced by fibroblasts regulates its own activity, and the role that GHRH signaling may play in physiological maintenance of wound healing could improve

The high concentration of glucose in diabetic patients inhibits the proliferation of fibroblasts and favors resistance to growth factors, decreasing wound healing. Interestingly, MR-409 enhances the survival of transplanted pancreatic islets and helps to lower blood glucose in diabetic SCID mice [100]; therefore, it would be interesting to investigate whether it might benefit diabetic wounds which are hard to cure, partially because of the special adverse bacterial environment. However, some other aspects of diabetic injuries should also be addressed; such is the case of the affectation of the neuropathic response, the true conductor in this process.

Despite these data, the precise physiologic and biochemical mechanism for GHRH accelerating wound healing remains unclear. Besides, the production of GHRH in dermal wounds still seems not to be clear. Moreover, given its short lifetime, it is unlikely that plasma GHRH

agonists do not stimulate tumor growth or neoplastic transformation [95].

on extra-pituitary cells and tissues [95].

blasts (**Figure 7**).

tion tissue formation [95].

with some GHRH agonists.

**Figure 6.** Local expression of IGF-1 in a wound. This expression may be induced by GH, but also IG-1 may proceed from platelets, macrophages, and fibroblasts. Local IGF-1 induces protein synthesis and cell proliferation by interacting with its receptor IGF-1R, and also has anti-inflammatory effects, although in this case IGF-1 seems to act via the estrogen receptor a (ERa).

### **4. Analogs of growth hormone-releasing hormone (GHRH) and wound healing**

The complexity of GH regulation seems to be related to the multiple roles that GH plays in the human body, very far than those classically thought [27]. Interestingly, some of these roles are played in conjunction with GH-stimulating factors.

As described, growth hormone-releasing hormone (GHRH) is an important neuroendocrine peptide secreted by the hypothalamus, regulating the synthesis and release of GH [87]. Classically, it was thought that the role of GHRH simply was the regulation of the synthesis and secretion of GH [88, 89]. However, the detection of GHRH and its receptors, as well as the expression of GHRH gene in several extra-hypothalamic tissues, including placenta, ovary, testis, digestive tract, and tumors [90, 91], suggests that GHRH plays a wider role than simply acting on the regulation of pituitary GH secretion; in fact, it seems to be particularly involved in conditions aimed at tissue regeneration and repair. The presence of the peptide in peripheral tissues highlights the possibility that locally produced GHRH might act as an autocrine growth factor playing a role in cell proliferation. In addition to its own actions in various tissues, several GHRH agonists have been developed showing that the effects of this neuropeptide could include direct actions on wound healing. For example, a pioneer work demonstrated that the GHRH agonist JI-38 stimulates the proliferation and migration of mouse embryonic fibroblasts (MEF) [92]. The upregulation of GHRH receptor (GHRH-R) and its splicing variant 1 (SV1) in GHRH-R negative 3T3 fibroblasts has been shown to promote its proliferation when GHRH and its analogs are given [93, 94]. Despite it is logical to think that fibroblast stimulation by GHRH agonist could be mediated by GH/IGF-1. Some authors have found that using MR-409 and MR-502 GHRH agonists, there was a promotion of wound healing by stimulating the proliferation and survival of dermal fibroblast through phosphorylation of the ERK1/2 and AKT pathways, although neither GH nor IGF-1 was found to be significantly increased in fibroblasts after 4 hours exposure to these agonists. Moreover, none of the agonists showed an effect on the expression levels of either IGF-1 receptor (IGF1-R) or its phosphorylated isoform. Thus, these findings imply direct effects of GHRH and its agonists on extra-pituitary cells and tissues [95].

GHRH affects the proliferation of fibroblasts as well as their migration and the expression of smooth muscle actin α (α-SMA) [92], which is organized into stress fibers and exerts contractile forces on the extracellular matrix [96]. Therefore, it seems that GHRH can regulate, simultaneously, both the kinetic profile and the differentiation of fibroblasts in myofibroblasts (**Figure 7**).

The suppression of growth of fibroblasts in not healing-wound environment is partially due to the decreased sensitivity of resident cells and rapid degradation of growth factors used in different therapies by proteases released from inflammatory cells and bacteria [97, 98]. Therefore, it would be necessary to have a factor that exerted a strong mitogen action on the fibroblasts, while being resistant to proteolytic degradation. In this sense, unlike the natural GHRH [95], the above mentioned MR agonists seem to have an increased resistance to degradation by proteases, because many of the coded amino acids in the peptide chain have been replaced with synthetic non-natural and/or non-coded amino acids which are much less susceptible to such degradation [99]. Consequently, these analogs have demonstrated a greatly prolonged half-life in vivo, making them promising agents for use in wound healing, where an environment rich in proteases is often found. Even more, it was found that MR class agonists do not stimulate tumor growth or neoplastic transformation [95].

**Figure 6.** Local expression of IGF-1 in a wound. This expression may be induced by GH, but also IG-1 may proceed from platelets, macrophages, and fibroblasts. Local IGF-1 induces protein synthesis and cell proliferation by interacting with its receptor IGF-1R, and also has anti-inflammatory effects, although in this case IGF-1 seems to act via the estrogen

The complexity of GH regulation seems to be related to the multiple roles that GH plays in the human body, very far than those classically thought [27]. Interestingly, some of these roles are

As described, growth hormone-releasing hormone (GHRH) is an important neuroendocrine peptide secreted by the hypothalamus, regulating the synthesis and release of GH [87]. Classically, it was thought that the role of GHRH simply was the regulation of the synthesis and secretion of GH [88, 89]. However, the detection of GHRH and its receptors, as well as the expression of GHRH gene in several extra-hypothalamic tissues, including placenta, ovary, testis, digestive tract, and tumors [90, 91], suggests that GHRH plays a wider role than simply acting on the regulation of pituitary GH secretion; in fact, it seems to be particularly involved in conditions aimed at tissue regeneration and repair. The presence of the peptide in peripheral tissues highlights the possibility that locally produced GHRH might act as an autocrine growth factor playing a role in cell proliferation. In addition to its own actions in various tissues, several GHRH agonists have been developed showing that the effects of this neuropeptide could include direct actions on wound healing. For example, a pioneer work demonstrated that the GHRH agonist JI-38 stimulates the proliferation and migration of mouse embryonic fibroblasts (MEF) [92]. The upregulation of GHRH receptor (GHRH-R) and its splicing variant 1 (SV1) in GHRH-R negative 3T3 fibroblasts has been shown to promote its proliferation when GHRH and its analogs are given [93, 94]. Despite it is logical to think

**4. Analogs of growth hormone-releasing hormone (GHRH) and** 

played in conjunction with GH-stimulating factors.

receptor a (ERa).

**wound healing**

128 Wound Healing - Current Perspectives

Another factor supporting the use of GHRH agonists has been found in human dermal microvascular endothelial cells (HDMEC), that seems to express both pituitary GHRH-R and its splicing variant 1 (SV1). HDMEC is responsible for angiogenesis, a critical event for granulation tissue formation [95].

The endogenous GHRH produced by fibroblasts regulates its own activity, and the role that GHRH signaling may play in physiological maintenance of wound healing could improve with some GHRH agonists.

The high concentration of glucose in diabetic patients inhibits the proliferation of fibroblasts and favors resistance to growth factors, decreasing wound healing. Interestingly, MR-409 enhances the survival of transplanted pancreatic islets and helps to lower blood glucose in diabetic SCID mice [100]; therefore, it would be interesting to investigate whether it might benefit diabetic wounds which are hard to cure, partially because of the special adverse bacterial environment. However, some other aspects of diabetic injuries should also be addressed; such is the case of the affectation of the neuropathic response, the true conductor in this process.

Despite these data, the precise physiologic and biochemical mechanism for GHRH accelerating wound healing remains unclear. Besides, the production of GHRH in dermal wounds still seems not to be clear. Moreover, given its short lifetime, it is unlikely that plasma GHRH

in tensile strength of wounds in animals, and rats fed with a protein-deficient diet showed a

Growth Hormone (GH) and Wound Healing http://dx.doi.org/10.5772/intechopen.80978 131

Burn injury induces acute and severe inflammation and a hypermetabolic state which are strongly correlated to the size of the burn [106]. The inflammatory process reaches a peak during the first week postburn and persists to a lesser extent throughout convalescence [107]. The hypermetabolic state begins 5 days after the burn, and may last up to 1 year after the injury,

GH is one of the most important anabolic hormones and, like other anabolic hormones, has an anti-cortisol activity, lowering the catabolic response of this steroid, without altering its protective anti-inflammatory activity. Many studies have demonstrated the usefulness of anabolic hormones in existing wounds in catabolic states. However, it remains difficult to determine whether the benefit is due to the increase in the systemic anabolic state or to a

Starvation and intense exercise, both being catabolic states, are potent stimuli of GH, while acute or chronic injury or illness inhibits GH release, especially in the elderly [109]. GH leads to an increased influx of amino acids into the cell, decreasing the flow of these from the same. The increase in fatty metabolism that GH produces is also beneficial, since it preserves the

Severe burns and injuries, people with HIV infection with wasting and elderly people, all of them catabolic states, are populations that could benefit from GH therapy. GH increases lean mass, muscle strength, and immune function in these states, but requires an intake of a high-

The skin is a target tissue for GH, and GHRs have been found on the surface of epidermal cells. Recent data indicate that IGF-1 and insulin also provide some of the anabolic effects of GH therapy in wounds [110, 111]. GH administered exogenously increases the thickness of the skin even in normal people [112]. It has been shown that GH can improve the re-epithelialization rate of sites where a skin graft has taken place in adults and children with severe burns or trauma [7, 10]. In addition, it has been seen, in experimental models, that GH also accelerates the healing by increasing wound collagen content, granulation tissue, and wound tensile strength, as well as the local production of IGF-1 by fibroblasts [109, 113].

A study conducted on burned children also supports the role of GH in catabolic states, since no differences were found in mortality, organ failure, or clinically significant morbidity between the groups, and the requirements for albumin supplementation were reduced by 65%, as well as episodes of hypocalcemia, an unexpected benefit of the hormone [114]. As it will be discussed at the end of the chapter, unlike it happens in children, it has been reported an increase of mortality in adult with burns when GH was used [115]. However, the authors of the study in pediatric population have been treating severely burned children with rhGH for more than 10 years, and they have reported that 0.2 mg/kg/day of rhGH in this catabolic state has some benefits, accelerating donor site wound healing by up to 30% and reducing a 25% the hospital stay and costs. They have also shown that GH increased protein synthesis by more than 25%. Another study has also found that GH causes significant serum elevations in

amino acids for the synthesis of proteins, instead of being used as an energy resource.

decrease in wound integrity and resistance as compared to control animals [105].

with energy requirements that reach 150–200% of the basal metabolic rate [108].

direct effect on the anabolic state of the wound [109].

protein, high-energy diet [109].

**Figure 7.** Possible effects of GHRH on wound healing. The possibility exists that GHRH is expressed in cells in a wound, since its short life in plasma does not explain its effects on wound healing. However, GHRH agonists do not suffer proteolytic degradation; therefore, they may mimic the effects of GHRH on the proliferation and migration of fibroblasts and its differentiation in myofibroblasts, as well as inducing the expression of smooth muscle a-actin which favors the appearance of contractile forces on the extracellular matrix (ECM). Blue arrows: induction or activation; red arrow: inhibition.

may reach adequate levels to contribute to wound healing. A possibility, not explored, is that some GHRH agonists produced in dermal wounds during healing might be responsible for the activity of GHRH on wound healing.

Whether this apparently novel function of GHRH is operational in a different kind of healing or it is indicative of the activity of a structurally related peptide(s), should be investigated more extensively to elucidate some of the basic aspects of skin biology and repair, as well as in view of its potential implications in therapeutic wound healing.

### **5. Wound healing in catabolic states: the role of growth hormone**

The balance between anabolic and catabolic states and hormones may affect wound healing, since the overall protein compartment status has a great influence on this process [101]. Protein synthesis restores and maintains lean body mass, composed of muscle, skin, and the immune system, all of them having a role during wound repair. When anabolic activity decreases, as occurs during stress, aging, or chronic disease, there is a derivation of proteins to the energy compartment and, therefore, affects wound healing as a result of protein depletion in the wound to restore lost lean mass. Impaired immunity and healing during catabolic states are directly proportional to the degree of lean mass loss [102, 103]. Protein depletion appears to delay wound healing by prolonging the inflammatory phase (inhibits fibroplasia, synthesis of collagen, and proteoglycans), affects the proliferation phase (neoangiogenesis) and inhibits wound remodeling [104]. It has been shown that protein depletion models produce a decrease in tensile strength of wounds in animals, and rats fed with a protein-deficient diet showed a decrease in wound integrity and resistance as compared to control animals [105].

Burn injury induces acute and severe inflammation and a hypermetabolic state which are strongly correlated to the size of the burn [106]. The inflammatory process reaches a peak during the first week postburn and persists to a lesser extent throughout convalescence [107]. The hypermetabolic state begins 5 days after the burn, and may last up to 1 year after the injury, with energy requirements that reach 150–200% of the basal metabolic rate [108].

GH is one of the most important anabolic hormones and, like other anabolic hormones, has an anti-cortisol activity, lowering the catabolic response of this steroid, without altering its protective anti-inflammatory activity. Many studies have demonstrated the usefulness of anabolic hormones in existing wounds in catabolic states. However, it remains difficult to determine whether the benefit is due to the increase in the systemic anabolic state or to a direct effect on the anabolic state of the wound [109].

Starvation and intense exercise, both being catabolic states, are potent stimuli of GH, while acute or chronic injury or illness inhibits GH release, especially in the elderly [109]. GH leads to an increased influx of amino acids into the cell, decreasing the flow of these from the same. The increase in fatty metabolism that GH produces is also beneficial, since it preserves the amino acids for the synthesis of proteins, instead of being used as an energy resource.

Severe burns and injuries, people with HIV infection with wasting and elderly people, all of them catabolic states, are populations that could benefit from GH therapy. GH increases lean mass, muscle strength, and immune function in these states, but requires an intake of a highprotein, high-energy diet [109].

may reach adequate levels to contribute to wound healing. A possibility, not explored, is that some GHRH agonists produced in dermal wounds during healing might be responsible for

**Figure 7.** Possible effects of GHRH on wound healing. The possibility exists that GHRH is expressed in cells in a wound, since its short life in plasma does not explain its effects on wound healing. However, GHRH agonists do not suffer proteolytic degradation; therefore, they may mimic the effects of GHRH on the proliferation and migration of fibroblasts and its differentiation in myofibroblasts, as well as inducing the expression of smooth muscle a-actin which favors the appearance of contractile forces on the extracellular matrix (ECM). Blue arrows: induction or activation; red arrow:

Whether this apparently novel function of GHRH is operational in a different kind of healing or it is indicative of the activity of a structurally related peptide(s), should be investigated more extensively to elucidate some of the basic aspects of skin biology and repair, as well as

The balance between anabolic and catabolic states and hormones may affect wound healing, since the overall protein compartment status has a great influence on this process [101]. Protein synthesis restores and maintains lean body mass, composed of muscle, skin, and the immune system, all of them having a role during wound repair. When anabolic activity decreases, as occurs during stress, aging, or chronic disease, there is a derivation of proteins to the energy compartment and, therefore, affects wound healing as a result of protein depletion in the wound to restore lost lean mass. Impaired immunity and healing during catabolic states are directly proportional to the degree of lean mass loss [102, 103]. Protein depletion appears to delay wound healing by prolonging the inflammatory phase (inhibits fibroplasia, synthesis of collagen, and proteoglycans), affects the proliferation phase (neoangiogenesis) and inhibits wound remodeling [104]. It has been shown that protein depletion models produce a decrease

in view of its potential implications in therapeutic wound healing.

**5. Wound healing in catabolic states: the role of growth hormone**

the activity of GHRH on wound healing.

inhibition.

130 Wound Healing - Current Perspectives

The skin is a target tissue for GH, and GHRs have been found on the surface of epidermal cells. Recent data indicate that IGF-1 and insulin also provide some of the anabolic effects of GH therapy in wounds [110, 111]. GH administered exogenously increases the thickness of the skin even in normal people [112]. It has been shown that GH can improve the re-epithelialization rate of sites where a skin graft has taken place in adults and children with severe burns or trauma [7, 10]. In addition, it has been seen, in experimental models, that GH also accelerates the healing by increasing wound collagen content, granulation tissue, and wound tensile strength, as well as the local production of IGF-1 by fibroblasts [109, 113].

A study conducted on burned children also supports the role of GH in catabolic states, since no differences were found in mortality, organ failure, or clinically significant morbidity between the groups, and the requirements for albumin supplementation were reduced by 65%, as well as episodes of hypocalcemia, an unexpected benefit of the hormone [114]. As it will be discussed at the end of the chapter, unlike it happens in children, it has been reported an increase of mortality in adult with burns when GH was used [115]. However, the authors of the study in pediatric population have been treating severely burned children with rhGH for more than 10 years, and they have reported that 0.2 mg/kg/day of rhGH in this catabolic state has some benefits, accelerating donor site wound healing by up to 30% and reducing a 25% the hospital stay and costs. They have also shown that GH increased protein synthesis by more than 25%. Another study has also found that GH causes significant serum elevations in other different parameters as total catecholamines, insulin, glucagon, or free fatty acids. GH therapy even showed a rise in blood flow of the leg [114].

inflammation was reduced in severity, with a more rapid regeneration of the pancreas, resulting in a reduction in the serum concentrations of interleukin 1-β pro-inflammatory (IL-1β) as well as the amylase and lipase activities. In addition, there was an increase in pancreatic blood flow, and DNA synthesis increased in this organ. This demonstrates that the possible role of Ghrelin during catabolic states needs an adequate functioning of the GH/IGF-1 axis [124]. This last statement has also been supported by models of colitis in which treatment with Ghrelin clearly improved the area of damage in the colonic mucosa in intact pituitary rats, but increased it in hypophysectomized animals. In addition, it was shown that rats with a normal production of GH-IGF-1 had improved blood flow in the colonic mucosa and increased mucosal cell proliferation while treated with Ghrelin, as well as reduced levels of IL1-1β and myeloperoxidase; just the opposite of what was found in hypophysectomized rats [125].

Growth Hormone (GH) and Wound Healing http://dx.doi.org/10.5772/intechopen.80978 133

The therapeutic effect of Ghrelin on wound healing has also been evaluated using a rat model in which the administration of radiation was combined with the induction of a wound. The altered healing of a wound caused by radiation often occurs in clinical practice and the exact mechanisms by which this occurs are not yet clear. In this wound model, the administration of Ghrelin promoted the healing of skin wounds, and also reduced the average time of wound closure [126]. Ghrelin inhibited the induction of serum pro-inflammatory mediators, especially TNFα, and promoted wound healing in a dose-dependent manner [127]. After the isolation and analysis of the granulation tissues, a greater synthesis of DNA, hexosamine, nitrate, and nitrite, a high content of collagen and an enhanced neovascularization was observed after treatment with Ghrelin. The hormone also increased the expression of VEGF and TGFβ, responsible for wound healing as described. Again, when a GH 1a secretagogue receptor blocker (GHS-R1a) was administered, all of these therapeutic effects of Ghrelin were affected [126]. These results identify Ghrelin as a peptide that could be used for the affected wound healing induced by radiation, although it is necessary that there is a normal secretion of GH

Cellular senescence is the consequence of DNA damage secondary to oxidative stress associated with aging or chronic morbid conditions such as diabetes. This seems to be an antitumor mechanism [128]. The number of senescent cells is low in young individuals, while it increases

At skin level, senescence has been reported in keratinocytes, melanocytes, endothelial cells,

This concept has emerged as a possible cause of general tissue dysfunction [134, 135], since, although senescent cells are unable to divide, they remain metabolically active. This high metabolic activity is associated with the release of a multitude of cytokines, chemokines, and pro-inflammatory growth factors, which leads to its denomination as the secretion phenotype associated with senescence (SASP) [136]. These factors would include interleukin (IL) 6 and IL-8, chemokines such as monocyte chemoattractant protein (MCPs), macrophage inflammatory proteins, and growth factors as VEGF, granulocyte/macrophage colony-stimulating factor (GMCSF), TGFβ, and proteinases such as matrix metalloproteinases [128, 137]. All these

so that its effects occur. These effects of Ghrelin are shown in **Figure 9**.

**5.2. Cellular senescence and wound healing: benefit of GH therapy**

epithelial cells, T-lymphocytes, and even in stem cells [131–133].

with age in all tissues, including the skin [129, 130].

In summary, the use of GH together with adequate nutrition and protein intake, at the appropriate doses, clearly improves anabolic activity and, as a consequence, positively impacts wound healing, even in patients with spinal cord injuries, as **Figure 8** shows. Although many data suggest that the effect of GH on wound healing can be direct, it is still unknown whether some other hormones could contribute to this positive effect.

### **5.1. Ghrelin, GH, and wound healing**

Ghrelin (GH-releasing peptide or GHRP) is a small peptide found in the gastrointestinal tract in 1999 [116]. Although it is mainly secreted by the stomach, it is known that Ghrelin is also produced in other territories, such as the intestine or placenta, for example.

In addition to its known actions on the regulation of appetite and energy expenditure, it has also been discovered that this hormone plays a role in the control of inflammation and metabolism, as do leptin and adiponectin. In fact, all three hormones are interrelated in chronic disease states [117–119]. Interestingly, in a study that addressed the relationship between these hormones in burns, the authors came to the surprising conclusion that they acted in two different ways: one in normal physiological conditions or chronic disease states, and another after severe acute stresses such as burn injury [120]. This can be an adaptive mechanism that depends on the physiological situation or the type of the pathological condition.

Recently, it was demonstrated that Ghrelin improves hemodynamic and metabolic alterations and attenuates cancer, heart affectations, and cachexia induced by burns, and also again protects the damage induced by burns and facilitates the healing of wounds [121].

In relation to the hemodynamic role of this hormone, receptors for Ghrelin have been found in the aorta, the left cardiac ventricle, and the left cardiac atrium in rats. In healthy humans, the intravenous infusion of Ghrelin decreases blood pressure, increases the cardiac index, and produces a greater volume of the pulse [122].

Ghrelin also has an anti-inflammatory effect, by inhibiting the secretion of IL-6 and TNFα from monocytes and T5 cells [119, 123]. The protective role of Ghrelin appears to depend on the integrity of GH/IGF-1 axis, since in studies of inflammation with pancreatitis, protection against inflammation did not occur in hypophysectomized rats unless they received IGF-1 in parallel with Ghrelin. In these studies, when normal GH secretion was reached, the

**Figure 8.** Evolution of a pressure ulcer in the foot of a quadriplegic patient (complete spinal cord injury, C5-C6) treated with GH applied topically (0.4 mg/day, 5 days/week), before the treatment (10/10/2012) and throughout it until the healing of the wound (02/27/2013).

inflammation was reduced in severity, with a more rapid regeneration of the pancreas, resulting in a reduction in the serum concentrations of interleukin 1-β pro-inflammatory (IL-1β) as well as the amylase and lipase activities. In addition, there was an increase in pancreatic blood flow, and DNA synthesis increased in this organ. This demonstrates that the possible role of Ghrelin during catabolic states needs an adequate functioning of the GH/IGF-1 axis [124]. This last statement has also been supported by models of colitis in which treatment with Ghrelin clearly improved the area of damage in the colonic mucosa in intact pituitary rats, but increased it in hypophysectomized animals. In addition, it was shown that rats with a normal production of GH-IGF-1 had improved blood flow in the colonic mucosa and increased mucosal cell proliferation while treated with Ghrelin, as well as reduced levels of IL1-1β and myeloperoxidase; just the opposite of what was found in hypophysectomized rats [125].

other different parameters as total catecholamines, insulin, glucagon, or free fatty acids. GH

In summary, the use of GH together with adequate nutrition and protein intake, at the appropriate doses, clearly improves anabolic activity and, as a consequence, positively impacts wound healing, even in patients with spinal cord injuries, as **Figure 8** shows. Although many data suggest that the effect of GH on wound healing can be direct, it is still unknown whether

Ghrelin (GH-releasing peptide or GHRP) is a small peptide found in the gastrointestinal tract in 1999 [116]. Although it is mainly secreted by the stomach, it is known that Ghrelin is also

In addition to its known actions on the regulation of appetite and energy expenditure, it has also been discovered that this hormone plays a role in the control of inflammation and metabolism, as do leptin and adiponectin. In fact, all three hormones are interrelated in chronic disease states [117–119]. Interestingly, in a study that addressed the relationship between these hormones in burns, the authors came to the surprising conclusion that they acted in two different ways: one in normal physiological conditions or chronic disease states, and another after severe acute stresses such as burn injury [120]. This can be an adaptive mechanism that

Recently, it was demonstrated that Ghrelin improves hemodynamic and metabolic alterations and attenuates cancer, heart affectations, and cachexia induced by burns, and also again

In relation to the hemodynamic role of this hormone, receptors for Ghrelin have been found in the aorta, the left cardiac ventricle, and the left cardiac atrium in rats. In healthy humans, the intravenous infusion of Ghrelin decreases blood pressure, increases the cardiac index, and

Ghrelin also has an anti-inflammatory effect, by inhibiting the secretion of IL-6 and TNFα from monocytes and T5 cells [119, 123]. The protective role of Ghrelin appears to depend on the integrity of GH/IGF-1 axis, since in studies of inflammation with pancreatitis, protection against inflammation did not occur in hypophysectomized rats unless they received IGF-1 in parallel with Ghrelin. In these studies, when normal GH secretion was reached, the

**Figure 8.** Evolution of a pressure ulcer in the foot of a quadriplegic patient (complete spinal cord injury, C5-C6) treated with GH applied topically (0.4 mg/day, 5 days/week), before the treatment (10/10/2012) and throughout it until the

produced in other territories, such as the intestine or placenta, for example.

depends on the physiological situation or the type of the pathological condition.

protects the damage induced by burns and facilitates the healing of wounds [121].

therapy even showed a rise in blood flow of the leg [114].

some other hormones could contribute to this positive effect.

**5.1. Ghrelin, GH, and wound healing**

132 Wound Healing - Current Perspectives

produces a greater volume of the pulse [122].

healing of the wound (02/27/2013).

The therapeutic effect of Ghrelin on wound healing has also been evaluated using a rat model in which the administration of radiation was combined with the induction of a wound. The altered healing of a wound caused by radiation often occurs in clinical practice and the exact mechanisms by which this occurs are not yet clear. In this wound model, the administration of Ghrelin promoted the healing of skin wounds, and also reduced the average time of wound closure [126]. Ghrelin inhibited the induction of serum pro-inflammatory mediators, especially TNFα, and promoted wound healing in a dose-dependent manner [127]. After the isolation and analysis of the granulation tissues, a greater synthesis of DNA, hexosamine, nitrate, and nitrite, a high content of collagen and an enhanced neovascularization was observed after treatment with Ghrelin. The hormone also increased the expression of VEGF and TGFβ, responsible for wound healing as described. Again, when a GH 1a secretagogue receptor blocker (GHS-R1a) was administered, all of these therapeutic effects of Ghrelin were affected [126]. These results identify Ghrelin as a peptide that could be used for the affected wound healing induced by radiation, although it is necessary that there is a normal secretion of GH so that its effects occur. These effects of Ghrelin are shown in **Figure 9**.

### **5.2. Cellular senescence and wound healing: benefit of GH therapy**

Cellular senescence is the consequence of DNA damage secondary to oxidative stress associated with aging or chronic morbid conditions such as diabetes. This seems to be an antitumor mechanism [128]. The number of senescent cells is low in young individuals, while it increases with age in all tissues, including the skin [129, 130].

At skin level, senescence has been reported in keratinocytes, melanocytes, endothelial cells, epithelial cells, T-lymphocytes, and even in stem cells [131–133].

This concept has emerged as a possible cause of general tissue dysfunction [134, 135], since, although senescent cells are unable to divide, they remain metabolically active. This high metabolic activity is associated with the release of a multitude of cytokines, chemokines, and pro-inflammatory growth factors, which leads to its denomination as the secretion phenotype associated with senescence (SASP) [136]. These factors would include interleukin (IL) 6 and IL-8, chemokines such as monocyte chemoattractant protein (MCPs), macrophage inflammatory proteins, and growth factors as VEGF, granulocyte/macrophage colony-stimulating factor (GMCSF), TGFβ, and proteinases such as matrix metalloproteinases [128, 137]. All these

study, the hormone significantly lowered plasma diacron-reactive oxygen metabolites and improved endothelial function, as measured by reactive hyperemia index [145]. This indicates that GH can exert a protective role in redox balance in GHD, in which predominates a pro-oxidant environment, corrected by short-term GH administration [146]. Klotho, a GH-releasing factor that currently is gaining in interest, also lowers the oxidative stress, decreasing apoptosis and senescence of the vascular system in an atherogenic risk rat model [147]. The hormone also affects the regulation of TRX and GRX, which are factors that regulate the post-translational modification of proteins and the redox balance, also influencing resistance to stress [41]. As a consequence of the antioxidant action of GH, the hormone produces a benefit in the inflammatory state associated with senescence [22]. It has been reported that this protection against oxidative stress is mediated by GH induction of the

Growth Hormone (GH) and Wound Healing http://dx.doi.org/10.5772/intechopen.80978 135

However, the exact role of GH in the redox equilibrium has not been fully understood, since in some cases of oxidative stress, overproduction, or administration of GH in excess may enhance oxidation [149]. Thus, both the overproduction of GH and its deficiency are closely

As described in the introduction, GH needs specific stimuli to exert its effects. In fact, there is a study carried out to determine the effect of rhGH on the rate of wound healing in normal individuals. In each subject was performed a split-thickness wound in one buttock and a fullthickness wound in the other. The full-thickness wound healed significantly more slowly in the group treated with rhGH compared to the control group treated with placebo, while no statistically significant difference was observed in the healing of the split-thickness wounds. This study concluded that rhGH may delay healing in normal patients with full-thickness wounds, although it could not be ruled out if the healing delay associated to rhGH group was due to the quality of the scab, thereby, appearing only as an alteration of the wound healing

In another trial, the serum levels of some hormones, GH, insulin, and cortisol were analyzed in normal and diabetic rats during wound healing. It was shown that the rate of wound healing in normal rats is faster than that of diabetics. The serum insulin concentrations were lower in the diabetic rats compared to the normal and control groups and showed a correlation with the wound healing process in diabetic rats. Serum cortisol concentrations decreased in the normal and diabetic groups during wound healing, but did not show a significant correlation with this process. Serum GH levels did not change significantly in any of the groups, nor did they show a significant correlation with the wound healing process [150]. As described above, a possible explanation for these findings is that the main effect of GH in this case could occur as a consequence of the local production of the hormone, something that was overlooked because it was not measured. A small wound on the back of the animal is not a stimulus strong enough to increase systemic GH, which seems to be related, as demonstrated, to more

A recent report from two prospective, randomized, double-blind, placebo-controlled Phase III trials conducted in Europe, which studied the effects of rhGH in critically ill burned adult patients, in

**5.3. Contrary studies not supporting a GH role in wound healing**

RAS/ERK pathways [148].

process [18].

intense catabolic states.

related to increased oxidative stress.

**Figure 9.** Ghrelin effects on a wound. While many positive effects appear at very different levels during wound healing, there is a need for a normal pituitary secretion of GH, so that these Ghrelin effects can occur. Therefore, it is not clear whether these effects depend on Ghrelin or on GH, although the possibility exists that GH could induce Ghrelin expression in the wound.

factors can act in an autocrine and paracrine way, also having effects on the surrounding cells and their environment. Therefore, the senescent cell itself could initiate a feedback mechanism by spreading this phenomenon to nearby cells [138].

Characteristically, the inflammation resulting from cellular senescence is sterile or is not associated with pathogens [137]. It has been suggested that chronic low-level inflammation that is often observed during aging in tissues without obvious infection is due to senescent cells and SASP [139]. In addition, a low number of senescent cells can have systemic effects, and it is already evident that the senescence process can be transmitted to normal cells by SASP in a paracrine or autocrine manner [128].

The basis of this senescence is mitochondrial dysfunction, which in turn causes oxidative stress, which has been implicated as a cause of aging [140].

Understanding this process would help develop different strategies that could mitigate chronic inflammation and, therefore, cellular senescence. These dysfunctional and destructive signs are also found in the wounds of diabetic or elderly patients, altering the normal healing process.

At this point, it is important to note that GH is a mitochondrial protector [141–143], therefore, playing a positive role in this process. GH restores the redox imbalance, improving the mitochondrial respiratory chain and the production of energy.

In situations of GH deficiency (GHD) there is an accelerated aging process. In mice with GHD, GH replacement therapy increases stress resistance by altering the functional capacity of the glutathione S-transferase system (GST) through the regulation of specific members of the GST family [144]. The hormone also affects the regulation of thioredoxins (TRX) and glutaredoxins (GRX), which are factors that regulate the post-translational modification of proteins and the redox balance, also influencing resistance to stress [144]. Patients with GHD show a decrease in their life expectancy with a twice higher risk of death from cardiovascular disease. In this regard, after 24 weeks of GH replacement therapy in the GREAT study, the hormone significantly lowered plasma diacron-reactive oxygen metabolites and improved endothelial function, as measured by reactive hyperemia index [145]. This indicates that GH can exert a protective role in redox balance in GHD, in which predominates a pro-oxidant environment, corrected by short-term GH administration [146]. Klotho, a GH-releasing factor that currently is gaining in interest, also lowers the oxidative stress, decreasing apoptosis and senescence of the vascular system in an atherogenic risk rat model [147]. The hormone also affects the regulation of TRX and GRX, which are factors that regulate the post-translational modification of proteins and the redox balance, also influencing resistance to stress [41]. As a consequence of the antioxidant action of GH, the hormone produces a benefit in the inflammatory state associated with senescence [22]. It has been reported that this protection against oxidative stress is mediated by GH induction of the RAS/ERK pathways [148].

However, the exact role of GH in the redox equilibrium has not been fully understood, since in some cases of oxidative stress, overproduction, or administration of GH in excess may enhance oxidation [149]. Thus, both the overproduction of GH and its deficiency are closely related to increased oxidative stress.

#### **5.3. Contrary studies not supporting a GH role in wound healing**

**Figure 9.** Ghrelin effects on a wound. While many positive effects appear at very different levels during wound healing, there is a need for a normal pituitary secretion of GH, so that these Ghrelin effects can occur. Therefore, it is not clear whether these effects depend on Ghrelin or on GH, although the possibility exists that GH could induce Ghrelin

factors can act in an autocrine and paracrine way, also having effects on the surrounding cells and their environment. Therefore, the senescent cell itself could initiate a feedback mecha-

Characteristically, the inflammation resulting from cellular senescence is sterile or is not associated with pathogens [137]. It has been suggested that chronic low-level inflammation that is often observed during aging in tissues without obvious infection is due to senescent cells and SASP [139]. In addition, a low number of senescent cells can have systemic effects, and it is already evident that the senescence process can be transmitted to normal cells by SASP in a

The basis of this senescence is mitochondrial dysfunction, which in turn causes oxidative

Understanding this process would help develop different strategies that could mitigate chronic inflammation and, therefore, cellular senescence. These dysfunctional and destructive signs are also found in the wounds of diabetic or elderly patients, altering the normal healing process. At this point, it is important to note that GH is a mitochondrial protector [141–143], therefore, playing a positive role in this process. GH restores the redox imbalance, improving the mito-

In situations of GH deficiency (GHD) there is an accelerated aging process. In mice with GHD, GH replacement therapy increases stress resistance by altering the functional capacity of the glutathione S-transferase system (GST) through the regulation of specific members of the GST family [144]. The hormone also affects the regulation of thioredoxins (TRX) and glutaredoxins (GRX), which are factors that regulate the post-translational modification of proteins and the redox balance, also influencing resistance to stress [144]. Patients with GHD show a decrease in their life expectancy with a twice higher risk of death from cardiovascular disease. In this regard, after 24 weeks of GH replacement therapy in the GREAT

nism by spreading this phenomenon to nearby cells [138].

stress, which has been implicated as a cause of aging [140].

chondrial respiratory chain and the production of energy.

paracrine or autocrine manner [128].

expression in the wound.

134 Wound Healing - Current Perspectives

As described in the introduction, GH needs specific stimuli to exert its effects. In fact, there is a study carried out to determine the effect of rhGH on the rate of wound healing in normal individuals. In each subject was performed a split-thickness wound in one buttock and a fullthickness wound in the other. The full-thickness wound healed significantly more slowly in the group treated with rhGH compared to the control group treated with placebo, while no statistically significant difference was observed in the healing of the split-thickness wounds. This study concluded that rhGH may delay healing in normal patients with full-thickness wounds, although it could not be ruled out if the healing delay associated to rhGH group was due to the quality of the scab, thereby, appearing only as an alteration of the wound healing process [18].

In another trial, the serum levels of some hormones, GH, insulin, and cortisol were analyzed in normal and diabetic rats during wound healing. It was shown that the rate of wound healing in normal rats is faster than that of diabetics. The serum insulin concentrations were lower in the diabetic rats compared to the normal and control groups and showed a correlation with the wound healing process in diabetic rats. Serum cortisol concentrations decreased in the normal and diabetic groups during wound healing, but did not show a significant correlation with this process. Serum GH levels did not change significantly in any of the groups, nor did they show a significant correlation with the wound healing process [150]. As described above, a possible explanation for these findings is that the main effect of GH in this case could occur as a consequence of the local production of the hormone, something that was overlooked because it was not measured. A small wound on the back of the animal is not a stimulus strong enough to increase systemic GH, which seems to be related, as demonstrated, to more intense catabolic states.

A recent report from two prospective, randomized, double-blind, placebo-controlled Phase III trials conducted in Europe, which studied the effects of rhGH in critically ill burned adult patients, in an intensive care unit, revealed a significant increase in mortality among catabolic patients treated with rhGH (42 vs. 18%) [115]. GH, in fact, can increase cell adhesion molecules (CAM), since the serum of healthy patients treated with GH significantly increased the expression of VCAM-1 in cultured umbilical vein endothelial cells [151], and this could be the mechanism involved, but it must be taken into account that in these studies high doses of GH were used (10–20 times greater than the usual treatment dose), which would facilitate the appearance of side effects produced by the hormone. In contrast to these data, when the same study was carried out in burned children, no differences were found in mortality, but other beneficial effects were found.

**Author details**

Diego Caicedo1

**References**

Santiago de Compostela, Spain

DOI: 10.1038/sj.jid.5700701

DOI: 10.1111/wrr.12205

scitranslmed.3009337

s-2007-979060

537-538

\* and Jesús Devesa2

2 Scientific Direction, Medical Center Foltra, Teo, Spain

\*Address all correspondence to: diego.caicedo.valdes@sergas.es

Nature. 2008;**453**:314-321. DOI: 10.1038/nature07039

2010;**63**:e364-e369. DOI: 10.1016/j.bjps.2009.10.027

2004;**28**:377-381. DOI: 10.1177/0148607104028006377

model. Plastic and Reconstructive Surgery. 1999;**104**:470-475

1 Service of Vascular Surgery, University Hospital of Santiago de Compostela,

[1] Eming SA, Krieg T, Davidson JM, Hall RP. Inflammation in wound repair: Molecular and cellular mechanisms. The Journal of Investigative Dermatology. 2007;**127**:514-525.

Growth Hormone (GH) and Wound Healing http://dx.doi.org/10.5772/intechopen.80978 137

[2] Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration.

[3] Barrientos S, Brem H, Stojadinovic O, Tomic-Canic M. Clinical application of growth factors and cytokines in wound healing. Wound Repair and Regeneration. 2014;**22**:569-578.

[4] Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: Mechanisms, signaling, and translation. Science Translational Medicine. 2014;**6**:265sr6. DOI: 10.1126/

[5] Bitar MS. Insulin-like growth factor-1 reverses diabetes-induced wound healing impairment in rats. Hormone and Metabolic Research. 1997;**29**:383-386. DOI: 10.1055/

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[8] Weiming Z, Ning L, Jieshou L. Effect of recombinant human growth hormone and enteral nutrition on short bowel syndrome. JPEN Journal of Parenteral and Enteral Nutrition.

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### **6. Conclusion**

Despite all data here presented, it is necessary to remember that the patient with a problem in the wound healing needs to be addressed in a holistic way. That is, "we do not treat a hole in the patient, but the whole patient". Normal wounds in healthy people are not a problem. However, a delaying wound always appears in a patient with a morbid condition, normally in an elderly patient or that with a catabolic state or a chronic disease as diabetes mellitus. Therefore, using only a topical wound treatment seems to be an unrealistic approach to healing. However, a total approach will be more beneficial to not only accelerate the healing process but also decrease the possibility of a new wound.

The knowledge of the molecular aspects related to wound repair and tissue regeneration, as well as the whole circumstances affecting also the patients is crucial to success dealing with this topic.

We cannot overlook the high amount of data regarding the role of GH and its secretagogues, not only in the healing process, but also improving the pro-oxidant state of the patients. GH therapy is a cheap and well known drug, and may increase many growth factors when is locally used in wounds. Maybe the combination of appropriate doses of systemic GH and topical application in the wound would be a good option. The combination of GH or its secretagogues and IGF-1 in a topical way, could be also a beneficious approach for wounds repair.

### **Acknowledgements**

This chapter has been funded by the Carlos III Health Institute and the European Regional Development Fund (ISCIII-FEDER), Madrid, Spain, (grant number PI 13-00790). We thank the Foltra Medical Center for the transfer of some images for this chapter.

Diego Caicedo and Jesús Devesa participated in a similar way in the realization of this work.

### **Conflict of interest**

The authors declare that no conflict of interest exists.

### **Author details**

an intensive care unit, revealed a significant increase in mortality among catabolic patients treated with rhGH (42 vs. 18%) [115]. GH, in fact, can increase cell adhesion molecules (CAM), since the serum of healthy patients treated with GH significantly increased the expression of VCAM-1 in cultured umbilical vein endothelial cells [151], and this could be the mechanism involved, but it must be taken into account that in these studies high doses of GH were used (10–20 times greater than the usual treatment dose), which would facilitate the appearance of side effects produced by the hormone. In contrast to these data, when the same study was carried out in burned children,

Despite all data here presented, it is necessary to remember that the patient with a problem in the wound healing needs to be addressed in a holistic way. That is, "we do not treat a hole in the patient, but the whole patient". Normal wounds in healthy people are not a problem. However, a delaying wound always appears in a patient with a morbid condition, normally in an elderly patient or that with a catabolic state or a chronic disease as diabetes mellitus. Therefore, using only a topical wound treatment seems to be an unrealistic approach to healing. However, a total approach will be more beneficial to not only accelerate the healing pro-

The knowledge of the molecular aspects related to wound repair and tissue regeneration, as well as the whole circumstances affecting also the patients is crucial to success dealing with

We cannot overlook the high amount of data regarding the role of GH and its secretagogues, not only in the healing process, but also improving the pro-oxidant state of the patients. GH therapy is a cheap and well known drug, and may increase many growth factors when is locally used in wounds. Maybe the combination of appropriate doses of systemic GH and topical application in the wound would be a good option. The combination of GH or its secretagogues and IGF-1 in a topical way, could be also a beneficious approach for wounds repair.

This chapter has been funded by the Carlos III Health Institute and the European Regional Development Fund (ISCIII-FEDER), Madrid, Spain, (grant number PI 13-00790). We thank the

Diego Caicedo and Jesús Devesa participated in a similar way in the realization of this work.

Foltra Medical Center for the transfer of some images for this chapter.

The authors declare that no conflict of interest exists.

no differences were found in mortality, but other beneficial effects were found.

cess but also decrease the possibility of a new wound.

**6. Conclusion**

136 Wound Healing - Current Perspectives

this topic.

**Acknowledgements**

**Conflict of interest**

Diego Caicedo1 \* and Jesús Devesa2

\*Address all correspondence to: diego.caicedo.valdes@sergas.es

1 Service of Vascular Surgery, University Hospital of Santiago de Compostela, Santiago de Compostela, Spain

2 Scientific Direction, Medical Center Foltra, Teo, Spain

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**Chapter 9**

**Provisional chapter**

**Autologous Platelet-Rich Plasma and Mesenchymal**

**Autologous Platelet-Rich Plasma and Mesenchymal** 

Emerging autologous cellular therapies, utilizing platelet-rich plasma and mesenchymal stem cell applications, have the potential to play an adjunctive role in a standardized wound care treatment plan in patients suffering from chronic and recalcitrant wounds. The use of platelet-rich plasma growth is based on the fact that platelet growth factors can support the three phases of wound healing and then ultimately contribute to full wound closure. Mesenchymal stem cell-based therapies are also an attractive approach for the treatment of these difficult-to-heal wounds. This field of regenerative medicine focuses primarily on stem cells, which are specialized cells with the ability to self-renew and differentiate into multiple cell types. Mesenchymal stem cells can be isolated from bone marrow and adipose tissue via minimally manipulative and cell-processing techniques, at point of care. Both platelet-rich plasma and mesenchymal stem cell applications have the potential to become an effective and ideal autologous biological cell-based therapy, which can be applied to chronic wounds to effectively change the wound bed

**Keywords:** chronic wounds, microenvironment, wound healing, clinical platelet-rich plasma, platelet-rich plasma gel, mesenchymal stem cells, bone marrow concentrate,

In the Western world, approximately 1–2% of the population will develop a chronic wound during their lifetime. These numbers will increase worldwide as a result of the aging population, increase in diabetes and obesity, and cardiovascular disease as well [1–3]. In particular, chronic leg wounds represent the largest fraction, with venous and diabetic foot ulcers (DFUs)

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.80502

**Stem Cells for the Treatment of Chronic Wounds**

**Stem Cells for the Treatment of Chronic Wounds**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

microenvironment to enable and accelerate wound closure.

http://dx.doi.org/10.5772/intechopen.80502

Peter A. Everts

Peter A. Everts

**Abstract**

adipose tissue

**1. Introduction**


#### **Autologous Platelet-Rich Plasma and Mesenchymal Stem Cells for the Treatment of Chronic Wounds Autologous Platelet-Rich Plasma and Mesenchymal Stem Cells for the Treatment of Chronic Wounds**

DOI: 10.5772/intechopen.80502

Peter A. Everts Peter A. Everts

[141] Ardail D, Debon A, Perret-Vivancos C, Biol-N'Garagba M-C, Krantic S, Lobie PE, et al. Growth hormone internalization in mitochondria decreases respiratory chain activity.

[142] Nylander E, Grönbladh A, Zelleroth S, Diwakarla S, Nyberg F, Hallberg M. Growth hormone is protective against acute methadone-induced toxicity by modulating the NMDA receptor complex. Neuroscience. 2016;**339**:538-547. DOI: 10.1016/j.

[143] Keane J, Tajouri L, Gray B. The effect of growth hormone administration on the regulation of mitochondrial apoptosis in-vivo. International Journal of Molecular Sciences.

[144] Rojanathammanee L, Rakoczy S, Brown-Borg HM. Growth hormone alters the glutathione S-transferase and mitochondrial thioredoxin systems in long-living Ames dwarf mice. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences.

[145] Suzuki K, Yanagi K, Shimizu M, Wakamatsu S, Niitani T, Hosonuma S, et al. Effect of growth hormone replacement therapy on plasma diacron-reactive oxygen metabolites and endothelial function in Japanese patients: The GREAT clinical study. Endocrine

[146] Scacchi M, Valassi E, Pincelli AI, Fatti LM, Pecori Giraldi F, Ascoli P, et al. Increased lipid peroxidation in adult GH-deficient patients: Effects of short-term GH administration. Journal of Endocrinological Investigation. 2006;**29**:899-904. DOI: 10.1007/BF03349194

[147] Ikushima M, Rakugi H, Ishikawa K, Maekawa Y, Yamamoto K, Ohta J, et al. Antiapoptotic and anti-senescence effects of Klotho on vascular endothelial cells. Biochemical and Biophysical Research Communications. 2006;**339**:827-832. DOI: 10.1016/j.

[148] Gu Y, Zou Y, Aikawa R, Hayashi D, Kudoh S, Yamauchi T, et al. Growth hormone signalling and apoptosis in neonatal rat cardiomyocytes. Molecular and Cellular

[149] Kokoszko A, Lewiński A, Karbownik-Lewińska M. The role of growth hormone and insulin-like growth factor I in oxidative processes. Endokrynologia Polska. 2008;

[150] Zadeh ZA, Kesmati M, Galehdari H, Rezai A, Seyednezhad SM, Torabi M. Evaluation of the relationship between serum levels of insulin, cortisol and growth hormones with wound healing in normal and diabetic rats. Physiology and Pharmacology.

[151] Hansen TK, Fisker S, Dall R, Ledet T, Jørgensen JOL, Rasmussen LM. Growth hormone increases vascular cell adhesion molecule 1 expression: In vivo and in vitro evidence. The Journal of Clinical Endocrinology and Metabolism. 2004;**89**:909-916. DOI: 10.1210/

Neuroendocrinology. 2010;**91**:16-26. DOI: 10.1159/000268289

2015;**16**:12753-12772. DOI: 10.3390/ijms160612753

2014;**69**:1199-1211. DOI: 10.1093/gerona/glt178

Journal. 2017;**65**:101-111. DOI: 10.1507/endocrj.EJ17-0330

Biochemistry. 2001;**223**:35-46. DOI: 10.1023/A:1017941625858

neuroscience.2016.10.019

148 Wound Healing - Current Perspectives

bbrc.2005.11.094

**59**:496-501

2014;**18**:92-100

jc.2003-030223

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80502

#### **Abstract**

Emerging autologous cellular therapies, utilizing platelet-rich plasma and mesenchymal stem cell applications, have the potential to play an adjunctive role in a standardized wound care treatment plan in patients suffering from chronic and recalcitrant wounds. The use of platelet-rich plasma growth is based on the fact that platelet growth factors can support the three phases of wound healing and then ultimately contribute to full wound closure. Mesenchymal stem cell-based therapies are also an attractive approach for the treatment of these difficult-to-heal wounds. This field of regenerative medicine focuses primarily on stem cells, which are specialized cells with the ability to self-renew and differentiate into multiple cell types. Mesenchymal stem cells can be isolated from bone marrow and adipose tissue via minimally manipulative and cell-processing techniques, at point of care. Both platelet-rich plasma and mesenchymal stem cell applications have the potential to become an effective and ideal autologous biological cell-based therapy, which can be applied to chronic wounds to effectively change the wound bed microenvironment to enable and accelerate wound closure.

**Keywords:** chronic wounds, microenvironment, wound healing, clinical platelet-rich plasma, platelet-rich plasma gel, mesenchymal stem cells, bone marrow concentrate, adipose tissue

### **1. Introduction**

In the Western world, approximately 1–2% of the population will develop a chronic wound during their lifetime. These numbers will increase worldwide as a result of the aging population, increase in diabetes and obesity, and cardiovascular disease as well [1–3]. In particular, chronic leg wounds represent the largest fraction, with venous and diabetic foot ulcers (DFUs)

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

accounting for 70–90% of these ulcers [4]. Concomitantly, the costs of wound care services are rising, with the market of wound care products surpassing \$15 billion annually.

**3.1. Platelet clot and degranulation**

wound healing process.

factor (EGF) [15, 16].

**3.3. Proliferative cell activity**

parameter of surgical wounds.

**3.2. Inflammatory cell mechanisms**

With wounds and also after surgical incisions, repair begins with platelet clot formation, activation of the coagulation cascade, and subsequent platelet degranulation, releasing PGFs. After tissue damage, specific growth factors, including platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), are already being produced by the injured tissue cells [8]. Once a platelet plug is in place, platelets will get trapped in the fibrin mesh and start to degranulate, releasing PGFs, among other molecular components. Different growth factors have different characteristics and thus biological activities. Chemotactic and mitogenic capabilities have been demonstrated with regard to inflammatory cells (i.e., neutrophils, monocytes, and macrophages) [9]. At wound sites, PDGF subunits AB and transforming growth factor-β (TGF-β) are the most important growth factors initiating the

Autologous Platelet-Rich Plasma and Mesenchymal Stem Cells for the Treatment of Chronic…

http://dx.doi.org/10.5772/intechopen.80502

151

During the first 2 days of wound healing, an inflammatory process is initiated by the migration of inflammatory cells (neutrophils, macrophages, and T-lymphocytes) to the wound site to accomplish phagocytosis with the removal of bacteria, cellular debris, and damaged tissue. After the early inflammatory phase subsides, the predominant macrophage population assumes a wound healing phenotype that is characterized by the production of numerous growth factors and cytokines, including PDGF, transforming growth factor β1 (TGF-β1), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor-a (VEGF-a), which promote cell proliferation and blood vessel development [10, 11]. Activated macrophages can be classified into different phenotypes. M1-type, with antimicrobial and antitumor properties, is activated upon wound formation by inflammatory signals from interferon-y (IFN-y) and tumor necrosis factor-α (TNF-α) or when pathogen-associated molecular patterns or endogenous danger signals are recognized. Their main role is hostdefense mechanisms in the early healing process, releasing IL-12, promoting pro-inflammatory Th1 immune responses [12]. Conversely, the M2 macrophage phenotype downregulates inflammation and initiates tissue repair by releasing anti-inflammatory cytokines, such as IL-10 [13, 14]. Apoptotic wound neutrophils are ingested by these M2 macrophages, which release cytokines to promote macrophage recruitment and synthesize mediators critical to remodeling and angiogenesis, including TGF-β, VEGF, and epidermal growth

Angiogenesis and fibroplasia are the next phases of wound healing, the proliferative phase. New blood vessel formation and the migration of fibroblasts, which deposit new ECM, are facilitated by EGF, keratinocyte growth factors (KGFs), and TGF-α [17, 18]. Keratinocytes migrate from the wound edges between the dermis facilitated by the production of collagenase and other proteases in the epidermis. Fibroblasts migrate, proliferate, and produce ECM in the wound bed, resulting in early granulation and tissue formation [19]. This process leads to an early increase in wound breaking strength, which is an important wound healing

There is an unmet need to stimulate the healing of acute and chronic wounds to a level that is not possible with the current standard care measures and therapy approaches.

An area of medicine that holds promise for the treatment of recalcitrant and difficult-to-heal wounds is regenerative medicine. Therefore, it is critical to use more effective and efficient treatment options from patient and cost perspectives. The use of autologous biologics, such as platelet-rich plasma (PRP)- and mesenchymal stem cell (MSC)-based therapies, holds substantial promise to enhance tissue regeneration and repair in many different diseases and could therefore also be potentially effective in chronic wound care management strategies.

This review aimed to describe the scientific rationale and clinical experiences of two different autologous biological therapies to support the healing of chronic and recalcitrant wounds. First is the use of clinical PRP, prepared at point of care using a dual spin buffy coat device, and second is the local application of MSCs, derived from either bone marrow concentrate or adipose tissue.

### **2. Skin layers**

The skin consists of three layers. The epidermis is the most outer layer, consisting of multilayered epithelium extending from the basement membrane, which separates the dermis from the air. The basement membrane contains progenitor stem cells, which undergo continuous self-renewal and differentiate into keratinocytes. The keratinocytes migrate towards the surface of the skin where they normally undergo terminal differentiation and maturation [5]. The dermis is the thickest layer, just below the epidermis. The dermis is a connective tissue, composed of the extracellular matrix (ECM), fibroblasts, vascular endothelial cells, and skin appendages such as sweat glands and hair follicles [6]. Fibroblasts are cells that secrete molecules including collagen and elastin, which provide mechanical strength and elasticity to the skin. The third layer is the hypodermis, which is underneath the dermis and composed of adipose tissue, providing insulation and cushioning between the skin and bone, muscle, tendon, and other skeletal structures [6]. A skin defect is repaired through cutaneous wound healing processes to recover loss of integrity, facilitate tensile strength, and provide a barrier for the skin [7]. Normal cutaneous wound repair is a multifaceted process.

### **3. Normal wound healing and cellular mechanisms**

Wound healing is a well-orchestrated and complex series of events involving cell-cell and cell-matrix interactions, with platelet growth factors (PGFs), their dedicated receptors, and stem cells serving as messengers to regulate the various processes involved. The "wound healing" process as a whole has to be considered from the point of view of the type of lesion, which will in turn dictate the degree of healing that can be obtained. A partial thickness skin abrasion heals almost entirely by epithelialization, whereas deep pressure chronic ulcers rely mainly on matrix synthesis, angiogenesis, fibroplasia, and wound contraction.

### **3.1. Platelet clot and degranulation**

accounting for 70–90% of these ulcers [4]. Concomitantly, the costs of wound care services are

There is an unmet need to stimulate the healing of acute and chronic wounds to a level that is

An area of medicine that holds promise for the treatment of recalcitrant and difficult-to-heal wounds is regenerative medicine. Therefore, it is critical to use more effective and efficient treatment options from patient and cost perspectives. The use of autologous biologics, such as platelet-rich plasma (PRP)- and mesenchymal stem cell (MSC)-based therapies, holds substantial promise to enhance tissue regeneration and repair in many different diseases and could therefore also be potentially effective in chronic wound care management strategies.

This review aimed to describe the scientific rationale and clinical experiences of two different autologous biological therapies to support the healing of chronic and recalcitrant wounds. First is the use of clinical PRP, prepared at point of care using a dual spin buffy coat device, and second is the local application of MSCs, derived from either bone marrow concentrate or adipose tissue.

The skin consists of three layers. The epidermis is the most outer layer, consisting of multilayered epithelium extending from the basement membrane, which separates the dermis from the air. The basement membrane contains progenitor stem cells, which undergo continuous self-renewal and differentiate into keratinocytes. The keratinocytes migrate towards the surface of the skin where they normally undergo terminal differentiation and maturation [5]. The dermis is the thickest layer, just below the epidermis. The dermis is a connective tissue, composed of the extracellular matrix (ECM), fibroblasts, vascular endothelial cells, and skin appendages such as sweat glands and hair follicles [6]. Fibroblasts are cells that secrete molecules including collagen and elastin, which provide mechanical strength and elasticity to the skin. The third layer is the hypodermis, which is underneath the dermis and composed of adipose tissue, providing insulation and cushioning between the skin and bone, muscle, tendon, and other skeletal structures [6]. A skin defect is repaired through cutaneous wound healing processes to recover loss of integrity, facilitate tensile strength, and provide a barrier

Wound healing is a well-orchestrated and complex series of events involving cell-cell and cell-matrix interactions, with platelet growth factors (PGFs), their dedicated receptors, and stem cells serving as messengers to regulate the various processes involved. The "wound healing" process as a whole has to be considered from the point of view of the type of lesion, which will in turn dictate the degree of healing that can be obtained. A partial thickness skin abrasion heals almost entirely by epithelialization, whereas deep pressure chronic ulcers rely

for the skin [7]. Normal cutaneous wound repair is a multifaceted process.

mainly on matrix synthesis, angiogenesis, fibroplasia, and wound contraction.

**3. Normal wound healing and cellular mechanisms**

rising, with the market of wound care products surpassing \$15 billion annually.

not possible with the current standard care measures and therapy approaches.

**2. Skin layers**

150 Wound Healing - Current Perspectives

With wounds and also after surgical incisions, repair begins with platelet clot formation, activation of the coagulation cascade, and subsequent platelet degranulation, releasing PGFs. After tissue damage, specific growth factors, including platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), are already being produced by the injured tissue cells [8]. Once a platelet plug is in place, platelets will get trapped in the fibrin mesh and start to degranulate, releasing PGFs, among other molecular components. Different growth factors have different characteristics and thus biological activities. Chemotactic and mitogenic capabilities have been demonstrated with regard to inflammatory cells (i.e., neutrophils, monocytes, and macrophages) [9]. At wound sites, PDGF subunits AB and transforming growth factor-β (TGF-β) are the most important growth factors initiating the wound healing process.

### **3.2. Inflammatory cell mechanisms**

During the first 2 days of wound healing, an inflammatory process is initiated by the migration of inflammatory cells (neutrophils, macrophages, and T-lymphocytes) to the wound site to accomplish phagocytosis with the removal of bacteria, cellular debris, and damaged tissue. After the early inflammatory phase subsides, the predominant macrophage population assumes a wound healing phenotype that is characterized by the production of numerous growth factors and cytokines, including PDGF, transforming growth factor β1 (TGF-β1), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor-a (VEGF-a), which promote cell proliferation and blood vessel development [10, 11]. Activated macrophages can be classified into different phenotypes. M1-type, with antimicrobial and antitumor properties, is activated upon wound formation by inflammatory signals from interferon-y (IFN-y) and tumor necrosis factor-α (TNF-α) or when pathogen-associated molecular patterns or endogenous danger signals are recognized. Their main role is hostdefense mechanisms in the early healing process, releasing IL-12, promoting pro-inflammatory Th1 immune responses [12]. Conversely, the M2 macrophage phenotype downregulates inflammation and initiates tissue repair by releasing anti-inflammatory cytokines, such as IL-10 [13, 14]. Apoptotic wound neutrophils are ingested by these M2 macrophages, which release cytokines to promote macrophage recruitment and synthesize mediators critical to remodeling and angiogenesis, including TGF-β, VEGF, and epidermal growth factor (EGF) [15, 16].

### **3.3. Proliferative cell activity**

Angiogenesis and fibroplasia are the next phases of wound healing, the proliferative phase. New blood vessel formation and the migration of fibroblasts, which deposit new ECM, are facilitated by EGF, keratinocyte growth factors (KGFs), and TGF-α [17, 18]. Keratinocytes migrate from the wound edges between the dermis facilitated by the production of collagenase and other proteases in the epidermis. Fibroblasts migrate, proliferate, and produce ECM in the wound bed, resulting in early granulation and tissue formation [19]. This process leads to an early increase in wound breaking strength, which is an important wound healing parameter of surgical wounds.

#### **3.4. Epithelialization**

The final phase of wound closure is epithelialization, characterized by the exit of inflammatory cells, a decrease in growth factor release, an increase in the ratio of collagen deposition to fibroblasts, and the cross-linking and organization of collagen molecules. Remodeling takes place over a much longer period of time in which the newly formed tissue is reorganized for higher tensile strength [17]. Hence, the normal wound healing process constitutes a delicate balance of cells secreting and regulating the many cytokines, chemokines, proteins, and growth factors.

To succeed in the reparative phase of wound healing, chronic wound care treatment strategies should have a dual approach. This includes the treatment of any underlying systemic disease and wound-microenvironmental tissue therapy. Evidence-based principles for local and systemic wound care management exist in the literature but are not further discussed in this chapter [28, 29]. In these traditional wound care treatment options, the application of autologous cellular biologics, such as platelet-rich plasma (PRP) growth factor therapy and

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PRP therapies have been used for a variety of indications, for more than 30 years. More than 10,000 references are currently in PubMed, using the search term platelet-rich plasma. These countless applications have given rise to considerable interest in the potential of autologous PRP in numerous regenerative medicine indications. In the last decade, numerous studies and reviews have been published on PRP therapies as a biological, adjunctive, therapy option

Platelets are formed from megakaryocytes and are synthesized in bone marrow by pinching off from their progenitor cell. Thereafter, platelets are released into the peripheral circulation. Platelets are small, anucleate, discoid blood cells (1–3 μm), with an in vivo half-life of

blood. Platelets have a ring of contractile microtubules (cytoskeleton) around their periphery, containing actin and myosin. Inside platelets, there are a number of intracellular structures, including α-granules comprising PGFs and angiogenesis regulators and dense granules containing ADP, ATP, serotonin, histamine, calcium, and mitochondria. Other complex platelet biological components include adhesins and coagulation and immunological molecules. These molecules serve a multitude of functions, first within the clotting cascade and finally as initiators of tissue-healing processes. Platelets are equipped with an extensively invaginated membrane with an intricate canalicular system, which is in constant contact with the extracellular fluid [30]. Normally, platelets are in a resting state, non-thrombogenic. They require a 'trigger' before they become a potent and an active player in hemostasis and an accelerator of

When PRP is indicated to treat recalcitrant wounds, in the vast majority of these applications, PRP is delivered as a topical semiviscous coagulum so that concentrated platelets and various cytokines can adhere to the surface of the wound bed. For platelets to stick to a prepared wound bed, the PRP sample needs to be activated, thereby changing from a resting, inactive state to an active form. The platelet discoid shape changes, with the development of pseudopodia (**Figure 1**). This change in platelet shape and configuration is facilitated by the

/mL of circulating

7 days. The average platelet count in adults ranges from 150 to 350 × 10<sup>6</sup>

the wound healing cascade, depending upon the microenvironmental effectors.

**5.2. Platelet-rich plasma gel, growth factors, and platelet receptors**

MSC applications, is not anticipated, but discussed in detail here below.

**5. Platelets in platelet-rich plasma therapy**

in the management of chronic wounds.

**5.1. Platelets and their intracellular content**

### **4. Chronic wound healing characteristics**

A vast majority of chronic wounds begin as minor traumatic injuries, such as penetrating injuries, insect bites, or even simple scratches of dry skin. Normally, these wounds heal within a few days/weeks. However, aging and underlying pathologies, such as diabetes-induced and nondiabetic neuropathies, can lead to the development of poor or non-healing wounds [20]. Furthermore, arterial and venous vascular pathologies with hyperglycemia could further complicate the wound healing process. Chronic wounds are chronically inflamed and can be characterized by dysfunctional cellular events and aberrant cytokine and growth factor activities, leading to failure of normal wound closure with the potential for infections [19, 21]. Wound infections trigger extensive recruitment of inflammatory cells, particularly resulting in high concentrations of neutrophils, serine elastase, and inflammatory macrophages, while cell extravasation is facilitated by disproportionate expression of vascular cell adhesion molecules and interstitial cell adhesion molecules by resident endothelial cells. The accumulated inflammatory cells in the wound bed lead to protease activity, with elevated levels of matrix metalloproteases (MMP) 2, 8, and 9, successively prolonging inflammation [22]. Moreover, tissue inhibitor of MMP 1 is decreased in non-healing wounds, thereby increasing collagenolytic activity. Furthermore, neutrophils also produce various reactive oxygen species (ROS), inducing considerable oxidative stress and thus damaging structural elements of the ECM and wound biochemical microenvironment [23]. Nonetheless, together with proinflammatory cytokines, an abnormally prolonged inflammatory phase will result in wound chronicity, which might lead to premature cell senescence [24]. Tissue hypoxia and repeated wound infections will continue to promote MMP enzyme activity, resulting in decreased growth factor functions, and fibrin deficits will transpire. It has been demonstrated that a chronically inflamed wound microenvironment subjects proteins and cytokines to degradation and sequestration, in particular the growth factors PDGF, EGF, and TGF-β [25, 26]. In addition, Cooper et al. demonstrated that a number of growth factors were markedly reduced in wound fluids from chronic wounds as compared to acute wounds [27]. Moreover, FGF and TGF-β concentrations were significantly downregulated in chronic wounds. Decreased growth factor levels and upregulation of proinflammatory cytokines and chemokines will worsen normal progression of wound healing and consequently the potential for full wound closure. In chronic wounds, the microenvironment must be modified to be an active and effective intervention, eliminating the factors that impede healing.

To succeed in the reparative phase of wound healing, chronic wound care treatment strategies should have a dual approach. This includes the treatment of any underlying systemic disease and wound-microenvironmental tissue therapy. Evidence-based principles for local and systemic wound care management exist in the literature but are not further discussed in this chapter [28, 29]. In these traditional wound care treatment options, the application of autologous cellular biologics, such as platelet-rich plasma (PRP) growth factor therapy and MSC applications, is not anticipated, but discussed in detail here below.

## **5. Platelets in platelet-rich plasma therapy**

**3.4. Epithelialization**

152 Wound Healing - Current Perspectives

growth factors.

**4. Chronic wound healing characteristics**

intervention, eliminating the factors that impede healing.

The final phase of wound closure is epithelialization, characterized by the exit of inflammatory cells, a decrease in growth factor release, an increase in the ratio of collagen deposition to fibroblasts, and the cross-linking and organization of collagen molecules. Remodeling takes place over a much longer period of time in which the newly formed tissue is reorganized for higher tensile strength [17]. Hence, the normal wound healing process constitutes a delicate balance of cells secreting and regulating the many cytokines, chemokines, proteins, and

A vast majority of chronic wounds begin as minor traumatic injuries, such as penetrating injuries, insect bites, or even simple scratches of dry skin. Normally, these wounds heal within a few days/weeks. However, aging and underlying pathologies, such as diabetes-induced and nondiabetic neuropathies, can lead to the development of poor or non-healing wounds [20]. Furthermore, arterial and venous vascular pathologies with hyperglycemia could further complicate the wound healing process. Chronic wounds are chronically inflamed and can be characterized by dysfunctional cellular events and aberrant cytokine and growth factor activities, leading to failure of normal wound closure with the potential for infections [19, 21]. Wound infections trigger extensive recruitment of inflammatory cells, particularly resulting in high concentrations of neutrophils, serine elastase, and inflammatory macrophages, while cell extravasation is facilitated by disproportionate expression of vascular cell adhesion molecules and interstitial cell adhesion molecules by resident endothelial cells. The accumulated inflammatory cells in the wound bed lead to protease activity, with elevated levels of matrix metalloproteases (MMP) 2, 8, and 9, successively prolonging inflammation [22]. Moreover, tissue inhibitor of MMP 1 is decreased in non-healing wounds, thereby increasing collagenolytic activity. Furthermore, neutrophils also produce various reactive oxygen species (ROS), inducing considerable oxidative stress and thus damaging structural elements of the ECM and wound biochemical microenvironment [23]. Nonetheless, together with proinflammatory cytokines, an abnormally prolonged inflammatory phase will result in wound chronicity, which might lead to premature cell senescence [24]. Tissue hypoxia and repeated wound infections will continue to promote MMP enzyme activity, resulting in decreased growth factor functions, and fibrin deficits will transpire. It has been demonstrated that a chronically inflamed wound microenvironment subjects proteins and cytokines to degradation and sequestration, in particular the growth factors PDGF, EGF, and TGF-β [25, 26]. In addition, Cooper et al. demonstrated that a number of growth factors were markedly reduced in wound fluids from chronic wounds as compared to acute wounds [27]. Moreover, FGF and TGF-β concentrations were significantly downregulated in chronic wounds. Decreased growth factor levels and upregulation of proinflammatory cytokines and chemokines will worsen normal progression of wound healing and consequently the potential for full wound closure. In chronic wounds, the microenvironment must be modified to be an active and effective PRP therapies have been used for a variety of indications, for more than 30 years. More than 10,000 references are currently in PubMed, using the search term platelet-rich plasma. These countless applications have given rise to considerable interest in the potential of autologous PRP in numerous regenerative medicine indications. In the last decade, numerous studies and reviews have been published on PRP therapies as a biological, adjunctive, therapy option in the management of chronic wounds.

### **5.1. Platelets and their intracellular content**

Platelets are formed from megakaryocytes and are synthesized in bone marrow by pinching off from their progenitor cell. Thereafter, platelets are released into the peripheral circulation. Platelets are small, anucleate, discoid blood cells (1–3 μm), with an in vivo half-life of 7 days. The average platelet count in adults ranges from 150 to 350 × 10<sup>6</sup> /mL of circulating blood. Platelets have a ring of contractile microtubules (cytoskeleton) around their periphery, containing actin and myosin. Inside platelets, there are a number of intracellular structures, including α-granules comprising PGFs and angiogenesis regulators and dense granules containing ADP, ATP, serotonin, histamine, calcium, and mitochondria. Other complex platelet biological components include adhesins and coagulation and immunological molecules. These molecules serve a multitude of functions, first within the clotting cascade and finally as initiators of tissue-healing processes. Platelets are equipped with an extensively invaginated membrane with an intricate canalicular system, which is in constant contact with the extracellular fluid [30]. Normally, platelets are in a resting state, non-thrombogenic. They require a 'trigger' before they become a potent and an active player in hemostasis and an accelerator of the wound healing cascade, depending upon the microenvironmental effectors.

### **5.2. Platelet-rich plasma gel, growth factors, and platelet receptors**

When PRP is indicated to treat recalcitrant wounds, in the vast majority of these applications, PRP is delivered as a topical semiviscous coagulum so that concentrated platelets and various cytokines can adhere to the surface of the wound bed. For platelets to stick to a prepared wound bed, the PRP sample needs to be activated, thereby changing from a resting, inactive state to an active form. The platelet discoid shape changes, with the development of pseudopodia (**Figure 1**). This change in platelet shape and configuration is facilitated by the

Following a PRP-G application on a debrided wound bed, fibrinolysis occurs over time and the platelets start to disintegrate, subsequently releasing their PGFs and other plasma proteins. This is the onset of PGF-mediated stimulation of cell proliferation, promotion of cell differentiation and chemotaxis, and induction of migration of various (stem) cells to the wound area [31, 32]. The rational for applying PRP-G to wound bed tissues is the delivery of a diversity of concentrated platelet-derived growth factors and other biological mediators (e.g., adhesive proteins, fibrinogen, fibronectin, vitronectin, and thrombospondin-1) to mimic, and accelerate, physiologic wound healing cascades and regenerative tissue repair

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After disintegration of the topical semiviscous coagulum, PGFs and other platelet molecules accumulate in the ECM and the released growth factors interact and bind with a specific platelet tyrosine kinase receptor (TKR), present on the outer surface of cell membranes (ligand-receptor interaction). TKRs are membrane spanning proteins that extend into the cytoplasm of the cell. After growth factors interact with their specific cell membrane TKR, activation of (inactive) messenger proteins in the cytoplasm occurs. The activated TKR cytoplasmic tail now serves as a binding site for the messenger proteins. An activated protein is generated through a signaling cascade, capable of entering the cell nucleus, where it triggers the genes responsible for controlling cell division. Subsequently, transcription of mRNA is induced, producing a biological response that initiates cascades that induce tissue repair and

**Figure 3.** Illustrative representation of the mechanisms involved in platelet growth factor binding to their receptor. Specific platelet growth factors find their dedicated cell membrane tyrosine kinase receptor on the outside cell membrane. Following coupling, active enzymatic intracellular signaling occurs, with transmission to the cell nucleus via messenger

processes (**Figure 2**) [33].

regeneration (**Figure 3**) [34, 35].

ribonucleic acid.

**Figure 1.** Graphic illustration of non-activated and an activated platelet. (A) A normal, discoid, resting platelet in a non-activated state, with platelet glycoprotein surface receptors on the outside of the platelet. (B) Following activation, the platelet shape is changed, with the development of pseudopods and the release of platelet granules and other intracellular storage vesicles via the opened canalicular system into the local microenvironmental milieu.

addition of platelet agonists (e.g., autologous or bovine thrombin, calcium, tissue factor, or other platelet-activating proteins) to a volume of PRP. Platelet activation and aggregation then leads to the creation of the semiviscous coagulum, that is, platelet clot, referred to in the literature as platelet-rich plasma gel (PRP-G). In this constitution, PRP-G can then be exogenously applied to soft tissues and chronic wounds.

**Figure 2.** Schematic illustration of the activities of platelet growth factors during the different stages of the wound healing cascade. The numbers indicate the sequence of the phased stages of the wound healing process in which platelet growth factors have pivotal roles (EGF, epidermal growth factor; FGF, fibroblast growth factor; PDGF, platelet derived growth factor; TGF-β, transforming growth factor beta; VEGF, vascular endothelial growth factor).

Following a PRP-G application on a debrided wound bed, fibrinolysis occurs over time and the platelets start to disintegrate, subsequently releasing their PGFs and other plasma proteins. This is the onset of PGF-mediated stimulation of cell proliferation, promotion of cell differentiation and chemotaxis, and induction of migration of various (stem) cells to the wound area [31, 32]. The rational for applying PRP-G to wound bed tissues is the delivery of a diversity of concentrated platelet-derived growth factors and other biological mediators (e.g., adhesive proteins, fibrinogen, fibronectin, vitronectin, and thrombospondin-1) to mimic, and accelerate, physiologic wound healing cascades and regenerative tissue repair processes (**Figure 2**) [33].

After disintegration of the topical semiviscous coagulum, PGFs and other platelet molecules accumulate in the ECM and the released growth factors interact and bind with a specific platelet tyrosine kinase receptor (TKR), present on the outer surface of cell membranes (ligand-receptor interaction). TKRs are membrane spanning proteins that extend into the cytoplasm of the cell. After growth factors interact with their specific cell membrane TKR, activation of (inactive) messenger proteins in the cytoplasm occurs. The activated TKR cytoplasmic tail now serves as a binding site for the messenger proteins. An activated protein is generated through a signaling cascade, capable of entering the cell nucleus, where it triggers the genes responsible for controlling cell division. Subsequently, transcription of mRNA is induced, producing a biological response that initiates cascades that induce tissue repair and regeneration (**Figure 3**) [34, 35].

**Figure 3.** Illustrative representation of the mechanisms involved in platelet growth factor binding to their receptor. Specific platelet growth factors find their dedicated cell membrane tyrosine kinase receptor on the outside cell membrane. Following coupling, active enzymatic intracellular signaling occurs, with transmission to the cell nucleus via messenger ribonucleic acid.

**Figure 2.** Schematic illustration of the activities of platelet growth factors during the different stages of the wound healing cascade. The numbers indicate the sequence of the phased stages of the wound healing process in which platelet growth factors have pivotal roles (EGF, epidermal growth factor; FGF, fibroblast growth factor; PDGF, platelet derived

addition of platelet agonists (e.g., autologous or bovine thrombin, calcium, tissue factor, or other platelet-activating proteins) to a volume of PRP. Platelet activation and aggregation then leads to the creation of the semiviscous coagulum, that is, platelet clot, referred to in the literature as platelet-rich plasma gel (PRP-G). In this constitution, PRP-G can then be exog-

intracellular storage vesicles via the opened canalicular system into the local microenvironmental milieu.

**Figure 1.** Graphic illustration of non-activated and an activated platelet. (A) A normal, discoid, resting platelet in a non-activated state, with platelet glycoprotein surface receptors on the outside of the platelet. (B) Following activation, the platelet shape is changed, with the development of pseudopods and the release of platelet granules and other

enously applied to soft tissues and chronic wounds.

154 Wound Healing - Current Perspectives

growth factor; TGF-β, transforming growth factor beta; VEGF, vascular endothelial growth factor).


**Table 1.** Comprehensive description of the most known platelet ɑ-granule components as they appear in PRP.

**6. PRP device technology and cellular formulations of clinical PRP**

**6.1. Autologous blood predonation and PRP processing devices**

**Proteins-chemokines-cytokines Biological activities** Adhesive proteins Cell contact interactions

Mitogenic factors Increases angiogenesis

Chemokines and cytokines Cellular interaction

Membrane glycoproteins Platelet aggregation

Granules Capillary permeability

**Table 2.** Non-platelet growth factor-related adjunctive effects of PRP therapy.

Proteases and anti-proteases Angiogenesis

PRP treatment protocols have evolved immensely over the past 20 years. Through laboratory, experimental, and clinical research, followed by more recent meta-analyses, physicians, medical practitioners, and scientists have gained a better understanding of platelets in PRP cellular physiology. The platelet secretome consists of all the proteins that are released upon platelet activation, which can be measured through proteomic-based techniques [49]. This proteomic profiling has increased our current understanding of the functional importance of the platelet granule contents [50], especially with regard to the biological cellular functions of the multifaceted platelet secretome and other plasma constituents, affecting PRP treatment outcomes.

Extracellular matrix composition

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Vascular remodeling Cellular regulation Cellular behavior

Cell proliferation Chemotaxis

Vascular remodeling Bone formation

Platelet adhesion Inflammation

Platelet and leukocyte interaction

Vascular local regulation

The starting point for any PRP preparation is whole blood. At point of care, a fresh unit of autologous blood is drawn via a phlebotomy, following standard operating procedures.

The median cubital vein is often used as this is an easily accessible and superficial vein, enabling the introduction of 18- to 21-gauge butterfly systems. Blood is collected in a syringe

A synopsis of the most well-known PRP growth factors is provided in **Table 1**, along with a description of the growth factor sources and their individual specific functions [36–47]. Besides the numerous activities of their growth factors, platelets also contribute to many adjunctive and supportive activities (**Table 2**) via paracrine, autocrine, and endocrine modes of actions [35, 37]. Because of these unique modes of action, PGFs are capable of exerting effects on multiple cell types, showing a series of morphometric and mitogenic functions. The morphometric growth factors, involved in bone growth, can turn undifferentiated multipotent MSCs into immature and mature osteoprogenitor cells through the presence of the so-called bone morphogenetic proteins (BMPs) [48]. Most PGFs have mitogenic actions that increase the population of healing cells and degranulate by mitogenesis.


**Table 2.** Non-platelet growth factor-related adjunctive effects of PRP therapy.

### **6. PRP device technology and cellular formulations of clinical PRP**

PRP treatment protocols have evolved immensely over the past 20 years. Through laboratory, experimental, and clinical research, followed by more recent meta-analyses, physicians, medical practitioners, and scientists have gained a better understanding of platelets in PRP cellular physiology. The platelet secretome consists of all the proteins that are released upon platelet activation, which can be measured through proteomic-based techniques [49]. This proteomic profiling has increased our current understanding of the functional importance of the platelet granule contents [50], especially with regard to the biological cellular functions of the multifaceted platelet secretome and other plasma constituents, affecting PRP treatment outcomes.

#### **6.1. Autologous blood predonation and PRP processing devices**

A synopsis of the most well-known PRP growth factors is provided in **Table 1**, along with a description of the growth factor sources and their individual specific functions [36–47]. Besides the numerous activities of their growth factors, platelets also contribute to many adjunctive and supportive activities (**Table 2**) via paracrine, autocrine, and endocrine modes of actions [35, 37]. Because of these unique modes of action, PGFs are capable of exerting effects on multiple cell types, showing a series of morphometric and mitogenic functions. The morphometric growth factors, involved in bone growth, can turn undifferentiated multipotent MSCs into immature and mature osteoprogenitor cells through the presence of the so-called bone morphogenetic proteins (BMPs) [48]. Most PGFs have mitogenic actions that

Interleukin-8, IL-8 Platelets, macrophage, epithelial cells Stimulates mitosis of epidermal cells and supports

**Table 1.** Comprehensive description of the most known platelet ɑ-granule components as they appear in PRP.

**Growth factor sources Biological activities**

Platelets, endothelial cells Increases angiogenesis and vessel permeability;

Platelets, macrophages, monocytes Stimulates endothelial chemotaxis/angiogenesis;

Mitogenic for mesenchymal cells and osteoblasts; stimulates chemotaxis and mitogenesis in fibroblast/ glial/smooth muscle cells; regulates collagenase secretion and collagen synthesis; stimulates macrophage and neutrophil chemotaxis

Stimulates undifferentiated mesenchymal cell proliferation; regulates endothelial, fibroblastic, and osteoblastic mitogenesis; regulates collagen synthesis and collagenase secretion; regulates mitogenic effects of other growth factors; stimulates endothelial chemotaxis and angiogenesis; inhibits macrophage and lymphocyte proliferation

stimulates mitogenesis for endothelial cells

regulates collagenase secretion; stimulates epithelial/mesenchymal mitogenesis

Promotes growth and differentiation of chondrocytes and osteoblasts; mitogenic for mesenchymal cells, chondrocytes, and osteoblasts

fibrosis, and platelet adhesion

and differentiation of osteoblasts

angiogenesis

Promotes angiogenesis, cartilage regeneration,

Chemotactic for fibroblasts and stimulates protein synthesis. Enhances bone formation by proliferation

Platelets, osteoblasts, endothelial cells, macrophages, monocytes, smooth

Platelets, extracellular matrix of bone, cartilage matrix, macrophages/ monocytes, and neutrophils

Platelets, macrophages, mesenchymal cells, chondrocytes, osteoblasts

Platelets through endocytosis from extracellular environment in bone

Plasma, epithelial cells, endothelial cells, fibroblasts, osteoblasts, bone matrix

marrow

muscle cells

increase the population of healing cells and degranulate by mitogenesis.

**Platelet growth factor**

156 Wound Healing - Current Perspectives

Platelet-derived growth factor, PDGF(a-b)

Transforming growth factor, TGF(ɑ-β)

Vascular endothelial growth factor, VEGF

Epidermal growth factor, EGF

Fibroblast growth factor, FGF(a-b)

Connective tissue growth factor, CTGF

Insulin-like growth factor-1, IGF-1

Adapted from Everts [130].

The starting point for any PRP preparation is whole blood. At point of care, a fresh unit of autologous blood is drawn via a phlebotomy, following standard operating procedures.

The median cubital vein is often used as this is an easily accessible and superficial vein, enabling the introduction of 18- to 21-gauge butterfly systems. Blood is collected in a syringe containing an anticoagulant to prevent clotting. The blood predonation volume depends on the PRP device of choice to prepare PRP and the volume needed for specific single, or multiple, wound care treatments in the same patient. Directly after blood collection, the PRP centrifugation process should be initiated in order to produce a sample of PRP.

Currently, physicians can choose from more than 30 PRP processing systems. However, a lack of consensus on standardizing PRP has contributed to the variation in PRP devices, which produce dissimilar platelet concentrations and cellular compositions [51, 52].

Optimal blood separation is best safeguarded by so-called double-spin PRP centrifuges with dedicated disposable platelet concentration devices. These double-spin PRP devices create a layered buffy coat stratum based on different centrifugal forces and specific gravities and densities of the individual blood components (**Figure 4**). Single-spin devices, or plasma-PRP

> devices, prepare a product from the acellular plasma layer, excluding erythrocytes and leukocytes, while collecting as many platelets as possible from the plasma layer [53]. These differences in cellular compositions, and thus PRP characteristics, have recently been recognized in the literature [54]. Marques et al. found that inferior treatment outcomes following PRP applications correlated directly with poor quality and inconsistent PRP products [55]. Therefore, PRP devices should be versatile and compliant to enable the production of different PRP formulations, while maintaining supraphysiologic platelet numbers (**Figure 5**). More specifically, the final cellular PRP treatment sample should be tailored to serve treatment protocols

> **Figure 5.** Whole blood and PRP smears with non-activated platelet. (A) Peripheral blood smear of whole blood inside the circle, platelets are visible (platelet count of the smear is 276,000/μL), next to white blood cells, erythrocytes, and fibrin. (B) Platelet-rich plasma smear. A high density of platelets inside the circle, with minimal leukocytes and erythrocytes

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contingent to wound bed condition, wound size and depth, and undermining tissue.

compared to baseline values, with minimal red blood cell contamination.

PRP can be characterized as a complex composition of autologous multicellular components in a small volume of plasma, with a substantial supraphysiologic concentration of platelets

Clinical PRP (C-PRP) contains a clinical dose of concentrated platelets in a treatment sample. Marx demonstrated enhancement of bone and soft tissue healing with a minimum platelet count of 1 ×

mL are needed for inducing a functional angiogenic response, via endothelial cell activity, in tissue repair mechanisms [57]. Therefore, to significantly induce an angiogenic response in circulatory compromised chronic wounds, C-PRP should contain at least 7.5 billion deliverable platelets in a 5-mL treatment sample. These platelets should then be able to release their entire content after (tissue) activation has occurred, as visualized in **Figure 6** by electron microscopic imaging. Furthermore, this platelet dose will correspondingly induce cell proliferation and cell migration. Ultimately, an increase in wound bed microcirculation would contribute to ECM remodeling and

platelets/

platelets/

/mL [56]. Furthermore, Giusti et al. revealed in an experimental study that 1.5 × 109

wound epithelialization [58]. Consequently, C-PRP containing a platelet dose of 1.5 × 109

mL has the ability to stimulate (neo)angiogenesis and elicit the healing of chronic wounds.

Leukocytes have a great impact on the intrinsic biology of chronic wounds because of their immune and host-defense mechanisms. Therefore, the presence of leukocytes in

**6.2. Definition of clinical-PRP and platelet dose**

(platelet count of 2,208,000/μL, prepared with the EmCyte System).

109

**6.3. Leukocytes in C-PRP**

**Figure 4.** Cellular density separation of whole blood by centrifugation. After the first centrifugation procedure, the whole blood components are separated in the PRP device from the plasma as a result of the different densities in two basic layers. The top layer is the platelet plasma suspension, consisting of plasma and the multicomponent buffy coat layer, containing platelets, monocytes, lymphocytes, and neutrophils. The second layer consists of erythrocyte pack. The range of the specific cell densities varies between individuals.

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**Figure 5.** Whole blood and PRP smears with non-activated platelet. (A) Peripheral blood smear of whole blood inside the circle, platelets are visible (platelet count of the smear is 276,000/μL), next to white blood cells, erythrocytes, and fibrin. (B) Platelet-rich plasma smear. A high density of platelets inside the circle, with minimal leukocytes and erythrocytes (platelet count of 2,208,000/μL, prepared with the EmCyte System).

devices, prepare a product from the acellular plasma layer, excluding erythrocytes and leukocytes, while collecting as many platelets as possible from the plasma layer [53]. These differences in cellular compositions, and thus PRP characteristics, have recently been recognized in the literature [54]. Marques et al. found that inferior treatment outcomes following PRP applications correlated directly with poor quality and inconsistent PRP products [55]. Therefore, PRP devices should be versatile and compliant to enable the production of different PRP formulations, while maintaining supraphysiologic platelet numbers (**Figure 5**). More specifically, the final cellular PRP treatment sample should be tailored to serve treatment protocols contingent to wound bed condition, wound size and depth, and undermining tissue.

### **6.2. Definition of clinical-PRP and platelet dose**

PRP can be characterized as a complex composition of autologous multicellular components in a small volume of plasma, with a substantial supraphysiologic concentration of platelets compared to baseline values, with minimal red blood cell contamination.

Clinical PRP (C-PRP) contains a clinical dose of concentrated platelets in a treatment sample. Marx demonstrated enhancement of bone and soft tissue healing with a minimum platelet count of 1 × 109 /mL [56]. Furthermore, Giusti et al. revealed in an experimental study that 1.5 × 109 platelets/ mL are needed for inducing a functional angiogenic response, via endothelial cell activity, in tissue repair mechanisms [57]. Therefore, to significantly induce an angiogenic response in circulatory compromised chronic wounds, C-PRP should contain at least 7.5 billion deliverable platelets in a 5-mL treatment sample. These platelets should then be able to release their entire content after (tissue) activation has occurred, as visualized in **Figure 6** by electron microscopic imaging. Furthermore, this platelet dose will correspondingly induce cell proliferation and cell migration. Ultimately, an increase in wound bed microcirculation would contribute to ECM remodeling and wound epithelialization [58]. Consequently, C-PRP containing a platelet dose of 1.5 × 109 platelets/ mL has the ability to stimulate (neo)angiogenesis and elicit the healing of chronic wounds.

#### **6.3. Leukocytes in C-PRP**

containing an anticoagulant to prevent clotting. The blood predonation volume depends on the PRP device of choice to prepare PRP and the volume needed for specific single, or multiple, wound care treatments in the same patient. Directly after blood collection, the PRP

Currently, physicians can choose from more than 30 PRP processing systems. However, a lack of consensus on standardizing PRP has contributed to the variation in PRP devices, which

Optimal blood separation is best safeguarded by so-called double-spin PRP centrifuges with dedicated disposable platelet concentration devices. These double-spin PRP devices create a layered buffy coat stratum based on different centrifugal forces and specific gravities and densities of the individual blood components (**Figure 4**). Single-spin devices, or plasma-PRP

**Figure 4.** Cellular density separation of whole blood by centrifugation. After the first centrifugation procedure, the whole blood components are separated in the PRP device from the plasma as a result of the different densities in two basic layers. The top layer is the platelet plasma suspension, consisting of plasma and the multicomponent buffy coat layer, containing platelets, monocytes, lymphocytes, and neutrophils. The second layer consists of erythrocyte pack. The

range of the specific cell densities varies between individuals.

centrifugation process should be initiated in order to produce a sample of PRP.

158 Wound Healing - Current Perspectives

produce dissimilar platelet concentrations and cellular compositions [51, 52].

Leukocytes have a great impact on the intrinsic biology of chronic wounds because of their immune and host-defense mechanisms. Therefore, the presence of leukocytes in

M1 macrophages are responsible for producing several inflammatory cytokines that support host defense through pathogen clearance, necrotic tissue clearance, and reactive oxygen species. Furthermore, the M1 phenotype produces growth factors such as VEGF and FGF. M2 macrophages have anti-inflammatory capacities and generate precursors for collagen and fibroblast stimulating factor, thus supporting their role in extracellular matrix deposition. Generally, the plasticity of monocytes is dependent on the microenvironment in which they are present. Monocytes and macrophages release additional pro-regenerative growth factors that lead to neovascularization, proliferation of myogenic precursor cells, and stimulation of the activity of

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Neutrophils have a clear function in healing cascades since they form a dense barrier against invading pathogens and counteract infections [63]. Their presence in PRP can be desirable in wound care treatment to functionally destroy and clear bacteria from the wound bed, in certain types of open surgical procedures to prevent wound infections, or within specific treatment protocols that require higher levels and longer periods of inflammation [64]. However, when PRP samples containing very high neutrophil concentrations are used, for example, in non-infected and granulating wound beds, this neutrophil-rich PRP poses a potential risk of progressive and persistent microenvironmental inflammation via the secretion of proteases and toxic oxygen metabolites. PRP products containing elevated levels of proinflammatory neutrophils facilitate a strong leukocytic chemotaxis to induce a phagocytic response, not

satellite cells, playing key roles in wound repair and inflammatory control [21, 62].

**6.4. Erythrocytes in C-PRP and effects of eryptosis on the wound microenvironment**

Detrimental consequences of erythrocytes or red blood cells (RBCs) on tissues have been studied by several groups. In a study by Hooiveld and coworkers, chondrocytes and synoviocytes were exposed to RBCs causing tissue degeneration and destruction, including apoptosis [67]. In another study, it was postulated that erythrocytes inhibit fibroblast proliferation in a collagen scaffold. These findings indicate potential negative effects on the healing of soft tissue cellular structures when using PRP that contains high concentrations of erythrocytes [68]. Indeed, the use of PRP containing RBCs should be avoided in wound healing strategies to

Another rare phenomenon occurs when a PRP preparation including RBCs is applied to tissues. Under normal physiological circumstances, erythrocytes are removed from the circulatory system by the process of senescence after approximately 120 days. In tissues treated with PRP-containing erythrocytes, natural mechanisms of erythrocyte elimination are no longer valid, and erythrocytes undergo eryptosis before they reach their full lifespan [69]. Typical features of eryptosis are similar compared to apoptosis: membrane blebbing and cell shrinkage, resulting in the release of platelet activating factor (PAF). PAF plays a role in control mechanisms of inflammation and stimulating ceramide release and intracellular stress response, while eryptotic RBCs bind to endothelial cells and impede microcirculation [70]. Therefore, the application of PRP containing RBCs in the chronic wound microenvironment finally leads to tissue inflammation and an intracellular stress response, causing oxidative

contributing to wound epithelialization [65, 66].

prevent wound breakdown.

destruction in the wound vasculature.

**Figure 6.** (A) Electron microscopic image of a single platelet in the circles. The internal platelet ɑ and dense granules (black and gray structures, respectively) and lysosomes are visible with intact cellular membranes. (B) Electron microscopic image of activated platelets in the circles. The platelet membranes are ruptured, and their granular content is no longer visible. The platelet growth factors and other vesicles have been released to the extracellular matrix.

re-establishing wound healing attempts in chronic non-healing wounds can be turntables in the wound healing process [59].

The eventual presence of leukocytes in PRP specimen depends on the operating and design principles of PRP devices. Most ideally, PRP processing devices should be able to produce different PRP cellular formulations, including leukocyte composition and concentration. PRP formulations should be based on a disease-specific pathology, medical condition, and tissue types.

Leukocytes develop from multipotential hemopoietic stem cells in the bone marrow and mature along several differentiation pathways. Via common myeloid progenitor cells and myeloblasts, they become differentiated granular (neutrophils, eosinophils, basophils) and a-granular cells (lymphocytes and monocytes) [60]. However, during PRP preparation, the cell membranes of eosinophils and basophils are destroyed following the centrifugation procedure. Interactive wound healing processes involve mediators, extracellular matrix components, resident cells, including platelets, and infiltrating leukocytes. They participate in the classical pathway of wound healing: hematoma, inflammation, tissue formation, and ultimately tissue remodeling.

In PRP, lymphocytes are more concentrated than other leukocytes. They produce insulin-like growth factors, and they may contribute to tissue remodeling [61].

Monocytes are non-inflammatory white blood cells and are the precursors to macrophages. Macrophages are important cells of the immune system that, similar to neutrophils, are formed to fight infection or engulf accumulating damaged or dead cells. Unlike neutrophils, monocytes do not lead to a prolonged inflammatory condition but play important roles in tissue healing. M1 macrophages are responsible for producing several inflammatory cytokines that support host defense through pathogen clearance, necrotic tissue clearance, and reactive oxygen species. Furthermore, the M1 phenotype produces growth factors such as VEGF and FGF. M2 macrophages have anti-inflammatory capacities and generate precursors for collagen and fibroblast stimulating factor, thus supporting their role in extracellular matrix deposition. Generally, the plasticity of monocytes is dependent on the microenvironment in which they are present. Monocytes and macrophages release additional pro-regenerative growth factors that lead to neovascularization, proliferation of myogenic precursor cells, and stimulation of the activity of satellite cells, playing key roles in wound repair and inflammatory control [21, 62].

Neutrophils have a clear function in healing cascades since they form a dense barrier against invading pathogens and counteract infections [63]. Their presence in PRP can be desirable in wound care treatment to functionally destroy and clear bacteria from the wound bed, in certain types of open surgical procedures to prevent wound infections, or within specific treatment protocols that require higher levels and longer periods of inflammation [64]. However, when PRP samples containing very high neutrophil concentrations are used, for example, in non-infected and granulating wound beds, this neutrophil-rich PRP poses a potential risk of progressive and persistent microenvironmental inflammation via the secretion of proteases and toxic oxygen metabolites. PRP products containing elevated levels of proinflammatory neutrophils facilitate a strong leukocytic chemotaxis to induce a phagocytic response, not contributing to wound epithelialization [65, 66].

#### **6.4. Erythrocytes in C-PRP and effects of eryptosis on the wound microenvironment**

re-establishing wound healing attempts in chronic non-healing wounds can be turntables in

**Figure 6.** (A) Electron microscopic image of a single platelet in the circles. The internal platelet ɑ and dense granules (black and gray structures, respectively) and lysosomes are visible with intact cellular membranes. (B) Electron microscopic image of activated platelets in the circles. The platelet membranes are ruptured, and their granular content is no longer

visible. The platelet growth factors and other vesicles have been released to the extracellular matrix.

The eventual presence of leukocytes in PRP specimen depends on the operating and design principles of PRP devices. Most ideally, PRP processing devices should be able to produce different PRP cellular formulations, including leukocyte composition and concentration. PRP formulations should be based on a disease-specific pathology, medical condition, and

Leukocytes develop from multipotential hemopoietic stem cells in the bone marrow and mature along several differentiation pathways. Via common myeloid progenitor cells and myeloblasts, they become differentiated granular (neutrophils, eosinophils, basophils) and a-granular cells (lymphocytes and monocytes) [60]. However, during PRP preparation, the cell membranes of eosinophils and basophils are destroyed following the centrifugation procedure. Interactive wound healing processes involve mediators, extracellular matrix components, resident cells, including platelets, and infiltrating leukocytes. They participate in the classical pathway of wound healing: hematoma, inflammation, tissue formation, and ultimately tissue remodeling. In PRP, lymphocytes are more concentrated than other leukocytes. They produce insulin-like

Monocytes are non-inflammatory white blood cells and are the precursors to macrophages. Macrophages are important cells of the immune system that, similar to neutrophils, are formed to fight infection or engulf accumulating damaged or dead cells. Unlike neutrophils, monocytes do not lead to a prolonged inflammatory condition but play important roles in tissue healing.

growth factors, and they may contribute to tissue remodeling [61].

the wound healing process [59].

160 Wound Healing - Current Perspectives

tissue types.

Detrimental consequences of erythrocytes or red blood cells (RBCs) on tissues have been studied by several groups. In a study by Hooiveld and coworkers, chondrocytes and synoviocytes were exposed to RBCs causing tissue degeneration and destruction, including apoptosis [67]. In another study, it was postulated that erythrocytes inhibit fibroblast proliferation in a collagen scaffold. These findings indicate potential negative effects on the healing of soft tissue cellular structures when using PRP that contains high concentrations of erythrocytes [68]. Indeed, the use of PRP containing RBCs should be avoided in wound healing strategies to prevent wound breakdown.

Another rare phenomenon occurs when a PRP preparation including RBCs is applied to tissues. Under normal physiological circumstances, erythrocytes are removed from the circulatory system by the process of senescence after approximately 120 days. In tissues treated with PRP-containing erythrocytes, natural mechanisms of erythrocyte elimination are no longer valid, and erythrocytes undergo eryptosis before they reach their full lifespan [69]. Typical features of eryptosis are similar compared to apoptosis: membrane blebbing and cell shrinkage, resulting in the release of platelet activating factor (PAF). PAF plays a role in control mechanisms of inflammation and stimulating ceramide release and intracellular stress response, while eryptotic RBCs bind to endothelial cells and impede microcirculation [70]. Therefore, the application of PRP containing RBCs in the chronic wound microenvironment finally leads to tissue inflammation and an intracellular stress response, causing oxidative destruction in the wound vasculature.

## **7. PRP preparation protocol to produce PurePRP®SP**

In this paragraph, a detailed and specific PRP preparation procedure is described to produce Pure Platelet-Rich Plasma-Supra Physiologic (PurePRP®SP, EmCyte Corporation, Fort Myers, FL, USA). This autologous cellular platform technology is able to generate C-PRP with high concentrations of platelets; there are protocol options to produce neutrophil-poor or -rich PRP, with minimal erythrocyte contamination (**Figure 7**). Furthermore, this platform technology enables clinicians to also concentrate bone marrow aspirate to retrieve, among other cells, concentrated mesenchymal cells [71]. Additionally, the same technology is capable of creating concentrated and viable adipose tissue complex. Both bone marrow and adipose biological tissue types will be discussed in another paragraph to emphasize the ability to use viable MSCs for wound care treatment.

#### **7.1. PRP preparation and procedural therapy application steps**

At point of care, 54 mL of fresh whole blood is predonated in a 60-mL syringe preloaded with 6 mL of 3.8% sodium citrate (anticoagulant). The PurePRP®SP device is loaded from the top and placed in a centrifuge with pre-programmed settings. Following a first centrifugation of 1.5 min, the whole blood is sequestered in a Platelet-Poor Plasma Suspension (PPS) containing a buffy coat layer and RBCs. Using a syringe, the PPS is aspirated until a band of RBCs, which holds mature platelets, is captured with the PPS. This volume is then transferred to the bottom part of the same device, the concentration chamber, and placed in the centrifuge for a 5-min second spin. During this period, a final cell PPS separation is achieved, with the concentrated platelets pelleted at the bottom of the chamber. Excessive platelet-poor plasma (PPP) is removed, leaving a PurePRP®SP volume, generally between 3 and 7 mL. This PPP volume is used to resuspend the platelets from the bottom of the device back into the PPP by gentle swirling of the device. When the bottom part is clear, all platelets are resuspended in

**Figure 8.** PurePRP®SP preparation procedure. (A) The PRP device is loaded with anticoagulated whole blood. (B) After first spin, the PPS is created following gravity centrifugal separation. (C) The PPS evacuated from the top chamber and meticulously injected in bottom part of the PRP device for the second spin procedure. (D) After the second spin, the PPS is further refined in a PPP fraction and concentrated platelets. (E) PPP has been softly removed with a syringe, leaving a desired volume behind to gently resuspend the supraphysiologic platelet concentrate, which is attached to the bottom of the second chamber of the device. (F) The platelets are aspirated in a small volume of plasma and the PurePRP®SP product is collected in a syringe prior to application (PPS, platelet plasma suspension; PRP, platelet-rich plasma; PPP,

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Depending on the biological wound treatment strategies, different PRP application techniques can be used. Before starting any PRP procedure, a meticulous sharp wound debridement should be done. Microfracturing the wound bed, with removal of cellular/plasma debris or dead tissue, allows PGFs to function more effectively while being resistant to rapid degrada-

The application of PRP, or PRP-G, can be done using different techniques. First, PRP can be injected intralesionally, including the wound edges [72]. This technique delivers the platelets directly into the deeper tissue structures. The objective of this technique is to stimulate tissue regeneration faster in more stagnant wounds and wound edges or to prepare the wound bed for a final reconstructive procedure [73]. Second, PRP mixed with bovine or autologous thrombin creates a PRP-G coagulum, where this topical "primary" biological wound dressing covers and sticks to the wound bed. The PRP-G can be applied to a wound bed via a single syringe technique or delivered using a double syringe spray device to ultimately generate a solid graft (**Figure 9**). Lastly, wounds with undermining can be filled with PRP-G using a single syringe and blunt needle approach. Furthermore, the same technique using a sharp needle is suitable for injecting the wound perimeter with adipose tissue, with or without PRP (**Figure 10**).

After PRP has been applied to the wound bed, wound undermining areas, and wound edges, platelets will slowly start to lyse, releasing their PGFs, cytokines, and other proteins; inducing

The literature is not clear on the number of PRP treatments needed to treat a wound and the associated outcomes. Several studies indicate multiple treatments over a period of time. Everts et al. performed initially two treatments weekly for 2 weeks. Thereafter, the procedure was bi-weekly, until final wound closure was expected. In the same study, the PRP procedure was followed by using a naturally derived porcine intestinal submucosa matrix graft to support building the

cell signaling processes; and initiating regeneration and tissue healing [74].

the plasma and the PurePRP®SP is then withdrawn with a 12-mL syringe (**Figure 8**).

platelet poor plasma; PurePRP®SP is a registered trademark of EmCyte corporation, Fort Myers FL, USA).

tion by proteolytic wound activities.

**Figure 7.** (A) Typical aspect of a neutrophil-poor PurePRP®SP sample, with a more yellow coloring. This PRP is intended to treat a wound that does not require proinflammatory PRP stimulation. (B) Typical aspect of a neutrophilrich PurePRP®SP sample. This formulation is defined as a full buffy coat PRP, containing a significant concentration of platelets, neutrophils, monocytes, and lymphocytes. The red color in this preparation is due to the erythrocytes present in the PRP, as the collected neutrophils are on top of the red cells when they are from the platelet-plasma suspension (PurePRP®SP, pure platelet-rich plasma supraphysiologic; PRP, platelet-rich plasma).

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**Figure 8.** PurePRP®SP preparation procedure. (A) The PRP device is loaded with anticoagulated whole blood. (B) After first spin, the PPS is created following gravity centrifugal separation. (C) The PPS evacuated from the top chamber and meticulously injected in bottom part of the PRP device for the second spin procedure. (D) After the second spin, the PPS is further refined in a PPP fraction and concentrated platelets. (E) PPP has been softly removed with a syringe, leaving a desired volume behind to gently resuspend the supraphysiologic platelet concentrate, which is attached to the bottom of the second chamber of the device. (F) The platelets are aspirated in a small volume of plasma and the PurePRP®SP product is collected in a syringe prior to application (PPS, platelet plasma suspension; PRP, platelet-rich plasma; PPP, platelet poor plasma; PurePRP®SP is a registered trademark of EmCyte corporation, Fort Myers FL, USA).

volume is used to resuspend the platelets from the bottom of the device back into the PPP by gentle swirling of the device. When the bottom part is clear, all platelets are resuspended in the plasma and the PurePRP®SP is then withdrawn with a 12-mL syringe (**Figure 8**).

Depending on the biological wound treatment strategies, different PRP application techniques can be used. Before starting any PRP procedure, a meticulous sharp wound debridement should be done. Microfracturing the wound bed, with removal of cellular/plasma debris or dead tissue, allows PGFs to function more effectively while being resistant to rapid degradation by proteolytic wound activities.

The application of PRP, or PRP-G, can be done using different techniques. First, PRP can be injected intralesionally, including the wound edges [72]. This technique delivers the platelets directly into the deeper tissue structures. The objective of this technique is to stimulate tissue regeneration faster in more stagnant wounds and wound edges or to prepare the wound bed for a final reconstructive procedure [73]. Second, PRP mixed with bovine or autologous thrombin creates a PRP-G coagulum, where this topical "primary" biological wound dressing covers and sticks to the wound bed. The PRP-G can be applied to a wound bed via a single syringe technique or delivered using a double syringe spray device to ultimately generate a solid graft (**Figure 9**). Lastly, wounds with undermining can be filled with PRP-G using a single syringe and blunt needle approach. Furthermore, the same technique using a sharp needle is suitable for injecting the wound perimeter with adipose tissue, with or without PRP (**Figure 10**).

After PRP has been applied to the wound bed, wound undermining areas, and wound edges, platelets will slowly start to lyse, releasing their PGFs, cytokines, and other proteins; inducing cell signaling processes; and initiating regeneration and tissue healing [74].

The literature is not clear on the number of PRP treatments needed to treat a wound and the associated outcomes. Several studies indicate multiple treatments over a period of time. Everts et al. performed initially two treatments weekly for 2 weeks. Thereafter, the procedure was bi-weekly, until final wound closure was expected. In the same study, the PRP procedure was followed by using a naturally derived porcine intestinal submucosa matrix graft to support building the

**Figure 7.** (A) Typical aspect of a neutrophil-poor PurePRP®SP sample, with a more yellow coloring. This PRP is intended to treat a wound that does not require proinflammatory PRP stimulation. (B) Typical aspect of a neutrophilrich PurePRP®SP sample. This formulation is defined as a full buffy coat PRP, containing a significant concentration of platelets, neutrophils, monocytes, and lymphocytes. The red color in this preparation is due to the erythrocytes present in the PRP, as the collected neutrophils are on top of the red cells when they are from the platelet-plasma suspension

(PurePRP®SP, pure platelet-rich plasma supraphysiologic; PRP, platelet-rich plasma).

**7. PRP preparation protocol to produce PurePRP®SP**

**7.1. PRP preparation and procedural therapy application steps**

viable MSCs for wound care treatment.

162 Wound Healing - Current Perspectives

In this paragraph, a detailed and specific PRP preparation procedure is described to produce Pure Platelet-Rich Plasma-Supra Physiologic (PurePRP®SP, EmCyte Corporation, Fort Myers, FL, USA). This autologous cellular platform technology is able to generate C-PRP with high concentrations of platelets; there are protocol options to produce neutrophil-poor or -rich PRP, with minimal erythrocyte contamination (**Figure 7**). Furthermore, this platform technology enables clinicians to also concentrate bone marrow aspirate to retrieve, among other cells, concentrated mesenchymal cells [71]. Additionally, the same technology is capable of creating concentrated and viable adipose tissue complex. Both bone marrow and adipose biological tissue types will be discussed in another paragraph to emphasize the ability to use

At point of care, 54 mL of fresh whole blood is predonated in a 60-mL syringe preloaded with 6 mL of 3.8% sodium citrate (anticoagulant). The PurePRP®SP device is loaded from the top and placed in a centrifuge with pre-programmed settings. Following a first centrifugation of 1.5 min, the whole blood is sequestered in a Platelet-Poor Plasma Suspension (PPS) containing a buffy coat layer and RBCs. Using a syringe, the PPS is aspirated until a band of RBCs, which holds mature platelets, is captured with the PPS. This volume is then transferred to the bottom part of the same device, the concentration chamber, and placed in the centrifuge for a 5-min second spin. During this period, a final cell PPS separation is achieved, with the concentrated platelets pelleted at the bottom of the chamber. Excessive platelet-poor plasma (PPP) is removed, leaving a PurePRP®SP volume, generally between 3 and 7 mL. This PPP

patient visits, the wounds were assessed according to the TIME wound grading system [78], which was designed for tissue evaluation, infectious condition, and moisture evaluation, and the condition of the wound edges was checked at every visit to monitor progress and

Autologous Platelet-Rich Plasma and Mesenchymal Stem Cells for the Treatment of Chronic…

The characteristics of biological PRP and PRP-G suggest that they might be a beneficial tool in the surgical armamentarium. PRP-G has been successfully used in maxillofacial surgery, orthopedics, cosmetic surgery, and dental implantology. Furthermore, several randomized controlled clinical trials studied the effect of PRP-G in wound rehabilitation and tissue engineering. Eleven studies were identified involving the use of different PRP formulations in venous and diabetic leg ulcers between 2007 and 2018 [79–89]. A summary of all the studies is shown in **Table 3**. A general comment from these studies is that some of them were underpowered [79, 81]. The PRP interventions were highly variable with regard to platelet dosing, formulations, the total number of PRP applications, and the interval between applications.

a platelet coagulum. The presence of leukocytes in PRPs and the platelet dose relative to peripheral blood were hardly described. The frequency of application varied between twice weekly and weekly. Time to wound healing or wound size reduction was the most common outcome measurement. Six trials involved predominantly diabetic patients [81–83, 87, 88], while mixed ulcer etiology was included in the other studies. Outcome results favored experimental treatments with PRP, in all studies presented. Furthermore, Carter et al. conducted a review in 2011, analyzing published prospective and retrospective studies and meta-analyzed the use of PRP and PRP-G in wound healing in acute and chronic conditions [90]. Their paper included 24 studies, from which 3 studies were systematic reviews and 9 studies were included in the meta-analysis. The systematic review and meta-analysis stated that PRP applications in cutaneous wounds exposed complete and partial wound healing when compared to control wound care. Furthermore, the presence of infection was reduced in acute wounds treated with PRP. Martinez-Zapata and co-workers presented their results from a systematic review, including10 randomized controlled trials (RCTs) in chronic wounds in their metaanalysis [91]. Three of these RCTs involved DFU and three studies involved venous leg ulcers. Their results indicated that autologous PRP can enhance DFU healing when compared with

A condensed summary review by Everts et al. revealed the efficacy and safety of PRP-G treatments when used by different institutions [92]. Picard et al. published a literature review, comprising 12 studies, to summarize evidence-based data regarding the treatment of diabetic chronic wounds with PRP. In 87.5% of controlled studies, they found a significant benefit for the use of PRP therapy to treat chronic diabetic wounds, which remained unhealed after standard wound care treatment [93]. However, more studies remain necessary to produce

or calcium gluconate to initiate

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165

**8. Overview of some of the most relevant studies using autologous** 

regression of wound healing.

standard care.

**PRP to treat chronic wounds**

PRP-G was produced using bovine thrombin and/or CaCl<sup>2</sup>

strong evidence eliminating poor design and high bias [90, 91].

**Figure 9.** PRP application techniques. (A) A semiviscous PRP-G coagulum is topically placed on a wound bed via a single syringe technique. PRP and thrombin are mixed in the same syringe and delivered via a blunt needle covering the entire wound bed. (B) Intralesional injection of PRP with a 30-gauge needle in the wound bed and/or wound edges. (C) Spray application using an aerosol delivery technique, with PRP and autologous thrombin in separate syringes. The content mixes at the tip of the spray catheter, where after PRP-G is formed at the tissue site (PRP-G, platelet-rich plasma gel; PRP, platelet-rich plasma).

**Figure 10.** (A) Filling an undermining of a wound with PRP-G, using a single syringe technique in which both PRP and thrombin have been mixed to a semiviscous coagulum (the black line indicates the direction of the undermining). (B) The wound perimeter of the chronic wound is injected with a mixture of PRP and concentrated adipose tissue to deliver PGFs and adipose tissue constituents like MSCs (PRP-G, platelet-rich plasma gel; PRP, platelet-rich plasma; PGFs, platelet growth factors; MSCs, mesenchymal cells).

ECM and limited permeability to keep the lysing platelet fluids in place (OASIS® Wound Matrix, Cook Biotech, Inc., West Lafayette, IN, USA), followed by a hydrocolloid secondary dressing (DuoDERM® Extra Thin Dressing, ConvaTec, Greensboro, NC, USA) [73]. Others have used, for example, a non-absorbent sterile transparent sheet (Tegaderm™, 3M Medical Inc.) or a knitted cellulose acetate non-adherent dressing impregnated with a petrolatum emulsion (Adaptic, Systagenix Wound Management Limited, North Yorkshire, UK) [75].

Recent review articles do not provide clear information on post-PRP treatment protocols [76, 77]. This author's experiences with PRP wound care treatments included no dressing changes for 5 days post-treatment. Thereafter, minimal wound cleaning and no sharp debridement are standard wound care activities, until the next PRP application. During all patient visits, the wounds were assessed according to the TIME wound grading system [78], which was designed for tissue evaluation, infectious condition, and moisture evaluation, and the condition of the wound edges was checked at every visit to monitor progress and regression of wound healing.

### **8. Overview of some of the most relevant studies using autologous PRP to treat chronic wounds**

The characteristics of biological PRP and PRP-G suggest that they might be a beneficial tool in the surgical armamentarium. PRP-G has been successfully used in maxillofacial surgery, orthopedics, cosmetic surgery, and dental implantology. Furthermore, several randomized controlled clinical trials studied the effect of PRP-G in wound rehabilitation and tissue engineering. Eleven studies were identified involving the use of different PRP formulations in venous and diabetic leg ulcers between 2007 and 2018 [79–89]. A summary of all the studies is shown in **Table 3**. A general comment from these studies is that some of them were underpowered [79, 81]. The PRP interventions were highly variable with regard to platelet dosing, formulations, the total number of PRP applications, and the interval between applications. PRP-G was produced using bovine thrombin and/or CaCl<sup>2</sup> or calcium gluconate to initiate a platelet coagulum. The presence of leukocytes in PRPs and the platelet dose relative to peripheral blood were hardly described. The frequency of application varied between twice weekly and weekly. Time to wound healing or wound size reduction was the most common outcome measurement. Six trials involved predominantly diabetic patients [81–83, 87, 88], while mixed ulcer etiology was included in the other studies. Outcome results favored experimental treatments with PRP, in all studies presented. Furthermore, Carter et al. conducted a review in 2011, analyzing published prospective and retrospective studies and meta-analyzed the use of PRP and PRP-G in wound healing in acute and chronic conditions [90]. Their paper included 24 studies, from which 3 studies were systematic reviews and 9 studies were included in the meta-analysis. The systematic review and meta-analysis stated that PRP applications in cutaneous wounds exposed complete and partial wound healing when compared to control wound care. Furthermore, the presence of infection was reduced in acute wounds treated with PRP. Martinez-Zapata and co-workers presented their results from a systematic review, including10 randomized controlled trials (RCTs) in chronic wounds in their metaanalysis [91]. Three of these RCTs involved DFU and three studies involved venous leg ulcers. Their results indicated that autologous PRP can enhance DFU healing when compared with standard care.

**Figure 10.** (A) Filling an undermining of a wound with PRP-G, using a single syringe technique in which both PRP and thrombin have been mixed to a semiviscous coagulum (the black line indicates the direction of the undermining). (B) The wound perimeter of the chronic wound is injected with a mixture of PRP and concentrated adipose tissue to deliver PGFs and adipose tissue constituents like MSCs (PRP-G, platelet-rich plasma gel; PRP, platelet-rich plasma;

**Figure 9.** PRP application techniques. (A) A semiviscous PRP-G coagulum is topically placed on a wound bed via a single syringe technique. PRP and thrombin are mixed in the same syringe and delivered via a blunt needle covering the entire wound bed. (B) Intralesional injection of PRP with a 30-gauge needle in the wound bed and/or wound edges. (C) Spray application using an aerosol delivery technique, with PRP and autologous thrombin in separate syringes. The content mixes at the tip of the spray catheter, where after PRP-G is formed at the tissue site (PRP-G, platelet-rich plasma

ECM and limited permeability to keep the lysing platelet fluids in place (OASIS® Wound Matrix, Cook Biotech, Inc., West Lafayette, IN, USA), followed by a hydrocolloid secondary dressing (DuoDERM® Extra Thin Dressing, ConvaTec, Greensboro, NC, USA) [73]. Others have used, for example, a non-absorbent sterile transparent sheet (Tegaderm™, 3M Medical Inc.) or a knitted cellulose acetate non-adherent dressing impregnated with a petrolatum emulsion (Adaptic,

Recent review articles do not provide clear information on post-PRP treatment protocols [76, 77]. This author's experiences with PRP wound care treatments included no dressing changes for 5 days post-treatment. Thereafter, minimal wound cleaning and no sharp debridement are standard wound care activities, until the next PRP application. During all

PGFs, platelet growth factors; MSCs, mesenchymal cells).

gel; PRP, platelet-rich plasma).

164 Wound Healing - Current Perspectives

Systagenix Wound Management Limited, North Yorkshire, UK) [75].

A condensed summary review by Everts et al. revealed the efficacy and safety of PRP-G treatments when used by different institutions [92]. Picard et al. published a literature review, comprising 12 studies, to summarize evidence-based data regarding the treatment of diabetic chronic wounds with PRP. In 87.5% of controlled studies, they found a significant benefit for the use of PRP therapy to treat chronic diabetic wounds, which remained unhealed after standard wound care treatment [93]. However, more studies remain necessary to produce strong evidence eliminating poor design and high bias [90, 91].


delayed healing, resulting in accelerated wound closure [94]. A stem cell is, by definition, the one cell capable of duplicating itself (self-renewal) and resuming its undifferentiated status, while also originating progeny that can differentiate into one or more final products that are physiologically defined by their specific functions. Stem cells can be classified on the basis of their origin and their potential to proliferate and differentiate. According to Wagers and Weissman, the classification of stem cells is based on their plasticity and potential for differentiation [95]: totipotent, able to give rise to all embryonic and extraembryonic cell types; pluripotent, able to give rise to all cell types of the embryo proper; multipotent, able to give rise to a subset of cell lineages; oligopotent, able to give rise to a restricted subset of cell lineages; and unipotent, able to contribute only one mature cell type. Adult stem cells have a multipotent lineage and are able to transdifferentiate into various progenies, forming cells of multipotent lineages, such as HSCs and MSCs [95]. HSCs are pluripotent cells that further differentiate via hematopoiesis into distinct progenitor cells which mature into blood cells of myeloid lineages (monocyte, granulocyte, erythrocyte, and megakaryocyte/platelets) and

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MSCs are multipotent adult stem cells and can be obtained from various adult tissues, including bone marrow stroma, adipose tissue, and other tissue types. According to the International Society of Cellular Therapy, MSCs are defined as those cells that are able to adhere to plastic and express a number of cell surface markers (including CD73, CD90, and CD105) while undergoing multilineage differentiation. Furthermore, MSCs should have the ability for self-renewal [97]. MSCs can also be identified as specialized populations of mural cells/pericytes. They provide a niche for HSCs and have the ability to differentiate into various mesodermal lineages. Under appropriate conditions and an optimal microenvironment, MSCs can differentiate into mesodermal lineage cells such as osteoblasts, endothelial cells, adipose tissue, and smooth muscle cells [4]. These capabilities have led to the use of MSC as a potential strategy for treating various diseases since they promote biological processes, such as angiogenesis and cell proliferation and differentiation [98]. Furthermore, they synthesize mediators (cytokines and trophic factors) that participate in tissue repair processes, immune modulation, and the regulation of inflammatory processes. [99]. The trophic effects are facilitated by the MSC secretion of reparative cytokines and growth factors, including TGF-β, VEGF, and EGF, to contribute to local tissue repair [100]. Caplan also suggested that the modulation of inflammation is instigated by the suppression of inflammatory T-cell proliferation and inhibition of monocyte and myeloid cell maturation [101]. Based on above characteristics, one can see that MSCs are able to establish a regenerative microenvironment at the site of release, which could improve the recruitment, activation, and differentiation of endogenous stem cells with the potential for repair in wound healing. Currently, clinical research is investigating MSCs as a therapy to treat difficult-to-heal wounds.

BM-MSCs from adult bone marrow tissue were first isolated by Pittenger et al. [102]. Since then, BM-MSCs are frequently used successfully as a biological product, like PRP, in regenerative

lymphoid cells (B, T and NK cells) [96].

**10.1. Bone marrow mesenchymal stem cells**

**10. Mesenchymal stem cells**

RCT, randomized controlled trial; CT, controlled trial; PT, prospective trial; DFU, diabetic foot ulcer; VLU, venous leg ulcer; PRP, platelet-rich plasma.

**Table 3.** Overview of some of the most relevant studies using autologous PRP technology to treat chronic wounds.

Presently, more studies are ongoing to clarify optimized PRP protocols to improve its angiogenic and regenerative properties to be implemented as a standard practice of care in advanced wound care treatment plans.

### **9. Comprehensive background on stem cells**

In any regenerative tissue microenvironment, there are essentially stem cells, growth factors, and a biological scaffold to provide the necessary biological milieu for cell-tissue regeneration and cell renewal. MSCs originating from either bone marrow or adipose tissue are now extensively being used in a variety of patients who have an indication for minimally invasive, regenerative medicine therapies to enhance tissue repair and regeneration. Traditionally, bone marrow aspirate (BMA) has been utilized as a source of bone marrowderived mesenchymal stem cells (BM-MSC), hematopoietic stem cells (HSCs), progenitor cells, and platelets. Lately, MSCs derived from adipose tissue have emerged in a variety of regenerative treatment protocols. However, in chronic wound care strategies, autologous, non-cultured, MSC therapies are rarely used. However, Hocking reported from preclinical and clinical trials that MSC therapy has the potential to effectively treat wounds with delayed healing, resulting in accelerated wound closure [94]. A stem cell is, by definition, the one cell capable of duplicating itself (self-renewal) and resuming its undifferentiated status, while also originating progeny that can differentiate into one or more final products that are physiologically defined by their specific functions. Stem cells can be classified on the basis of their origin and their potential to proliferate and differentiate. According to Wagers and Weissman, the classification of stem cells is based on their plasticity and potential for differentiation [95]: totipotent, able to give rise to all embryonic and extraembryonic cell types; pluripotent, able to give rise to all cell types of the embryo proper; multipotent, able to give rise to a subset of cell lineages; oligopotent, able to give rise to a restricted subset of cell lineages; and unipotent, able to contribute only one mature cell type. Adult stem cells have a multipotent lineage and are able to transdifferentiate into various progenies, forming cells of multipotent lineages, such as HSCs and MSCs [95]. HSCs are pluripotent cells that further differentiate via hematopoiesis into distinct progenitor cells which mature into blood cells of myeloid lineages (monocyte, granulocyte, erythrocyte, and megakaryocyte/platelets) and lymphoid cells (B, T and NK cells) [96].

### **10. Mesenchymal stem cells**

Presently, more studies are ongoing to clarify optimized PRP protocols to improve its angiogenic and regenerative properties to be implemented as a standard practice of care in

**Table 3.** Overview of some of the most relevant studies using autologous PRP technology to treat chronic wounds.

In any regenerative tissue microenvironment, there are essentially stem cells, growth factors, and a biological scaffold to provide the necessary biological milieu for cell-tissue regeneration and cell renewal. MSCs originating from either bone marrow or adipose tissue are now extensively being used in a variety of patients who have an indication for minimally invasive, regenerative medicine therapies to enhance tissue repair and regeneration. Traditionally, bone marrow aspirate (BMA) has been utilized as a source of bone marrowderived mesenchymal stem cells (BM-MSC), hematopoietic stem cells (HSCs), progenitor cells, and platelets. Lately, MSCs derived from adipose tissue have emerged in a variety of regenerative treatment protocols. However, in chronic wound care strategies, autologous, non-cultured, MSC therapies are rarely used. However, Hocking reported from preclinical and clinical trials that MSC therapy has the potential to effectively treat wounds with

advanced wound care treatment plans.

leg ulcer; PRP, platelet-rich plasma.

**Year; author [reference]**

2011; Saad Setta

2017; Obolensky

[84]

[79]

**Study design**

166 Wound Healing - Current Perspectives

2007; Kakagi [77] RCT 51; foot tissue

2016; Pravin [82] RCT 31; 22 VLU and

**N patients in study; indication**

2010; Jeong [77] RCT 100; DFU >4 weeks Complete wound healing

defects

DFU; 9 others

CT 100; non-healing, mixed etiology

**Duration of wound**

2015; Karimi [80] RCT 50; DFU No limit PRP significantly reduced wound surface

2015; Li [81] RCT 117; DFU >2 weeks PRP significant better healing than

2017; Moneib [83] RCT 40; venous ulcers >6 months Significant ulcer reduction

2017; Babaei [85] PT 150; DFU >3 weeks Full closure after 8.8 weeks

2017; Milek [86] CT 100; DFU >6 months Full wound closure treatment group

2018; Etugov [87] PT 23; VLU >4 weeks Significant ulcer size reduction compared

RCT, randomized controlled trial; CT, controlled trial; PT, prospective trial; DFU, diabetic foot ulcer; VLU, venous

RCT 24; non-healing DFU >8 weeks PRP treated group healed significantly

**Outcomes**

faster

>3 months Ulcer reduction in treatment group

and depth in 3 weeks

>8 weeks Leukocyte free PRP healed better, 86% ulcer healing

hospitalization; less total costs

controls only small wounds

standard care

>6 weeks Earlier epithelialization; shorter

to control

**9. Comprehensive background on stem cells**

MSCs are multipotent adult stem cells and can be obtained from various adult tissues, including bone marrow stroma, adipose tissue, and other tissue types. According to the International Society of Cellular Therapy, MSCs are defined as those cells that are able to adhere to plastic and express a number of cell surface markers (including CD73, CD90, and CD105) while undergoing multilineage differentiation. Furthermore, MSCs should have the ability for self-renewal [97]. MSCs can also be identified as specialized populations of mural cells/pericytes. They provide a niche for HSCs and have the ability to differentiate into various mesodermal lineages. Under appropriate conditions and an optimal microenvironment, MSCs can differentiate into mesodermal lineage cells such as osteoblasts, endothelial cells, adipose tissue, and smooth muscle cells [4]. These capabilities have led to the use of MSC as a potential strategy for treating various diseases since they promote biological processes, such as angiogenesis and cell proliferation and differentiation [98]. Furthermore, they synthesize mediators (cytokines and trophic factors) that participate in tissue repair processes, immune modulation, and the regulation of inflammatory processes. [99]. The trophic effects are facilitated by the MSC secretion of reparative cytokines and growth factors, including TGF-β, VEGF, and EGF, to contribute to local tissue repair [100]. Caplan also suggested that the modulation of inflammation is instigated by the suppression of inflammatory T-cell proliferation and inhibition of monocyte and myeloid cell maturation [101]. Based on above characteristics, one can see that MSCs are able to establish a regenerative microenvironment at the site of release, which could improve the recruitment, activation, and differentiation of endogenous stem cells with the potential for repair in wound healing. Currently, clinical research is investigating MSCs as a therapy to treat difficult-to-heal wounds.

#### **10.1. Bone marrow mesenchymal stem cells**

BM-MSCs from adult bone marrow tissue were first isolated by Pittenger et al. [102]. Since then, BM-MSCs are frequently used successfully as a biological product, like PRP, in regenerative

**Figure 11.** Aspire bone marrow aspiration from the posterior superior iliac spine area. (A) The introducer and aspiration needle are placed through the skin, sub cutaneous layer, and cortical bone into the marrow cavity. (B) The BMA device is placed in the PSIS. Bone marrow is meticulously aspirated via suction vacuum applied to a syringe, through the aspirator needle. (C) Bone marrow cells, including purified mesenchymal stem cell, hematopoietic stem cells, total nucleated cells, platelets, and progenitor cells, are collected through the fenestrated aspirator needle with a blunt tip from the cancellous bone. (D) The final BMC sample is produced following a 2-step proprietary centrifugation protocol. Inside the blue circle, concentrated bone marrow cells are visible, on the top of the erythrocyte layer (BMA, bone marrow aspirate; PSIS, posterior superior iliac spine; BMC, bone marrow concentrate; Aspire™ bone marrow aspiration system is trademark of EmCyte Corporation, Fort Myers FL, USA).

of eryptosis on the wound, as this will cause profound inflammation and compromise the

**Table 4.** Effects of bone marrow aspirate concentration on cell counts, hematocrit, and the elimination of hemolytic red

BMA, bone marrow aspirate; BMC, bone marrow concentrate; × BL, effects times baseline values; TNC, total nucleated cells; −nRBCs, minus red blood cells; CD34+, stem cell marker/expression on hematopoietic progenitor cells found in bone marrow; CFU-f, fibroblast colony-forming units: assay for bone marrow mesenchymal stem cell analysis; MSCs,

Similar to BM-MSCs, adipose-derived mesenchymal stem cell (AD-MSC) has been used in regenerative medicine applications. AD-MSCs can be isolated following an adipose tissue

Various preparation techniques, including centrifugation, exist to collect, wash, and rinse adipose tissue to generate a concentrated adipose tissue concentrate (ATC). Adipocytes constitute almost 90% of adipose tissue volume and nearly 65% of the total cell number [107]. When enzymatically digested, adipose tissue yields a heterogeneous population of many cell types (pre-adipocytes, fibroblasts, vascular smooth muscle cells, endothelial cells, resident monocytes/macrophages, and lymphocytes), which upon isolation are termed the stromal vascular fraction (SVF) [108]. AD-MSCs have a multilineage cell differentiation potential, that is, they are capable of differentiating into adipogenic, chondrogenic, myogenic, osteogenic, and neurogenic cells [109]. Thus, AD-MSCs might be indicated in clinical applications for the repair of damaged tissues, as well as for angiogenic therapy to improve

The popularity of AD-MSCs in regenerative medicine treatment protocols and recently in a biological wound care treatment protocol as well is due to an abundance of MSCs, with a high proliferation capacity and differentiation potential, when compared to MSCs derived from bone marrow [111, 112]. Furthermore, Yun et al. described AD-MSC-mediated effects on the reduction of proinflammatory cytokines, chemokines, cellular apoptosis, and collagenases

[113]. Moreover, AD-MSCs have been shown to be immune-privileged [114].

(mini) liposuction procedure of subcutaneous fat tissue, mostly from the abdomen.

**Laboratory parameters BMA BMC Concentrating effect**

/mL 96 614 6.4 × BL

Hematocrit % 40.7 6.8 −83% × BL Hemolysis % 6.3 1.8 −73% × BL Cell viability % 95.9 97.3 + 0.6% × BL

/mL 1.68 9.2 5.5 × BL

/mL 1.05 5.59 5.3 × BL

/mL 28 142.8 5.1 × BL

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microcirculation [69].

mesenchymal stem cells.

cells.

TNC (−nRBCs) × 10<sup>6</sup>

Platelets × 10<sup>6</sup>

CD34+ cells × 105

CFU-f (MSCs) × 10<sup>3</sup>

neovascularization [110].

**10.2. Adipose mesenchymal stem cells**

medicine therapies to treat a variety of musculoskeletal disorders, such as chondral defects, osteoarthritis, and rotator cuff lesions [103, 104]. BM-MSCs are relatively easy to acquire via a BMA procedure. Bone marrow can be harvested from a variety of anatomic sites during a surgical procedure in the operating room, or an office setting, with minimal morbidity. A variety of donor locations are available, including the anterior or posterior iliac crest, calcaneus, tibia, distal femur, and proximal humerus. The iliac crest is used frequently and known to be a rich source of BM-MSCs (**Figure 11**). BM-MSCs are transplanted autologously, therefore avoiding any ethical issues. Furthermore, the relatively simple preparation and separation and high genetic stability of BM-MSCs allow for their easy use in vitro and as an injectate. Imperative for an effective BM-MSC injection is the quality of the initial bone marrow aspiration procedure with regard to minimizing trauma to cellular content of the bone marrow niche, such as platelets, progenitor cells, and leukocytes, while maximizing cellular yields and minimizing peripheral blood infiltration [105].

Furthermore, the collected bone marrow cells should be viable, with no presence of disintegrated erythrocytes (hemolysis), as this would have a profound negative effect on tissue regeneration [106]. The author believes that a BMA sample should always be preceded by a 2-step centrifugation procedure to concentrate the sample to a bone marrow concentrate (BMC). This will concentrate the indispensable cellular content, such as MSCs (measured by CFU-f), HSCs, total nucleated cells, and platelets, above the baseline counts of these cells. Nonetheless, the centrifugation procedure will decrease hemolytic parameters as well as RBC levels. The effects of concentrating BMA with regard to some of the most important constituents and factors are shown in **Table 4**. Erythrocytes should also be avoided in a BMC specimen, for the same reasons as discussed in the above paragraph on C-PRP and effects


BMA, bone marrow aspirate; BMC, bone marrow concentrate; × BL, effects times baseline values; TNC, total nucleated cells; −nRBCs, minus red blood cells; CD34+, stem cell marker/expression on hematopoietic progenitor cells found in bone marrow; CFU-f, fibroblast colony-forming units: assay for bone marrow mesenchymal stem cell analysis; MSCs, mesenchymal stem cells.

**Table 4.** Effects of bone marrow aspirate concentration on cell counts, hematocrit, and the elimination of hemolytic red cells.

of eryptosis on the wound, as this will cause profound inflammation and compromise the microcirculation [69].

#### **10.2. Adipose mesenchymal stem cells**

medicine therapies to treat a variety of musculoskeletal disorders, such as chondral defects, osteoarthritis, and rotator cuff lesions [103, 104]. BM-MSCs are relatively easy to acquire via a BMA procedure. Bone marrow can be harvested from a variety of anatomic sites during a surgical procedure in the operating room, or an office setting, with minimal morbidity. A variety of donor locations are available, including the anterior or posterior iliac crest, calcaneus, tibia, distal femur, and proximal humerus. The iliac crest is used frequently and known to be a rich source of BM-MSCs (**Figure 11**). BM-MSCs are transplanted autologously, therefore avoiding any ethical issues. Furthermore, the relatively simple preparation and separation and high genetic stability of BM-MSCs allow for their easy use in vitro and as an injectate. Imperative for an effective BM-MSC injection is the quality of the initial bone marrow aspiration procedure with regard to minimizing trauma to cellular content of the bone marrow niche, such as platelets, progenitor cells, and leukocytes, while maximizing cellular yields and minimizing

aspiration system is trademark of EmCyte Corporation, Fort Myers FL, USA).

**Figure 11.** Aspire bone marrow aspiration from the posterior superior iliac spine area. (A) The introducer and aspiration needle are placed through the skin, sub cutaneous layer, and cortical bone into the marrow cavity. (B) The BMA device is placed in the PSIS. Bone marrow is meticulously aspirated via suction vacuum applied to a syringe, through the aspirator needle. (C) Bone marrow cells, including purified mesenchymal stem cell, hematopoietic stem cells, total nucleated cells, platelets, and progenitor cells, are collected through the fenestrated aspirator needle with a blunt tip from the cancellous bone. (D) The final BMC sample is produced following a 2-step proprietary centrifugation protocol. Inside the blue circle, concentrated bone marrow cells are visible, on the top of the erythrocyte layer (BMA, bone marrow aspirate; PSIS, posterior superior iliac spine; BMC, bone marrow concentrate; Aspire™ bone marrow

Furthermore, the collected bone marrow cells should be viable, with no presence of disintegrated erythrocytes (hemolysis), as this would have a profound negative effect on tissue regeneration [106]. The author believes that a BMA sample should always be preceded by a 2-step centrifugation procedure to concentrate the sample to a bone marrow concentrate (BMC). This will concentrate the indispensable cellular content, such as MSCs (measured by CFU-f), HSCs, total nucleated cells, and platelets, above the baseline counts of these cells. Nonetheless, the centrifugation procedure will decrease hemolytic parameters as well as RBC levels. The effects of concentrating BMA with regard to some of the most important constituents and factors are shown in **Table 4**. Erythrocytes should also be avoided in a BMC specimen, for the same reasons as discussed in the above paragraph on C-PRP and effects

peripheral blood infiltration [105].

168 Wound Healing - Current Perspectives

Similar to BM-MSCs, adipose-derived mesenchymal stem cell (AD-MSC) has been used in regenerative medicine applications. AD-MSCs can be isolated following an adipose tissue (mini) liposuction procedure of subcutaneous fat tissue, mostly from the abdomen.

Various preparation techniques, including centrifugation, exist to collect, wash, and rinse adipose tissue to generate a concentrated adipose tissue concentrate (ATC). Adipocytes constitute almost 90% of adipose tissue volume and nearly 65% of the total cell number [107]. When enzymatically digested, adipose tissue yields a heterogeneous population of many cell types (pre-adipocytes, fibroblasts, vascular smooth muscle cells, endothelial cells, resident monocytes/macrophages, and lymphocytes), which upon isolation are termed the stromal vascular fraction (SVF) [108]. AD-MSCs have a multilineage cell differentiation potential, that is, they are capable of differentiating into adipogenic, chondrogenic, myogenic, osteogenic, and neurogenic cells [109]. Thus, AD-MSCs might be indicated in clinical applications for the repair of damaged tissues, as well as for angiogenic therapy to improve neovascularization [110].

The popularity of AD-MSCs in regenerative medicine treatment protocols and recently in a biological wound care treatment protocol as well is due to an abundance of MSCs, with a high proliferation capacity and differentiation potential, when compared to MSCs derived from bone marrow [111, 112]. Furthermore, Yun et al. described AD-MSC-mediated effects on the reduction of proinflammatory cytokines, chemokines, cellular apoptosis, and collagenases [113]. Moreover, AD-MSCs have been shown to be immune-privileged [114].

## **11. MSCs in cutaneous wound healing**

Currently, cell-base therapy is an attractive approach for the treatment of recalcitrant chronic wounds. MSCs from adipose and bone marrow tissues are being investigated as a therapeutic strategy for a distinct group of pathological conditions, including chronic hard-to-heal wounds [115]. The orchestrated process of wound healing entails cellular and hormonal physiological processes of inflammation, epithelialization, proliferation, collagen matrix formation, and particular neoangiogenesis, regulated by various growth factors such as TGF-β, VEGF, PDGF, granulocyte macrophage colony-stimulating factor, the interleukin family, EGF, FGF, and TNF-α [116, 117]. However, the activity of these cytokines in chronic wounds is often reduced due to a prolonged inflammatory state, decreasing the neoangiogenic potential.

subset of patients, promoting the regeneration of impaired endothelium and neoangiogenesis in ischemic tissues [126, 127]. The effects of several types of bone marrow cell therapy (e.g., bone marrow-derived mononuclear cells, CD34+ bone marrow cells, and mesenchymal stromal cells) have been studied in CLI patients. The outcomes of several cell-based therapy trials demonstrated that the rate of major amputation was significantly decreased [128]. It can be concluded that MSC application can be considered a promising target for future biological

Autologous Platelet-Rich Plasma and Mesenchymal Stem Cells for the Treatment of Chronic…

http://dx.doi.org/10.5772/intechopen.80502

171

Regenerative medicine technologies offer solutions to a number of compelling clinical problems that have not been able to adequately result in a solution through the use of drugs,

The purpose of this chapter was to review multiple aspects of both PRP and MSC biocellular therapies as part of a wound care treatment plan to support in the healing of chronic and

Numerous significant aspects that are still not well understood or standardized have been discussed, as well as the rationale for cell-based therapies. For platelet-rich plasma preparations, specific formulations, platelet dosing, processing, and the differences between systems were discussed. With regard to bone marrow and adipose tissue, as cell sources for obtaining

Among both tissue-based cellular therapies, bone marrow mesenchymal cells have been the most frequently employed and reported on. In this review, evidence is shown on results from several clinical studies in which autologous biologics have been applied in patients with chronic wounds. The outcomes of these studies suggested that the application of biocellular products can reverse the microenvironment in chronic wounds, achieving the ultimate goal: full wound epithelialization in the shortest possible time. Furthermore, it was revealed that

high quality mesenchymal cells, some technicalities were provided.

these treatments are safely executed without adverse effects for patients.

The author served also as Chief Scientific Officer of EmCyte Corporation.

Address all correspondence to: peter@gulfcoastbiologics.com

Gulf Coast Biologics, Fort Myers, FL, USA

therapies in CLI patients [129].

surgery, or permanent replacement devices.

**12. Conclusions**

recalcitrant wounds.

**Conflict of interest**

**Author details**

Peter A. Everts

BM-MSCs and AD-MSCs have been studied as potential solutions for these major issues. Both types of MSCs have been shown to be effective in augmenting wound healing by modulating the immune response and secreting paracrine factors which promote therapeutic (neo) angiogenesis and thereby providing biological ingredients for wound tissue regeneration, and they are ultimately capable of inducing full wound closure (**Figure 11**; [118–121]).

Optimal wound bed preparation encompasses not only debridement and proper management of the bacterial load but also correction of the wound matrix and reconditioning of phenotypically altered resident cells which are present in chronic wounds. Based on their characteristics and biological activity, MSCs are capable of interacting with resident wound cells to transform resident cells to functional matrix building cells [122]. This might be of particular importance for the dermal rebuilding process to stimulate keratinocytes to accomplish epithelialization.

Given their higher isolation yield, ease of harvesting, and abundance of adipose tissue, some groups believe that AD-MSCs might be more clinically attractive. Not only because of their angiogenic capability, but they may also function in situ as pericytes providing vascular stability and they might communicate with endothelial cells in response to environmental stimuli [123, 124]. However, experienced clinicians may dispute the cited potential risk for complications with BMA, as they feel comfortable in performing BMA procedures in medical-office settings using local anesthetics and imaging to perform the aspiration. Shapiro and coworkers performed a prospective, single-blind, placebo-controlled trial on 25 patients with bilateral knee osteoarthritis and reported that the BMA, production, and use of BMC is a safe procedure [125].

### **11.1. Critical limb ischemia**

BM-MSCs are frequently being studied in patients with critical limb ischemia, who also might suffer from chronic wounds and who are not eligible for the revascularization procedure due to several comorbidities, namely high operative risk, multiple failures of revascularization, and high rate of restenosis. These patients are suitable for biological cell-based therapy with MSCs. In particular, BM-MSCs protocols are newly emerging therapies to treat CLI in this subset of patients, promoting the regeneration of impaired endothelium and neoangiogenesis in ischemic tissues [126, 127]. The effects of several types of bone marrow cell therapy (e.g., bone marrow-derived mononuclear cells, CD34+ bone marrow cells, and mesenchymal stromal cells) have been studied in CLI patients. The outcomes of several cell-based therapy trials demonstrated that the rate of major amputation was significantly decreased [128]. It can be concluded that MSC application can be considered a promising target for future biological therapies in CLI patients [129].

### **12. Conclusions**

**11. MSCs in cutaneous wound healing**

genic potential.

170 Wound Healing - Current Perspectives

epithelialization.

procedure [125].

**11.1. Critical limb ischemia**

Currently, cell-base therapy is an attractive approach for the treatment of recalcitrant chronic wounds. MSCs from adipose and bone marrow tissues are being investigated as a therapeutic strategy for a distinct group of pathological conditions, including chronic hard-to-heal wounds [115]. The orchestrated process of wound healing entails cellular and hormonal physiological processes of inflammation, epithelialization, proliferation, collagen matrix formation, and particular neoangiogenesis, regulated by various growth factors such as TGF-β, VEGF, PDGF, granulocyte macrophage colony-stimulating factor, the interleukin family, EGF, FGF, and TNF-α [116, 117]. However, the activity of these cytokines in chronic wounds is often reduced due to a prolonged inflammatory state, decreasing the neoangio-

BM-MSCs and AD-MSCs have been studied as potential solutions for these major issues. Both types of MSCs have been shown to be effective in augmenting wound healing by modulating the immune response and secreting paracrine factors which promote therapeutic (neo) angiogenesis and thereby providing biological ingredients for wound tissue regeneration, and they

Optimal wound bed preparation encompasses not only debridement and proper management of the bacterial load but also correction of the wound matrix and reconditioning of phenotypically altered resident cells which are present in chronic wounds. Based on their characteristics and biological activity, MSCs are capable of interacting with resident wound cells to transform resident cells to functional matrix building cells [122]. This might be of particular importance for the dermal rebuilding process to stimulate keratinocytes to accomplish

Given their higher isolation yield, ease of harvesting, and abundance of adipose tissue, some groups believe that AD-MSCs might be more clinically attractive. Not only because of their angiogenic capability, but they may also function in situ as pericytes providing vascular stability and they might communicate with endothelial cells in response to environmental stimuli [123, 124]. However, experienced clinicians may dispute the cited potential risk for complications with BMA, as they feel comfortable in performing BMA procedures in medical-office settings using local anesthetics and imaging to perform the aspiration. Shapiro and coworkers performed a prospective, single-blind, placebo-controlled trial on 25 patients with bilateral knee osteoarthritis and reported that the BMA, production, and use of BMC is a safe

BM-MSCs are frequently being studied in patients with critical limb ischemia, who also might suffer from chronic wounds and who are not eligible for the revascularization procedure due to several comorbidities, namely high operative risk, multiple failures of revascularization, and high rate of restenosis. These patients are suitable for biological cell-based therapy with MSCs. In particular, BM-MSCs protocols are newly emerging therapies to treat CLI in this

are ultimately capable of inducing full wound closure (**Figure 11**; [118–121]).

Regenerative medicine technologies offer solutions to a number of compelling clinical problems that have not been able to adequately result in a solution through the use of drugs, surgery, or permanent replacement devices.

The purpose of this chapter was to review multiple aspects of both PRP and MSC biocellular therapies as part of a wound care treatment plan to support in the healing of chronic and recalcitrant wounds.

Numerous significant aspects that are still not well understood or standardized have been discussed, as well as the rationale for cell-based therapies. For platelet-rich plasma preparations, specific formulations, platelet dosing, processing, and the differences between systems were discussed. With regard to bone marrow and adipose tissue, as cell sources for obtaining high quality mesenchymal cells, some technicalities were provided.

Among both tissue-based cellular therapies, bone marrow mesenchymal cells have been the most frequently employed and reported on. In this review, evidence is shown on results from several clinical studies in which autologous biologics have been applied in patients with chronic wounds. The outcomes of these studies suggested that the application of biocellular products can reverse the microenvironment in chronic wounds, achieving the ultimate goal: full wound epithelialization in the shortest possible time. Furthermore, it was revealed that these treatments are safely executed without adverse effects for patients.

### **Conflict of interest**

The author served also as Chief Scientific Officer of EmCyte Corporation.

### **Author details**

Peter A. Everts

Address all correspondence to: peter@gulfcoastbiologics.com

Gulf Coast Biologics, Fort Myers, FL, USA

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**Chapter 10**

Provisional chapter

**The Wound Healing Responses and Corneal**

DOI: 10.5772/intechopen.81886

The Wound Healing Responses and Corneal

**Biomechanics after Keratorefractive Surgery**

Keywords: wound healing, refractive surgery, corneal biomechanics

Corneal biomechanics have been concerned recently since it is not only found to play an important role in the wound healing process after corneal refractive surgeries, but also essential to improve the predictability and safety of refractive procedures. Corneal biomechanics and wound healing responses are linked in time and space and may also cause complications of keratectasia, haze formation, and regression. This review focuses on wound healing and biomechanics of the corneal refractive procedures. Identifying corneal wound healing from the biomechanical point of view is mandatory to improve the out-

Over the past 30 years, corneal refractive surgery has successfully corrected the refractive error for millions of patients. The spread of laser corneal refractive surgery is increasing the interest in the study of the safety and predictability. Many studies showed that the wound healing process influences the predictability and safety. Corneal wound healing is a major contributor to the success of refractive surgeries. Biological differences in wound healing responses are thought to be a major factor limiting the predictability of refractive surgery [1]. In some cases, mechanical instability or an abnormal wound healing process can lead to serious complica-

Hence, it is important to investigate and understand the corneal biomechanics and wound

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Biomechanics after Keratorefractive Surgery

Wenjing Wu and Yan Wang

Wenjing Wu and Yan Wang

Abstract

1. Introduction

http://dx.doi.org/10.5772/intechopen.81886

comes and reduce the complications.

tions such as keratectasia or severe haze.

healing process for a better vision after corneal refractive surgery.

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter


#### **The Wound Healing Responses and Corneal Biomechanics after Keratorefractive Surgery** The Wound Healing Responses and Corneal Biomechanics after Keratorefractive Surgery

DOI: 10.5772/intechopen.81886

Wenjing Wu and Yan Wang Wenjing Wu and Yan Wang

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81886

#### Abstract

[126] Uccioli L, Meloni M, Izzo V, Giurato L, Merolla S, et al. Critical limb ischemia: Current challenges and future prospects. Vascular Health and Risk Management. 2018;**14**:63-74

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Vascular Surgery. 2014;**27**:23-31

Utrecht: Utrecht University; 2007

2017;**81**:1713-1720

180 Wound Healing - Current Perspectives

Corneal biomechanics have been concerned recently since it is not only found to play an important role in the wound healing process after corneal refractive surgeries, but also essential to improve the predictability and safety of refractive procedures. Corneal biomechanics and wound healing responses are linked in time and space and may also cause complications of keratectasia, haze formation, and regression. This review focuses on wound healing and biomechanics of the corneal refractive procedures. Identifying corneal wound healing from the biomechanical point of view is mandatory to improve the outcomes and reduce the complications.

Keywords: wound healing, refractive surgery, corneal biomechanics

### 1. Introduction

Over the past 30 years, corneal refractive surgery has successfully corrected the refractive error for millions of patients. The spread of laser corneal refractive surgery is increasing the interest in the study of the safety and predictability. Many studies showed that the wound healing process influences the predictability and safety. Corneal wound healing is a major contributor to the success of refractive surgeries. Biological differences in wound healing responses are thought to be a major factor limiting the predictability of refractive surgery [1]. In some cases, mechanical instability or an abnormal wound healing process can lead to serious complications such as keratectasia or severe haze.

Hence, it is important to investigate and understand the corneal biomechanics and wound healing process for a better vision after corneal refractive surgery.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## 2. The wound healing responses and corneal biomechanics after keratorefractive surgery

#### 2.1. Corneal refractive surgery

Corneal refractive surgery changes the corneal curvature to correct the refractive error. The most common laser refractive procedures performed today are small incision lenticule extraction surgery (SMILE) [2, 3], femtosecond laser in situ keratomileusis (FS-LASIK), and surface ablation procedures, i.e., photorefractive keratectomy (PRK), laser epithelial keratomileusis (LASEK), and epi-LASIK [4]. With the development of the femtosecond laser, the SMILE surgery and FS-LASIK have become the most commonly used procedures in China for myopic subjects.

2.2.3. Descemet's membrane and biomechanics

2.2.4. Endothelium and biomechanics

tion by the corneal stroma [8, 9].

more stable visual results.

2.3.1. Epithelial wound healing

are regenerated [9].

ing rigidity of the cornea through the Finite element evaluation [5].

Descemet's membrane is approximately 10-nm thick and considered as a secretion of endothelial cells. The membrane is comprised of type IV collagen fibers. It is highly elastic and represents a barrier against punctures. Descemet's membrane serves as an endothelial basement membrane. Bowman's layer and Descemet's membrane accounted for 20% of the bend-

The Wound Healing Responses and Corneal Biomechanics after Keratorefractive Surgery

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183

The endothelium is composed of one layer of cells, which adhere to the Descemet's membrane. The endothelium cells cannot regenerate after damage or aging, but can spread and enlarge to maintain the cornea clear and transparent, and further prevent the cornea from becoming hydrated. The corneal endothelium may indirectly affect the corneal stiffness by regulating corneal hydration. The loss of corneal endothelial cells will result in increased water absorp-

It is noteworthy that the corneal biomechanics and wound healing responses are linked in time and space. Specifically, the corneal biomechanics involves the stromal healing responses; the better stromal healing process will contribute to better corneal biomechanics after surgery and

Epithelial wound healing involves three main steps: sliding, proliferation, and stratification of epithelial cells. Specifically, the epithelium cells migrate to the wound surface; then the cells increase and divide; lastly the cells cover the wound area and multiple layers of the epithelium

Some studies suggest that many cytokines are involved in the healing process including the epithelial growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and transforming growth factor β (TGF-β). [9] These changes permit cells to migrate, establishing dynamic adhesion with other epithelial cells and extracellular matrix components. In the epithelial cells surrounding the wound edge, there is an increased expression of CD44.

When the basal membrane is damaged, cytokines, neuropeptides, growth factors, chemokines, and matrix metalloproteinases can diffuse into the stroma and interact with keratocytes. These factors could stimulate the transformation of the keratocytes into myofibroblast cells [9]. One recent study [10] used the exosomes extracted from the epithelial cells, and cultured these exosomes with fibroblast cells; they found that the stroma cells transformed into myofibroblast

After the migration of epithelial cells, the phase of proliferation begins.

2.3.2. Corneal epithelial and stromal interactions

2.3. Corneal biomechanics and corneal wound healing after refractive surgeries

#### 2.2. Corneal structure and biomechanics

The cornea is a highly specialized transparent avascular tissue and is composed of five layers. They are epithelium, stroma, Descemet's membrane, and endothelium. Stroma is the main part of the cornea, and any factor that changes the corneal structure may obviously influence the biomechanical properties of the cornea [5, 6].

### 2.2.1. Epithelium, Bowman's membrane, and biomechanics

The epithelium's contribution to corneal biomechanics was significantly lower than that of the stroma with respect to the stiffness. Bowman's membrane tissue is a transparent sheet of approximately 12 μm. It is acellular and is composed of densely packed collagen fibrils that are in random direction. The fibrils are continuous with those in the stroma, which is believed to stabilize the corneal curvature [5].

#### 2.2.2. Corneal stroma and biomechanics

The stroma constitutes nearly 90% of the corneal thickness. And its biomechanical properties are influenced by the collagen fibers and extracellular matrix (ECM), which further determine the corneal strength, shape, and transparency [7, 8].

The stroma is a fibrous layer of lamellae made up of connective tissue. Interlamellar branching is more extensive in the anterior stroma than in the posterior stroma. The density of the collagen lamellae is higher, and their arrangement and directionality are more complicated anteriorly than posteriorly. The collagen lamellae in the corneal stroma are organized into a complex, highly intertwined three-dimensional meshwork of transversely oriented fibers, which contributes to the corneal shape and stromal stiffness. Another critical component for corneal stromal biomechanics is the ECM. The ECM is mostly composed of proteoglycans (PGs), which comprise a core protein and are located in the spaces among the collagen fibers in the corneal stroma. PGs play a critical role in collagen fibril assembly and spacing, and their mechanical importance may be greater than currently recognized [9].

### 2.2.3. Descemet's membrane and biomechanics

2. The wound healing responses and corneal biomechanics after

Corneal refractive surgery changes the corneal curvature to correct the refractive error. The most common laser refractive procedures performed today are small incision lenticule extraction surgery (SMILE) [2, 3], femtosecond laser in situ keratomileusis (FS-LASIK), and surface ablation procedures, i.e., photorefractive keratectomy (PRK), laser epithelial keratomileusis (LASEK), and epi-LASIK [4]. With the development of the femtosecond laser, the SMILE surgery and FS-

The cornea is a highly specialized transparent avascular tissue and is composed of five layers. They are epithelium, stroma, Descemet's membrane, and endothelium. Stroma is the main part of the cornea, and any factor that changes the corneal structure may obviously influence the

The epithelium's contribution to corneal biomechanics was significantly lower than that of the stroma with respect to the stiffness. Bowman's membrane tissue is a transparent sheet of approximately 12 μm. It is acellular and is composed of densely packed collagen fibrils that are in random direction. The fibrils are continuous with those in the stroma, which is believed

The stroma constitutes nearly 90% of the corneal thickness. And its biomechanical properties are influenced by the collagen fibers and extracellular matrix (ECM), which further determine

The stroma is a fibrous layer of lamellae made up of connective tissue. Interlamellar branching is more extensive in the anterior stroma than in the posterior stroma. The density of the collagen lamellae is higher, and their arrangement and directionality are more complicated anteriorly than posteriorly. The collagen lamellae in the corneal stroma are organized into a complex, highly intertwined three-dimensional meshwork of transversely oriented fibers, which contributes to the corneal shape and stromal stiffness. Another critical component for corneal stromal biomechanics is the ECM. The ECM is mostly composed of proteoglycans (PGs), which comprise a core protein and are located in the spaces among the collagen fibers in the corneal stroma. PGs play a critical role in collagen fibril assembly and spacing, and their

LASIK have become the most commonly used procedures in China for myopic subjects.

keratorefractive surgery

182 Wound Healing - Current Perspectives

2.1. Corneal refractive surgery

2.2. Corneal structure and biomechanics

biomechanical properties of the cornea [5, 6].

to stabilize the corneal curvature [5].

2.2.2. Corneal stroma and biomechanics

2.2.1. Epithelium, Bowman's membrane, and biomechanics

the corneal strength, shape, and transparency [7, 8].

mechanical importance may be greater than currently recognized [9].

Descemet's membrane is approximately 10-nm thick and considered as a secretion of endothelial cells. The membrane is comprised of type IV collagen fibers. It is highly elastic and represents a barrier against punctures. Descemet's membrane serves as an endothelial basement membrane. Bowman's layer and Descemet's membrane accounted for 20% of the bending rigidity of the cornea through the Finite element evaluation [5].

### 2.2.4. Endothelium and biomechanics

The endothelium is composed of one layer of cells, which adhere to the Descemet's membrane. The endothelium cells cannot regenerate after damage or aging, but can spread and enlarge to maintain the cornea clear and transparent, and further prevent the cornea from becoming hydrated. The corneal endothelium may indirectly affect the corneal stiffness by regulating corneal hydration. The loss of corneal endothelial cells will result in increased water absorption by the corneal stroma [8, 9].

### 2.3. Corneal biomechanics and corneal wound healing after refractive surgeries

It is noteworthy that the corneal biomechanics and wound healing responses are linked in time and space. Specifically, the corneal biomechanics involves the stromal healing responses; the better stromal healing process will contribute to better corneal biomechanics after surgery and more stable visual results.

### 2.3.1. Epithelial wound healing

Epithelial wound healing involves three main steps: sliding, proliferation, and stratification of epithelial cells. Specifically, the epithelium cells migrate to the wound surface; then the cells increase and divide; lastly the cells cover the wound area and multiple layers of the epithelium are regenerated [9].

Some studies suggest that many cytokines are involved in the healing process including the epithelial growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and transforming growth factor β (TGF-β). [9] These changes permit cells to migrate, establishing dynamic adhesion with other epithelial cells and extracellular matrix components. In the epithelial cells surrounding the wound edge, there is an increased expression of CD44. After the migration of epithelial cells, the phase of proliferation begins.

### 2.3.2. Corneal epithelial and stromal interactions

When the basal membrane is damaged, cytokines, neuropeptides, growth factors, chemokines, and matrix metalloproteinases can diffuse into the stroma and interact with keratocytes. These factors could stimulate the transformation of the keratocytes into myofibroblast cells [9]. One recent study [10] used the exosomes extracted from the epithelial cells, and cultured these exosomes with fibroblast cells; they found that the stroma cells transformed into myofibroblast cells with higher expression of TGF-β, CD63, and PDGF-B. This study indicates that the epithelial cells are very important for stromal wound healing, and they may use exosomes to transmit the wound healing signals in order to regulate the process [10].

2.4. Complications relevant to the corneal wound healing and biomechanics after laser

instability could cause some complications after corneal refractive surgery.

cases where the biomechanical status of the cornea is abnormal.

Corneal wound healing is important for the predictability and safety of corneal refractive surgery. The refractive outcome and its stability over time are strongly influenced by the corneal biomechanics and wound healing process. And the abnormal healing process or biomechanical

The Wound Healing Responses and Corneal Biomechanics after Keratorefractive Surgery

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185

Refractive regression is defined as a gradual loss of the attempted correction that limits prediction. Many studies showed loss of surgical outcome and the main cause seems to be the regression. The regression is mainly due to epithelial hyperplasia and stromal remodeling, two processes related to corneal wound healing. Refractive regression is a major challenge for myopia, especially for high levels of correction. Apoptosis, keratocyte proliferation, and myofibroblast cellular density have proved to be more intense following treatment for high myopia compared to treatments for mild myopia. Myofibroblasts are important effectors of regression. The changes of corneal biomechanics also induce the changes of the corneal shape

Corneal ectasia is a rare complication induced by the corneal refractive surgery. It may occur due to an insufficient residual stromal thickness or unidentified subclinical keratoconus. Many advanced examinations have been used clinically to exclude the potential subclinical keratoconus. Moreover, surgeons have also used many ways to preserve as thicker corneal thickness as possible. However, it is still difficult to avoid the onset of keratectasia. It may be because the corneal stiffness or biomechanics is different among individuals [16]. And the postoperative stromal tensile strength is different for each procedure. This indicates that the risk evaluations for ectasia should take the residual stromal bed thickness and corneal biomechanical properties into account. Moreover, biomechanical changes can manifest clinically as changes of the corneal shape and increased sensitivity to shape changes. The role of biomechanics is therefore important to consider in routine refractive procedures and in special

Corneal haze refers to the cornea opacity. It is commonly seen in surface ablation surgeries. Haze can potentially form in the interface between the LASIK flap and the stromal bed or directly underneath the newly formed epithelium overlying the stromal tissue after PRK surgery [17]. In modern refractive surgery, haze tends to be mild and resolves very quickly. In

Abnormal regulation of the wound healing process can result in the formation of stromal haze with decreased corneal crystalline expression, increased light scattering, and production of a

extremely rare instances, haze can cause decreased visual acuity and increased glare.

refractive surgery

2.4.1. Regression

and cause regression.

2.4.1.1. Keratectasia

2.4.2. Haze

After the epithelial and stromal damage, soluble mediators could be secreted through the epithelium and move to the stroma area. These molecules, like TGF-β and TSP-1, could stimulate the wound healing process and make keratocytes transform into myofibroblasts. The myofibroblasts lay down the ECM and generate alpha-smooth muscle actin (α-SMA) to close the wound. However, abnormal wound healing synthesizes excessive α-SMA, exerts traction forces across the ECM, and causes unorganized tissue architecture, haze, and regression after corneal refractive surgery [11, 12]. Only when the EBM is appropriately re-established, proper stromal levels of TGF-β and PDGF cause myofibroblast apoptosis, keratocyte repopulation, clearing of the abnormal ECM, and restoring of corneal transparency [13]. A delay in the regeneration of the EBM, due to damage, dystrophy, or elevated levels of MMP-2 and MMP-9, causes TGF-β and PDGF to continue entering the corneal stroma.

#### 2.3.3. Stromal wound healing and corneal biomechanics

The wound healing of the stroma is end when the collagen fibrils fully connected the wound edge. Activated cells migrate to the wound area. The keratocytes are changed through the reorganization of the cytoskeleton and the development of stress fibers and focal adhesion structures. Genes that encode fibronectin, metalloproteinases, and integrins are activated. The early matrix consists of fibronectin [14], which was conducive to cell migration and proliferation. Then the matrix is converted to a collagen and proteoglycan matrix that increases the tissue tensile strength and resilience. Growth factors increased stiffness and enhanced mechanical load through enhanced collagen fiber formation and cross-linking. The geometry of the collagen network will determine the mechanical properties of the wound. The collagen fiber diameter increases with time during the wound healing process and is related to tensile strength. Interweaving of collagen bundles between neighboring lamellae provides an important structural foundation for shear resistance and transfer of tensile loads between lamellae.

The transformation of keratocytes into myofibroblasts is curial in the wound healing process. These cells are characterized by the expression of α-smooth muscle actin, stress fibers, and focal adhesion complexes. The microfilament bundles of myofibroblasts form stress fibers, and they contract and remodel the adjacent ECM. Myofibroblasts extend from the anterior stroma to the posterior stroma in a progressive manner. These cells develop fibrotic tissue for repair. Besides that, deposition of the ECM is beneficial for the matrix stiffening and global cellular stress. However, excess myofibroblasts cause the deposition of disorganized collagen and glycosaminoglycan [15]. The underlying mechanism for the interaction between myofibroblast cells and matrix is the focal adhesions. They play the role of a mechanotransduction system, transmitting the force generated by stress fibers to the surrounding ECM and also transducing the extracellular mechanical signals into the intracellular signaling. Further investigations are needed to find whether we could regulate the mechanotransduction system to influence the corneal wound healing process.

### 2.4. Complications relevant to the corneal wound healing and biomechanics after laser refractive surgery

Corneal wound healing is important for the predictability and safety of corneal refractive surgery. The refractive outcome and its stability over time are strongly influenced by the corneal biomechanics and wound healing process. And the abnormal healing process or biomechanical instability could cause some complications after corneal refractive surgery.

### 2.4.1. Regression

cells with higher expression of TGF-β, CD63, and PDGF-B. This study indicates that the epithelial cells are very important for stromal wound healing, and they may use exosomes to

After the epithelial and stromal damage, soluble mediators could be secreted through the epithelium and move to the stroma area. These molecules, like TGF-β and TSP-1, could stimulate the wound healing process and make keratocytes transform into myofibroblasts. The myofibroblasts lay down the ECM and generate alpha-smooth muscle actin (α-SMA) to close the wound. However, abnormal wound healing synthesizes excessive α-SMA, exerts traction forces across the ECM, and causes unorganized tissue architecture, haze, and regression after corneal refractive surgery [11, 12]. Only when the EBM is appropriately re-established, proper stromal levels of TGF-β and PDGF cause myofibroblast apoptosis, keratocyte repopulation, clearing of the abnormal ECM, and restoring of corneal transparency [13]. A delay in the regeneration of the EBM, due to damage, dystrophy, or elevated levels of MMP-2 and MMP-9,

The wound healing of the stroma is end when the collagen fibrils fully connected the wound edge. Activated cells migrate to the wound area. The keratocytes are changed through the reorganization of the cytoskeleton and the development of stress fibers and focal adhesion structures. Genes that encode fibronectin, metalloproteinases, and integrins are activated. The early matrix consists of fibronectin [14], which was conducive to cell migration and proliferation. Then the matrix is converted to a collagen and proteoglycan matrix that increases the tissue tensile strength and resilience. Growth factors increased stiffness and enhanced mechanical load through enhanced collagen fiber formation and cross-linking. The geometry of the collagen network will determine the mechanical properties of the wound. The collagen fiber diameter increases with time during the wound healing process and is related to tensile strength. Interweaving of collagen bundles between neighboring lamellae provides an important structural foundation for shear resistance and transfer of tensile loads between lamellae. The transformation of keratocytes into myofibroblasts is curial in the wound healing process. These cells are characterized by the expression of α-smooth muscle actin, stress fibers, and focal adhesion complexes. The microfilament bundles of myofibroblasts form stress fibers, and they contract and remodel the adjacent ECM. Myofibroblasts extend from the anterior stroma to the posterior stroma in a progressive manner. These cells develop fibrotic tissue for repair. Besides that, deposition of the ECM is beneficial for the matrix stiffening and global cellular stress. However, excess myofibroblasts cause the deposition of disorganized collagen and glycosaminoglycan [15]. The underlying mechanism for the interaction between myofibroblast cells and matrix is the focal adhesions. They play the role of a mechanotransduction system, transmitting the force generated by stress fibers to the surrounding ECM and also transducing the extracellular mechanical signals into the intracellular signaling. Further investigations are needed to find whether we could regulate the mechanotransduction system to influence the

transmit the wound healing signals in order to regulate the process [10].

causes TGF-β and PDGF to continue entering the corneal stroma.

2.3.3. Stromal wound healing and corneal biomechanics

184 Wound Healing - Current Perspectives

corneal wound healing process.

Refractive regression is defined as a gradual loss of the attempted correction that limits prediction. Many studies showed loss of surgical outcome and the main cause seems to be the regression. The regression is mainly due to epithelial hyperplasia and stromal remodeling, two processes related to corneal wound healing. Refractive regression is a major challenge for myopia, especially for high levels of correction. Apoptosis, keratocyte proliferation, and myofibroblast cellular density have proved to be more intense following treatment for high myopia compared to treatments for mild myopia. Myofibroblasts are important effectors of regression. The changes of corneal biomechanics also induce the changes of the corneal shape and cause regression.

### 2.4.1.1. Keratectasia

Corneal ectasia is a rare complication induced by the corneal refractive surgery. It may occur due to an insufficient residual stromal thickness or unidentified subclinical keratoconus. Many advanced examinations have been used clinically to exclude the potential subclinical keratoconus. Moreover, surgeons have also used many ways to preserve as thicker corneal thickness as possible. However, it is still difficult to avoid the onset of keratectasia. It may be because the corneal stiffness or biomechanics is different among individuals [16]. And the postoperative stromal tensile strength is different for each procedure. This indicates that the risk evaluations for ectasia should take the residual stromal bed thickness and corneal biomechanical properties into account. Moreover, biomechanical changes can manifest clinically as changes of the corneal shape and increased sensitivity to shape changes. The role of biomechanics is therefore important to consider in routine refractive procedures and in special cases where the biomechanical status of the cornea is abnormal.

#### 2.4.2. Haze

Corneal haze refers to the cornea opacity. It is commonly seen in surface ablation surgeries. Haze can potentially form in the interface between the LASIK flap and the stromal bed or directly underneath the newly formed epithelium overlying the stromal tissue after PRK surgery [17]. In modern refractive surgery, haze tends to be mild and resolves very quickly. In extremely rare instances, haze can cause decreased visual acuity and increased glare.

Abnormal regulation of the wound healing process can result in the formation of stromal haze with decreased corneal crystalline expression, increased light scattering, and production of a disorganized extracellular matrix. Myofibroblasts are major contributors to corneal opacity with reduced expression of crystallin, greater secretion of type III collagen, and spread morphology. Over a period of time ranging from several weeks to several months, the myofibroblasts tend to gradually disappear through a series of remodeling processes. This process may be closely related to the expression of matrix metalloproteinases. These proteins are a family of proteolysis enzymes, which could degrade abnormal collagen fibrils. The cytokines, growth factors, and inflammatory mediators could also regulate the synthesis of metalloproteinase [18–20].

[5] Ma J, Wang Y, Wei P, Jhanji V. Biomechanics and structure of the cornea: Implications and association with corneal disorders. Survey of Ophthalmology. 2018;63(6):851-861. DOI:

The Wound Healing Responses and Corneal Biomechanics after Keratorefractive Surgery

http://dx.doi.org/10.5772/intechopen.81886

187

[6] Roberts CJ. Importance of accurately assessing biomechanics of the cornea. Current Opinion in Ophthalmology. 2016;27(4):285-291. DOI: 10.1097/ICU.0000000000000282

[7] Roberts CJ, Dupps WJ Jr. Biomechanics of corneal ectasia and biomechanical treatments. Journal of Cataract and Refractive Surgery. 2014;40(6):991-998. DOI: 10.1016/j.jcrs.2014.04.013

[8] Spadea L, Giammaria D, Trabucco P. Corneal wound healing after laser vision correction. The British Journal of Ophthalmology. 2016;100(1):28-33. DOI: 10.1136/bjophthalmol-

[9] Baldwin HC, Marshall J. Growth factors in corneal wound healing following refractive surgery: A review. Acta Ophthalmologica Scandinavica. 2002;80(3):238-247. Review

[10] Han KY, Tran JA, Chang JH, Azar DT, Zieske JD. Potential role of corneal epithelial cellderived exosomes in corneal wound healing and neovascularization. Scientific Reports.

[11] Kling S, Hafezi F. Corneal biomechanics—A review. Ophthalmic & Physiological Optics.

[12] Shu DY, Lovicu FJ. Myofibroblast transdifferentiation: The dark force in ocular wound healing and fibrosis. Progress in Retinal and Eye Research. 2017;60:44-65. DOI: 10.1016/j.

[13] Kivanany PB, Grose KC, Petroll WM. Temporal and spatial analysis of stromal cell and extracellular matrix patterning following lamellar keratectomy. Experimental Eye

[14] Raghunathan VK, Thomasy SM, Strøm P, Yañez-Soto B, Garland SP, Sermeno J, et al. Tissue and cellular biomechanics during corneal wound injury and repair. Acta Bio-

[15] Ljubimov AV, Saghizadeh M. Progress in corneal wound healing. Progress in Retinal and

[16] Moshirfar MD, Desautels JD, Walker BS, Murri MC, Birdsong OC, Hoopes PSr. Optical regression following corneal laser refractive surgery: Epithelial and stromal responses.

[17] Anitua E, Muruzabal F, Alcalde I, Merayo-Lloves J, Orive G. Plasma rich in growth factors (PRGF-Endoret) stimulates corneal wound healing and reduces haze formation after PRK surgery. Experimental Eye Research. 2013;115:153-161. DOI: 10.1016/j.exer.2013.07.007

[18] Daniels JT, Schultz GS, Blalock TD, Garrett Q, Grotendorst GR, Dean NM, et al. Mediation of transforming growth factor-beta(1)-stimulated matrix contraction by fibroblasts: A role

Medical Hypothesis, Discovery and Innovation in Ophthalmology. 2018;7(1):1-9

10.1016/j.survophthal.2018.05.004

2017;37(3):240-252. DOI: 10.1111/opo.12345

Research. 2016;153:56-64. DOI: 10.1016/j.exer.2016.10.009

materialia. 2017;58:291-301. DOI: 10.1016/j.actbio.2017.05.051

Eye Research. 2015;49:17-45. DOI: 10.1016/j.preteyeres.2015.07.002

2015-306770

2017;7:40548

preteyeres.2017.08.001

### 3. Conclusions

Laser refractive surgeries are effective for the correction of refractive errors. A better understanding of corneal wound healing from the biomechanical point of view is mandatory if refractive surgery is ever to achieve more predictable and safer refractive results.

### Author details

Wenjing Wu and Yan Wang\*

\*Address all correspondence to: wangyan7143@vip.sina.com

Tianjin Eye Hospital, Tianjin Eye Institute, Tianjin Key Laboratory of Ophthalmology and Visual Science, Nankai University, Tianjin, China

### References


[5] Ma J, Wang Y, Wei P, Jhanji V. Biomechanics and structure of the cornea: Implications and association with corneal disorders. Survey of Ophthalmology. 2018;63(6):851-861. DOI: 10.1016/j.survophthal.2018.05.004

disorganized extracellular matrix. Myofibroblasts are major contributors to corneal opacity with reduced expression of crystallin, greater secretion of type III collagen, and spread morphology. Over a period of time ranging from several weeks to several months, the myofibroblasts tend to gradually disappear through a series of remodeling processes. This process may be closely related to the expression of matrix metalloproteinases. These proteins are a family of proteolysis enzymes, which could degrade abnormal collagen fibrils. The cytokines, growth factors, and inflammatory mediators could also regulate the synthesis of

Laser refractive surgeries are effective for the correction of refractive errors. A better understanding of corneal wound healing from the biomechanical point of view is mandatory if

Tianjin Eye Hospital, Tianjin Eye Institute, Tianjin Key Laboratory of Ophthalmology and

[1] Azar DT, Chang JH, Han KY. Wound healing after keratorefractive surgery: Review of biological and optical considerations. Cornea. 2012;31(Suppl 1):S9-S19. DOI: 10.1097/

[2] Sekundo W, Kunert KS, Blum M. Small incision corneal refractive surgery using the small incision lenticuleextraction (SMILE) procedure for the correction of myopia and myopic astigmatism: Results of a 6 month prospective study. The British Journal of Ophthalmol-

[3] Shah R, Shah S, Sengupta S. Results of small incision lenticule extraction: All-in-one femtosecond laser refractive surgery. Journal of Cataract and Refractive Surgery. 2011;37(1):

[4] Murueta-Goyena A, Cañadas P. Visual outcomes and management after corneal refractive surgery: A review. Journal of Optometry. 2018;11(2):121-129. DOI: 10.1016/j.optom.2017.

refractive surgery is ever to achieve more predictable and safer refractive results.

\*Address all correspondence to: wangyan7143@vip.sina.com

ogy. 2011;95(3):335-339. DOI: 10.1136/bjo.2009.174284

127-137. DOI: 10.1016/j.jcrs.2010.07.033

Visual Science, Nankai University, Tianjin, China

metalloproteinase [18–20].

186 Wound Healing - Current Perspectives

3. Conclusions

Author details

References

09.002

Wenjing Wu and Yan Wang\*

ICO.0b013e31826ab0a7


for connective tissue growth factor in contractile scarring. The American Journal of Pathology. 2003;163(5):2043-2052

**Chapter 11**

**Provisional chapter**

**Open Abdomen: The Surgeons' Challenge**

**Open Abdomen: The Surgeons' Challenge**

DOI: 10.5772/intechopen.81428

An open abdomen is defined as purposely foregoing fascial closure of the abdomen after the cavity is opened. Management of complex abdominal problems with the open abdomen and temporary abdominal closure techniques has become a common and valuable tool in surgery. Several challenging clinical situations can necessitate leaving the abdominal cavity open after surgery, resulting in an open abdomen. The indications for open abdomen are as follows: Damage control for life-threatening intraabdominal bleeding, severe acute pancreatitis, severe abdominal sepsis, and prevention and treatment of the abdominal compartment syndrome. Damage control surgery is based on a rapid control of bleeding and focuses on reversing physiologic exhaustion in a critically ill or injured patient. In severe abdominal sepsis, the intervention should be abbreviated due to suboptimal local conditions for healing and global susceptibility to spiraling organ failure. Abdominal compartment syndrome (ACS) is commonly encountered and the only solution is decreasing the pressure by decompressive laparotomy. Open abdomen is associated with significant complications, including wound infection, fluid and protein loss, a catabolic state, loss of abdominal wall domain, and development of enteroatmospheric fistula; however, if the indications are clear, it can become a most valuable resource in treating these conditions.

**Keywords:** open abdomen, laparostoma, damage control, abdominal compartment

The open abdomen is the most challenging of the wounds that a surgeon faces, that is because of the metabolic, physiological, and dynamic implications that this condition entails. An open abdomen is defined as a purposely foregoing fascial closure of the abdomen after the

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Juan José Santivañez Palomino, Arturo Vergara and

Juan José Santivañez Palomino, Arturo Vergara

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81428

syndrome, abdominal sepsis

**1. General aspects**

Manuel Cadena

and Manuel Cadena

**Abstract**


#### **Open Abdomen: The Surgeons' Challenge Open Abdomen: The Surgeons' Challenge**

DOI: 10.5772/intechopen.81428

José Santivañez Palomino, Arturo Vergara and Manuel Cadena José Santivañez Palomino, Arturo Vergara and Manuel Cadena

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81428

#### **Abstract**

for connective tissue growth factor in contractile scarring. The American Journal of Pathol-

[19] Torricelli AA, Santhanam A, Wu J, Singh V, Wilson SE. The corneal fibrosis response to epithelial-stromal injury. Experimental Eye Research. 2016;142:110-118. DOI: 10.1016/j.

[20] Wilson SE. Corneal myofibroblast biology and pathobiology: Generation, persistence, and transparency. Experimental Eye Research. 2012;99:78-88. DOI: 10.1016/j.exer.2012.03.018

ogy. 2003;163(5):2043-2052

exer.2014.09.012

188 Wound Healing - Current Perspectives

An open abdomen is defined as purposely foregoing fascial closure of the abdomen after the cavity is opened. Management of complex abdominal problems with the open abdomen and temporary abdominal closure techniques has become a common and valuable tool in surgery. Several challenging clinical situations can necessitate leaving the abdominal cavity open after surgery, resulting in an open abdomen. The indications for open abdomen are as follows: Damage control for life-threatening intraabdominal bleeding, severe acute pancreatitis, severe abdominal sepsis, and prevention and treatment of the abdominal compartment syndrome. Damage control surgery is based on a rapid control of bleeding and focuses on reversing physiologic exhaustion in a critically ill or injured patient. In severe abdominal sepsis, the intervention should be abbreviated due to suboptimal local conditions for healing and global susceptibility to spiraling organ failure. Abdominal compartment syndrome (ACS) is commonly encountered and the only solution is decreasing the pressure by decompressive laparotomy. Open abdomen is associated with significant complications, including wound infection, fluid and protein loss, a catabolic state, loss of abdominal wall domain, and development of enteroatmospheric fistula; however, if the indications are clear, it can become a most valuable resource in treating these conditions.

**Keywords:** open abdomen, laparostoma, damage control, abdominal compartment syndrome, abdominal sepsis

### **1. General aspects**

The open abdomen is the most challenging of the wounds that a surgeon faces, that is because of the metabolic, physiological, and dynamic implications that this condition entails. An open abdomen is defined as a purposely foregoing fascial closure of the abdomen after the

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

cavity has been opened [1]. Throughout the years, management of complex abdominal problems dealing with an open abdomen and techniques that handle the temporary closure of the abdominal wall have become common and valuable tools for the surgeon [2]. Several challenging clinical situations force the surgeon in leaving the abdominal cavity open after surgery, resulting in an open abdomen or laparostoma [3].

involves the stabilization of the physiological parameters in the intensive care unit, followed by the final stage of definitive surgical care in the operating room; this usually occurs within 24–48 h of the initial operation (preferably following the reversal of the lethal triad) [9].

Open Abdomen: The Surgeons' Challenge http://dx.doi.org/10.5772/intechopen.81428 191

• Open abdomen has become the preferred surgical method for patients whose physiological

• Persistent hypotension, acidosis (pH < 7.2), hypothermia (T < 34°C), and coagulopathy are strong predictors of the need for damage control and open abdomen in trauma patients. • Control of active hemorrhage must be the primary goal of the trauma surgeon during dam-

• The abdomen should never be closed because of the high risk of intra-abdominal

*The role of an open abdomen in the management of severe secondary peritonitis has been a controversial issue throughout time* [2]. In severe secondary peritonitis, some patients may experience disease progression from severe sepsis and septic shock to progressive organ dysfunction, hypotension, myocardial depression, and coagulopathy, where a staged approach might be

*If the patient is not in a condition where he can undergo definitive repair* and/or abdominal wall closure (such as instability, elevated requirements of inotropics, etc.), *the intervention should be cut short* because of the suboptimal local conditions for healing [11]. In addition, *peritonitis and intra-abdominal sepsis can influence the intra-abdominal pressure* because of bowel distension,

When facing the inability to completely control contamination in a single operation, it is recommended to postpone definitive intervention or anastomosis [13]. Extensive visceral edema and decreased abdominal wall compliance may increase the risk of developing abdominal compartment syndrome; therefore, primary fascial closure should not be attempted and the abdomen should be left open [12]. *Following the first* 24–48 h *after the initial surgery, the patient should be taken back to the operating room* for reoperation, lavage, drainage, source control, and

The CIAOW study reports that patients with abdominal sepsis have been shown to have worse outcomes after an open abdomen, with an increased incidence of fistula formation, intra-abdominal abscesses, and a higher-delayed primary closure rate [14, 15]. However,

derangements do not allow the completion of an intended operation.

• Open abdomen is still associated with serious complications.

**2.1. Key points**

age control laparotomy.

**3. Severe abdominal sepsis**

ascites, or parietal muscle contraction [12].

if its feasible [13] the closure of the abdominal wall.

hypertension.

required [10].

There are several indications for open abdomen, some of which are severe acute pancreatitis [4], damage control for life-threatening intra-abdominal bleeding (with a need for a "second look"), severe abdominal sepsis, and finally, prevention and treatment of an abdominal compartment syndrome [2]. In our recent experience, we have found that peritoneal failure, as the result of the imbalance between the mechanisms of defense of the guest and the peritoneal injury, is the clear indication of the need for the open abdomen.

### **2. Damage control**

Damage control surgery is based on a rapid control of bleeding and focuses on reversing physiologic exhaustion in a critically ill or injured patient [5]. Initially, it was introduced in the field as a temporizing measure used to salvage trauma patients very near death. Through time, damage control surgery has evolved to become the *preferred method for those general surgical patients whose physiological derangements do not allow the completion of an intended operation* [6].

About 10–15% of all laparotomies performed specifically for a trauma patient are managed with damage control techniques [7]. *Persistent hypotension, acidosis* (*pH <* 7.2), *hypothermia* (*T <* 34°C), *and coagulopathy are strong predictors of the need to use damage control and open abdomen in trauma patients* [8]. However, damage control should not be an afterthought; it should be considered early in the decision process before the patient reaches a point of no return (before reaching the triad of death). Therefore before the surgery begins, there are many factors that should be considered: the available resources, the nature of the injuries, the experience of the surgeon, the clinical condition of the patient, and any comorbid conditions the patient might have [2].

*During the damage control laparotomy, the primary goal of the trauma surgeon should be control of active hemorrhage* (vascular shunting or ligation, direct packing, resection, etc.), followed by a strict control of contamination, and lastly temporary abdominal closure [6].

Despite the advancement of supportive care and the development of new sophisticated commercial devices for temporary abdominal closure, an open abdomen is still highly associated with serious postoperatively complications such as nutritional problems dealing with fluid and protein loss, loss of abdominal domain secondary to fascial retraction, frozen abdomen, and enteroatmospheric fistulas [4].

Following a damage control surgery, the abdomen should never be closed because of the high risk of intra-abdominal hypertension. The second stage within damage control procedures involves the stabilization of the physiological parameters in the intensive care unit, followed by the final stage of definitive surgical care in the operating room; this usually occurs within 24–48 h of the initial operation (preferably following the reversal of the lethal triad) [9].

### **2.1. Key points**

cavity has been opened [1]. Throughout the years, management of complex abdominal problems dealing with an open abdomen and techniques that handle the temporary closure of the abdominal wall have become common and valuable tools for the surgeon [2]. Several challenging clinical situations force the surgeon in leaving the abdominal cavity open after

There are several indications for open abdomen, some of which are severe acute pancreatitis [4], damage control for life-threatening intra-abdominal bleeding (with a need for a "second look"), severe abdominal sepsis, and finally, prevention and treatment of an abdominal compartment syndrome [2]. In our recent experience, we have found that peritoneal failure, as the result of the imbalance between the mechanisms of defense of the guest and the peritoneal

Damage control surgery is based on a rapid control of bleeding and focuses on reversing physiologic exhaustion in a critically ill or injured patient [5]. Initially, it was introduced in the field as a temporizing measure used to salvage trauma patients very near death. Through time, damage control surgery has evolved to become the *preferred method for those general surgical patients whose physiological derangements do not allow the completion of an* 

About 10–15% of all laparotomies performed specifically for a trauma patient are managed with damage control techniques [7]. *Persistent hypotension, acidosis* (*pH <* 7.2), *hypothermia* (*T <* 34°C), *and coagulopathy are strong predictors of the need to use damage control and open abdomen in trauma patients* [8]. However, damage control should not be an afterthought; it should be considered early in the decision process before the patient reaches a point of no return (before reaching the triad of death). Therefore before the surgery begins, there are many factors that should be considered: the available resources, the nature of the injuries, the experience of the surgeon, the clinical condition of the patient, and any comorbid conditions the patient might

*During the damage control laparotomy, the primary goal of the trauma surgeon should be control of active hemorrhage* (vascular shunting or ligation, direct packing, resection, etc.), followed by a

Despite the advancement of supportive care and the development of new sophisticated commercial devices for temporary abdominal closure, an open abdomen is still highly associated with serious postoperatively complications such as nutritional problems dealing with fluid and protein loss, loss of abdominal domain secondary to fascial retraction, frozen abdomen,

Following a damage control surgery, the abdomen should never be closed because of the high risk of intra-abdominal hypertension. The second stage within damage control procedures

strict control of contamination, and lastly temporary abdominal closure [6].

surgery, resulting in an open abdomen or laparostoma [3].

injury, is the clear indication of the need for the open abdomen.

**2. Damage control**

190 Wound Healing - Current Perspectives

*intended operation* [6].

and enteroatmospheric fistulas [4].

have [2].


### **3. Severe abdominal sepsis**

*The role of an open abdomen in the management of severe secondary peritonitis has been a controversial issue throughout time* [2]. In severe secondary peritonitis, some patients may experience disease progression from severe sepsis and septic shock to progressive organ dysfunction, hypotension, myocardial depression, and coagulopathy, where a staged approach might be required [10].

*If the patient is not in a condition where he can undergo definitive repair* and/or abdominal wall closure (such as instability, elevated requirements of inotropics, etc.), *the intervention should be cut short* because of the suboptimal local conditions for healing [11]. In addition, *peritonitis and intra-abdominal sepsis can influence the intra-abdominal pressure* because of bowel distension, ascites, or parietal muscle contraction [12].

When facing the inability to completely control contamination in a single operation, it is recommended to postpone definitive intervention or anastomosis [13]. Extensive visceral edema and decreased abdominal wall compliance may increase the risk of developing abdominal compartment syndrome; therefore, primary fascial closure should not be attempted and the abdomen should be left open [12]. *Following the first* 24–48 h *after the initial surgery, the patient should be taken back to the operating room* for reoperation, lavage, drainage, source control, and if its feasible [13] the closure of the abdominal wall.

The CIAOW study reports that patients with abdominal sepsis have been shown to have worse outcomes after an open abdomen, with an increased incidence of fistula formation, intra-abdominal abscesses, and a higher-delayed primary closure rate [14, 15]. However, there is no definitive data or strong recommendation regarding the use of open abdomen in the face of severe peritonitis. Therefore, *when using an open abdomen approach under these circumstances, caution and individualization of patients should be the priority* [8].

development of abdominal compartment syndrome in the absence of a primary abdomino-

Open Abdomen: The Surgeons' Challenge http://dx.doi.org/10.5772/intechopen.81428 193

The organ dysfunction that can be seen with abdominal compartment syndrome is usually recognized by the changes in lung and renal function. As abdominal compartment syndrome develops, the pulmonary dynamics change, tidal volumes decrease or, if mechanical ventilation is being used, an increase in peak pressure can be observed with similar tidal volumes. Renal dysfunction can be seen when there is a decrease in urine output caused by decreased renal perfusion as the renal vein is compressed due to the increased abdominal pressure. Other organs can display changes after abdominal compartment syndrome including but not limited to the heart and brain [1]. Intra-abdominal hypertension and abdominal compartment

*All patients in the intensive care unit should have measurements of their intra-abdominal pressure because the real incidence of abdominal compartment syndrome in the intensive care unit remains sub-diagnosed, and in some cases it is still unknown*. When abdominal compartment syndrome is suspected, bladder pressures should be measured. This is accomplished by instilling a small amount of sterile saline into the bladder and attaching a Foley tube to a pressure transducer [1]; according to the findings, the following steps will be decided and a treatment will be administered (**Table 1**).

Management of this condition requires a multidisciplinary approach by the surgeon and the

There are four main principles when it comes to the management of intra-abdominal hypertension: first of all, serial monitoring of intra-abdominal pressure should be taken every 4–6 h; optimization of systemic perfusion and organ function in the patient with an increased intraabdominal pressure; medical procedures to reduce intra-abdominal pressure that are institution of specific such as sedation, analgesia, or neuromuscular blockade, and prompt surgical

Medical interventions include sedation to improve abdominal wall compliance, as well as the placing of a nasogastric tube for gastric drainage, removing intraperitoneal fluid collections if they are present, limiting intravenous fluids if possible, diuresis, and also allowing hypercarbia by reducing tidal volumes. *Although all these interventions are promising, the only solution for* 

intensive care unit team, taking in account a specific staged process [4] (**Figure 1**).

decompressive laparotomy for refractory intra-abdominal hypertension [2] (**Figure 2**).

*ACS is decreasing the pressure by performing a decompressive laparotomy* [1, 2, 16, 19].

**Table 1.** Final 2013 consensus definitions of the World Society of the Abdominal Compartment Syndrome [19].

syndrome can also generate changes in other intra-abdominal organs [18].

pelvic process [4].

**Intra-abdominal pressure (IAP)**

Intra-abdominal hypertension grade I 12–15 mm Hg Intra-abdominal hypertension grade II 16–20 mm Hg Intra-abdominal hypertension grade III 21–25 mm Hg Intra-abdominal hypertension grade IV > 25 mm Hg

Normal 5–7 mm Hg

### **3.1. Key points**


### **4. Abdominal compartment syndrome**

*Intra-abdominal hypertension and abdominal compartment syndrome are commonly encountered among surgical and nonsurgical critically ill patients*. Intra-abdominal hypertension is defined as a sustained pathologic increase in intra-abdominal pressure greater than or equal to 12 mm Hg. Abdominal compartment syndrome is defined as a sustained increase in intra-abdominal tension ≥20 mm Hg that is associated with new organ dysfunction or failure [2, 16].

$$\begin{array}{c} \text{Abdomainal performs pressure (APP) = mean arrival pressure (MAP)}\\ \text{ - intra-abdomainal pressure (MAP)} \end{array} \tag{1}$$

*Intra-abdominal hypertension can lead to tissue hypoperfusion, especially of the abdominal viscera, as well as organ dysfunction*. Uncontrolled intra-abdominal hypertension that exceeds 25 mm Hg can cause abdominal compartment syndrome, which is a potentially lethal complication. It is characterized by cardiorespiratory and renal dysfunction, as well as bacterial and toxin intestinal translocation and intracranial hypertension [17].

*Abdominal compartment syndrome develops as a result of alterations in perfusion related to intraabdominal hypertension*. It can be classified as primary if it is the result of a pathophysiologic process within the abdominopelvic cavity. It can be caused by bleeding, acute accumulation of ascites, a rapidly growing tumor or another type of mass, retroperitoneal edema, even the packing of visceral injuries, etc. Secondary abdominal compartment syndrome refers to the development of abdominal compartment syndrome in the absence of a primary abdominopelvic process [4].

The organ dysfunction that can be seen with abdominal compartment syndrome is usually recognized by the changes in lung and renal function. As abdominal compartment syndrome develops, the pulmonary dynamics change, tidal volumes decrease or, if mechanical ventilation is being used, an increase in peak pressure can be observed with similar tidal volumes. Renal dysfunction can be seen when there is a decrease in urine output caused by decreased renal perfusion as the renal vein is compressed due to the increased abdominal pressure. Other organs can display changes after abdominal compartment syndrome including but not limited to the heart and brain [1]. Intra-abdominal hypertension and abdominal compartment syndrome can also generate changes in other intra-abdominal organs [18].

*All patients in the intensive care unit should have measurements of their intra-abdominal pressure because the real incidence of abdominal compartment syndrome in the intensive care unit remains sub-diagnosed, and in some cases it is still unknown*. When abdominal compartment syndrome is suspected, bladder pressures should be measured. This is accomplished by instilling a small amount of sterile saline into the bladder and attaching a Foley tube to a pressure transducer [1]; according to the findings, the following steps will be decided and a treatment will be administered (**Table 1**).

Management of this condition requires a multidisciplinary approach by the surgeon and the intensive care unit team, taking in account a specific staged process [4] (**Figure 1**).

There are four main principles when it comes to the management of intra-abdominal hypertension: first of all, serial monitoring of intra-abdominal pressure should be taken every 4–6 h; optimization of systemic perfusion and organ function in the patient with an increased intraabdominal pressure; medical procedures to reduce intra-abdominal pressure that are institution of specific such as sedation, analgesia, or neuromuscular blockade, and prompt surgical decompressive laparotomy for refractory intra-abdominal hypertension [2] (**Figure 2**).

Medical interventions include sedation to improve abdominal wall compliance, as well as the placing of a nasogastric tube for gastric drainage, removing intraperitoneal fluid collections if they are present, limiting intravenous fluids if possible, diuresis, and also allowing hypercarbia by reducing tidal volumes. *Although all these interventions are promising, the only solution for ACS is decreasing the pressure by performing a decompressive laparotomy* [1, 2, 16, 19].

#### **Intra-abdominal pressure (IAP)**

there is no definitive data or strong recommendation regarding the use of open abdomen in the face of severe peritonitis. Therefore, *when using an open abdomen approach under these* 

• The role of an open abdomen in the management of severe secondary peritonitis has been

• If the patient is not in a condition to undergo a definitive repair, the intervention should be cut short (hemodynamic instability, elevated requirements of inotropics, or insulated

• Peritonitis and intra-abdominal sepsis can influence the intra-abdominal hypertension.

• Following 24–48 h after the initial surgery, the patient should be taken back to the operat-

• Caution and individualization of patients should be exercised when using open abdomen

*Intra-abdominal hypertension and abdominal compartment syndrome are commonly encountered among surgical and nonsurgical critically ill patients*. Intra-abdominal hypertension is defined as a sustained pathologic increase in intra-abdominal pressure greater than or equal to 12 mm Hg. Abdominal compartment syndrome is defined as a sustained increase in intra-abdominal tension ≥20 mm Hg that is associated with new organ dysfunction or

Abdominal perfusion pressure (APP) = mean arterial pressure (MAP)

*Intra-abdominal hypertension can lead to tissue hypoperfusion, especially of the abdominal viscera, as well as organ dysfunction*. Uncontrolled intra-abdominal hypertension that exceeds 25 mm Hg can cause abdominal compartment syndrome, which is a potentially lethal complication. It is characterized by cardiorespiratory and renal dysfunction, as well as bacterial and toxin

*Abdominal compartment syndrome develops as a result of alterations in perfusion related to intraabdominal hypertension*. It can be classified as primary if it is the result of a pathophysiologic process within the abdominopelvic cavity. It can be caused by bleeding, acute accumulation of ascites, a rapidly growing tumor or another type of mass, retroperitoneal edema, even the packing of visceral injuries, etc. Secondary abdominal compartment syndrome refers to the

− intra-abdominal pressure (IAP) (1)

*circumstances, caution and individualization of patients should be the priority* [8].

**3.1. Key points**

ing room.

failure [2, 16].

a controversial issue.

192 Wound Healing - Current Perspectives

multi-organic failure).

in severe abdominal sepsis.

**4. Abdominal compartment syndrome**

intestinal translocation and intracranial hypertension [17].

Normal 5–7 mm Hg Intra-abdominal hypertension grade I 12–15 mm Hg Intra-abdominal hypertension grade II 16–20 mm Hg Intra-abdominal hypertension grade III 21–25 mm Hg Intra-abdominal hypertension grade IV > 25 mm Hg

**Table 1.** Final 2013 consensus definitions of the World Society of the Abdominal Compartment Syndrome [19].

**Figure 1.** Intra-abdominal hypertension (IAH)/abdominal compartment syndrome (ACS) management algorithm. IAP, intra-abdominal pressure [16].

The main goals of decompressive laparotomy include reduction of the increased IAP in order to stop organ dysfunction, allow for a continued expansion of abdominal viscera during ongoing resuscitation, provide temporary abdominal coverage until the disease process resolves, prevent fascial retraction, and to allow a means for continued evacuation of fluid [2].

**4.1. Key points**

algorithm. IAP, intra-abdominal pressure [16].

ill patients.

• IAH and ACS are commonly encountered among both surgical and nonsurgical critically

**Figure 2.** Intra-abdominal hypertension (IAH)/abdominal compartment syndrome (ACS) medical management

Open Abdomen: The Surgeons' Challenge http://dx.doi.org/10.5772/intechopen.81428 195


**Figure 2.** Intra-abdominal hypertension (IAH)/abdominal compartment syndrome (ACS) medical management algorithm. IAP, intra-abdominal pressure [16].

#### **4.1. Key points**

The main goals of decompressive laparotomy include reduction of the increased IAP in order to stop organ dysfunction, allow for a continued expansion of abdominal viscera during ongoing resuscitation, provide temporary abdominal coverage until the disease process resolves, prevent fascial retraction, and to allow a means for continued evacuation of fluid [2].

**Figure 1.** Intra-abdominal hypertension (IAH)/abdominal compartment syndrome (ACS) management algorithm. IAP,

intra-abdominal pressure [16].

194 Wound Healing - Current Perspectives

• IAH and ACS are commonly encountered among both surgical and nonsurgical critically ill patients.


the plastic silo, also known as the Bogotá bag, with a nonadherent plastic sheet, usually from

Open Abdomen: The Surgeons' Challenge http://dx.doi.org/10.5772/intechopen.81428 197

In 1995 the vacuum pack technique was described, where a perforated plastic sheet is used to cover the viscera and then sterile surgical towels are placed on the wound; a surgical drain is then connected to a continuous negative pressure that is placed on the towels, and everything is covered by an airtight seal; the dressing should be changed every 2–3 days in the operative

The vacuum pack was then developed with using a negative-pressure dressing system that includes a polyurethane foam covered with a protective fenestrated nonadherent layer tubing, a canister, and a computerized pump [2]. It has a few advantages, such as a reduced need for frequent dressing changes, increased vascularity of the wound, decreased bacterial

In their systematic review and meta-analysis, Cirocchi et al. support the use of negativepressure wound therapy in the temporary abdominal closure technique used to care for an open abdomen, concluding that negative-pressure wound therapy is associated with a better

There is a new strategy that combines negative-pressure wound therapy with a mesh-mediated fascial traction tension. In a systematic review with 4358 patients, Atema et al. reported that negative-pressure wound therapy was the most frequently described temporary abdominal closure technique. The highest weighted fascial closure rate was found in series describing negative-pressure wound therapy with continuous mesh or suture-mediated fascial traction and dynamic retention sutures. Additionally, in a series applying negative-pressure wound therapy without fascial traction, a weighted fistula rate of 14.6% was seen, but when negativepressure wound therapy was combined with continuous suture or mesh-mediated fascial

Another implementation of the system was introduced by the Abthera™; it consists of a fenestrated plastic sheet with foam sponges that extend in a circular pattern, which is then placed over the viscera encompassing the paracolic gutters and the pelvis; foam sponges are placed on top of the protective layer. Furthermore, an adhesive drape covers the wound

a sterile 3 liter urology irrigation bag, sutured to the edges of the skin [4].

counts, and an extended opportunity for definitive fascial closure [4].

outcome than no negative-pressure wound therapy [22].

traction, the fistula risk dropped to 5.7% [3].

room but could also be changed in the ICU [4, 21].

1. Skin approximation with towel clips or running suture

4. Velcro or zipper-type synthetic materials (Wittmann patch, Starsurgical)

2. Bogota bag 3. Synthetic meshes

5. Negative-pressure dressing

c. Abthera™ system (KCI)

a. Vacuum pack (Barker technique)

b. Vacuum-assisted closure (VAC Therapy, KCI)

**Table 2.** Techniques for temporary abdominal wall closure [2].

### **5. Management of the open abdomen**

After the clinical scenarios that were just reviewed, *life-saving, decompressive laparotomy and temporary abdominal closure with future restoration of anatomic continuity of the abdominal wall* [20] *are frequently needed*. The chance of achieving one of the most important outcomes, the delayed primary fascial closure, depends on the severity of the underlying etiology [3]. While the management of an open abdomen has surely evolved over the last years, numerous strategies for temporary abdominal closure of an open abdomen have been described in the literature.

*Besides prevention of evisceration, temporary abdominal closure can also facilitate subsequent access to the abdominal cavity and prevents retraction of the skin and fascia* [3]. The ideal temporary abdominal closure should have some very specific qualities: it should be easy to apply and remove, it should allow rapid access to a surgical second look, it should drain secretions, it should ease primary closure and should have acceptable morbidity and mortality, it should allow easy nursing, and last but not least, it should be readily available and cheap [4] (**Table 2**).

Since the late 1970s and during the 1980s, abdominal dressings for an open abdomen were quite simple, and the treatment was centered only on the protection and control of the bowel that can be found outside the abdomen (nonabsorbable meshes were used, but these led to a high rate of intestinal fistulation) [4]. In the mid-1980s, a zipper was added to the mesh in order to make the process of re-exploration easier [21].

Throughout the years, the surgeons moved on from protection of the ileus to the preservation of the peritoneal space and the prevention of lateral retraction of the fascia, which are the most critical obstacles when dealing with the reconstruction of the abdominal wall at the end of the treatment [4].

For quick abdominal closure in damage control procedures, skin approximation with towel clips or running suture has been suggested in patients in extremis [2]. Another easy method is


2. Bogota bag

• Abdominal perfusion pressure (APP), mean arterial pressure (MAP), intra-abdominal

• IAH can lead to tissue hypoperfusion, especially of the abdominal viscera, and organ

• Abdominal compartment syndrome develops as a result of alterations in perfusion related

• All patients in the intensive care unit should have measurements of intra-abdominal pressure because the incidence of this entity remains sub-diagnosed and still unknown in some

• The challenging situation to manage requires a multidisciplinary approach by the surgeon

• Although medical interventions are possible, the only solution for ACS is decreasing the

After the clinical scenarios that were just reviewed, *life-saving, decompressive laparotomy and temporary abdominal closure with future restoration of anatomic continuity of the abdominal wall* [20] *are frequently needed*. The chance of achieving one of the most important outcomes, the delayed primary fascial closure, depends on the severity of the underlying etiology [3]. While the management of an open abdomen has surely evolved over the last years, numerous strategies for temporary abdominal closure of an open abdomen have been described in the literature. *Besides prevention of evisceration, temporary abdominal closure can also facilitate subsequent access to the abdominal cavity and prevents retraction of the skin and fascia* [3]. The ideal temporary abdominal closure should have some very specific qualities: it should be easy to apply and remove, it should allow rapid access to a surgical second look, it should drain secretions, it should ease primary closure and should have acceptable morbidity and mortality, it should allow easy

nursing, and last but not least, it should be readily available and cheap [4] (**Table 2**).

Since the late 1970s and during the 1980s, abdominal dressings for an open abdomen were quite simple, and the treatment was centered only on the protection and control of the bowel that can be found outside the abdomen (nonabsorbable meshes were used, but these led to a high rate of intestinal fistulation) [4]. In the mid-1980s, a zipper was added to the mesh in

Throughout the years, the surgeons moved on from protection of the ileus to the preservation of the peritoneal space and the prevention of lateral retraction of the fascia, which are the most critical obstacles when dealing with the reconstruction of the abdominal wall at the end of the

For quick abdominal closure in damage control procedures, skin approximation with towel clips or running suture has been suggested in patients in extremis [2]. Another easy method is

pressure (IAP).

196 Wound Healing - Current Perspectives

dysfunction.

to IAH.

cases.

treatment [4].

and the ICU team in a specific staged process.

pressure by decompressive laparotomy.

**5. Management of the open abdomen**

order to make the process of re-exploration easier [21].

	- a. Vacuum pack (Barker technique)
	- b. Vacuum-assisted closure (VAC Therapy, KCI)
	- c. Abthera™ system (KCI)

#### **Table 2.** Techniques for temporary abdominal wall closure [2].

the plastic silo, also known as the Bogotá bag, with a nonadherent plastic sheet, usually from a sterile 3 liter urology irrigation bag, sutured to the edges of the skin [4].

In 1995 the vacuum pack technique was described, where a perforated plastic sheet is used to cover the viscera and then sterile surgical towels are placed on the wound; a surgical drain is then connected to a continuous negative pressure that is placed on the towels, and everything is covered by an airtight seal; the dressing should be changed every 2–3 days in the operative room but could also be changed in the ICU [4, 21].

The vacuum pack was then developed with using a negative-pressure dressing system that includes a polyurethane foam covered with a protective fenestrated nonadherent layer tubing, a canister, and a computerized pump [2]. It has a few advantages, such as a reduced need for frequent dressing changes, increased vascularity of the wound, decreased bacterial counts, and an extended opportunity for definitive fascial closure [4].

In their systematic review and meta-analysis, Cirocchi et al. support the use of negativepressure wound therapy in the temporary abdominal closure technique used to care for an open abdomen, concluding that negative-pressure wound therapy is associated with a better outcome than no negative-pressure wound therapy [22].

There is a new strategy that combines negative-pressure wound therapy with a mesh-mediated fascial traction tension. In a systematic review with 4358 patients, Atema et al. reported that negative-pressure wound therapy was the most frequently described temporary abdominal closure technique. The highest weighted fascial closure rate was found in series describing negative-pressure wound therapy with continuous mesh or suture-mediated fascial traction and dynamic retention sutures. Additionally, in a series applying negative-pressure wound therapy without fascial traction, a weighted fistula rate of 14.6% was seen, but when negativepressure wound therapy was combined with continuous suture or mesh-mediated fascial traction, the fistula risk dropped to 5.7% [3].

Another implementation of the system was introduced by the Abthera™; it consists of a fenestrated plastic sheet with foam sponges that extend in a circular pattern, which is then placed over the viscera encompassing the paracolic gutters and the pelvis; foam sponges are placed on top of the protective layer. Furthermore, an adhesive drape covers the wound and extends over the skin. Suction tubing is attached to a portable suction device to create negative pressure [23].

• We count with different techniques for temporary abdominal closure like skin approximation with towel clips or running suture, Bogota bag, synthetic meshes, velcro or zipper-type

Open Abdomen: The Surgeons' Challenge http://dx.doi.org/10.5772/intechopen.81428 199

• The best and the correct management of a patient with open abdomen is still unclear: the technique is relatively new, and in the literature, the data and the casuistic reported are too

• Definitive closure of the abdominal wall has to be obtained as soon as possible. Different

Although the OA has addressed some serious and potentially lethal problems related to early closure of the abdomen, this technique is also *associated with significant complications, including wound infection, fluid and protein loss, a catabolic state, loss of abdominal wall domain, and develop-*

The appearance of enteric contents from an abdominal incision is a devastating complication and can be emotionally distressing for both the patient and the surgeon. *Enteroatmospheric fistulas range from easily controlled low-output colocutaneous fistulas to high-output enteroatmospheric fistulas* that require a prolonged nutritional support, specialized wound care, and complex reoperative surgery [26]. The overall incidence of this complication is about 5%. However, in

Preemptive measures to prevent enteroatmospheric fistula and frozen abdomen are crucial (i.e., early abdominal wall closure, bowel coverage with plastic sheets, omentum or skin, no direct application of synthetic prosthesis over bowel loops, no direct application of negativepressure wound therapy on the viscera, and deep burying of intestinal anastomoses under

In some cases, numerous enteroatmospheric fistulas may develop, and the constant leak of enteric contents on the open abdomen aggravates the inflammation and encourages the formation of new fistulas [2]. Enteroatmospheric fistula management should be tailored according to patient condition, fistula output and position, and anatomical features (Grade

Enteric fistula management is composed of three phases: recognition and stabilization of the

Enteroatmospheric fistula is a life-threatening condition requiring longitudinal care for many months. A spectrum of vexing clinical problems ranging from hypovolemic shock to malnutrition to complex abdominal wall reconstruction challenges the skill of even highly experienced surgeons. *High-output fistulas and EAFs are best managed in centers providing comprehensive care* 

patient, anatomical definition and decision-making, and definitive operation [27].

the chronically open abdomen, the incidence increases to about 15% [2].

synthetic materials, or negative-pressure dressing.

various and too heterogeneous to assess.

**6. Complications**

bowel loops) [11].

*of intestinal failure* [28].

1C) [8, 11].

*ment of enteroatmospheric fistula* [9].

techniques can be applied for different settings.

The three main negative-pressure therapy modalities (Barker, VAC abdominal dressing system, Abthera™) have different mechanical properties, which may affect treatment outcomes. The most important difference between all of these modalities is the distribution pattern of the preset negative pressure [2]. Sammons applied a negative pressure of 125 mmHg to these three systems and measured the pressures in different areas of the dressing, concluding that pressure distribution of Abthera™ therapy was significantly superior to that of the Barker vacuum packing in all three measure zones and in medial and distal zones when comparing with the VAC system [24].

In the World Journal of Emergency Surgery Guidelines (2018), they recommend that negativepressure wound therapy along with continuous fascial traction is the preferred method for temporary abdominal closure (Grade 1B). Temporary abdominal closure without negativepressure wound therapy (e.g., mesh alone, Bogota bag) should NOT be used for the purpose of temporary abdominal closure, because of the low-delayed fascial closure rate and the significant intestinal fistula rate that often accompanies the method (Grade 1B) [8, 11].

The best and the right way to manage a patient with an open abdomen is still unclear: the technique is relatively new, and in the literature, the data and the casuistic reported are too varied and too heterogeneous to assess properly [4].

Early fascial and/or abdominal definitive closure should be the strategy for managing an open abdomen once any requirements for ongoing resuscitation have ceased, the source control has been definitively reached, there are no concerns regarding intestinal viability, no further surgical re-exploration is needed, and there are no concerns for abdominal compartment syndrome (Grade 1B) [8].

In many patients, early definitive fascial closure may not be possible because of the persistent bowel edema or intra-abdominal sepsis. In these cases, progressive closure should be attempted when there is a return to the operating room for a washout or dressing change, by placing a few interrupted sutures at the top and bottom of the fascia defect [2] with each new procedure.

*Definitive closure of the abdominal wall has to be achieved as soon as possible. Different techniques can be applied in different settings*: direct closure with dynamic traction techniques in early closure with little fascial gap, component separation, rotational flaps, the use of prosthetic or biologic mesh, etc.; nevertheless, a planned ventral hernia has to be considered if severe and persistent contamination of the peritoneal cavity is present [25].

### **5.1. Key points**


### **6. Complications**

and extends over the skin. Suction tubing is attached to a portable suction device to create

The three main negative-pressure therapy modalities (Barker, VAC abdominal dressing system, Abthera™) have different mechanical properties, which may affect treatment outcomes. The most important difference between all of these modalities is the distribution pattern of the preset negative pressure [2]. Sammons applied a negative pressure of 125 mmHg to these three systems and measured the pressures in different areas of the dressing, concluding that pressure distribution of Abthera™ therapy was significantly superior to that of the Barker vacuum packing in all three measure zones and in medial and distal zones when comparing

In the World Journal of Emergency Surgery Guidelines (2018), they recommend that negativepressure wound therapy along with continuous fascial traction is the preferred method for temporary abdominal closure (Grade 1B). Temporary abdominal closure without negativepressure wound therapy (e.g., mesh alone, Bogota bag) should NOT be used for the purpose of temporary abdominal closure, because of the low-delayed fascial closure rate and the sig-

The best and the right way to manage a patient with an open abdomen is still unclear: the technique is relatively new, and in the literature, the data and the casuistic reported are too

Early fascial and/or abdominal definitive closure should be the strategy for managing an open abdomen once any requirements for ongoing resuscitation have ceased, the source control has been definitively reached, there are no concerns regarding intestinal viability, no further surgical re-exploration is needed, and there are no concerns for abdominal compartment syn-

In many patients, early definitive fascial closure may not be possible because of the persistent bowel edema or intra-abdominal sepsis. In these cases, progressive closure should be attempted when there is a return to the operating room for a washout or dressing change, by placing a few interrupted sutures at the top and bottom of the fascia defect [2] with each new procedure. *Definitive closure of the abdominal wall has to be achieved as soon as possible. Different techniques can be applied in different settings*: direct closure with dynamic traction techniques in early closure with little fascial gap, component separation, rotational flaps, the use of prosthetic or biologic mesh, etc.; nevertheless, a planned ventral hernia has to be considered if severe and persistent

• Life-saving decompressive laparotomy and temporary abdominal closure with later resto-

• Besides prevention of evisceration, temporary abdominal closure can facilitate regaining

ration of anatomic continuity of the abdominal wall are frequently needed.

access to the abdominal cavity and prevents retraction of the skin and fascia.

nificant intestinal fistula rate that often accompanies the method (Grade 1B) [8, 11].

varied and too heterogeneous to assess properly [4].

contamination of the peritoneal cavity is present [25].

negative pressure [23].

198 Wound Healing - Current Perspectives

with the VAC system [24].

drome (Grade 1B) [8].

**5.1. Key points**

Although the OA has addressed some serious and potentially lethal problems related to early closure of the abdomen, this technique is also *associated with significant complications, including wound infection, fluid and protein loss, a catabolic state, loss of abdominal wall domain, and development of enteroatmospheric fistula* [9].

The appearance of enteric contents from an abdominal incision is a devastating complication and can be emotionally distressing for both the patient and the surgeon. *Enteroatmospheric fistulas range from easily controlled low-output colocutaneous fistulas to high-output enteroatmospheric fistulas* that require a prolonged nutritional support, specialized wound care, and complex reoperative surgery [26]. The overall incidence of this complication is about 5%. However, in the chronically open abdomen, the incidence increases to about 15% [2].

Preemptive measures to prevent enteroatmospheric fistula and frozen abdomen are crucial (i.e., early abdominal wall closure, bowel coverage with plastic sheets, omentum or skin, no direct application of synthetic prosthesis over bowel loops, no direct application of negativepressure wound therapy on the viscera, and deep burying of intestinal anastomoses under bowel loops) [11].

In some cases, numerous enteroatmospheric fistulas may develop, and the constant leak of enteric contents on the open abdomen aggravates the inflammation and encourages the formation of new fistulas [2]. Enteroatmospheric fistula management should be tailored according to patient condition, fistula output and position, and anatomical features (Grade 1C) [8, 11].

Enteric fistula management is composed of three phases: recognition and stabilization of the patient, anatomical definition and decision-making, and definitive operation [27].

Enteroatmospheric fistula is a life-threatening condition requiring longitudinal care for many months. A spectrum of vexing clinical problems ranging from hypovolemic shock to malnutrition to complex abdominal wall reconstruction challenges the skill of even highly experienced surgeons. *High-output fistulas and EAFs are best managed in centers providing comprehensive care of intestinal failure* [28].

#### **6.1. Key points**

• Open abdomen is associated with significant postsurgical complications, including wound infection, fluid and protein loss, a catabolic state, loss of abdominal wall domain, and development of enteroatmospheric fistula.

[7] Teixeira P, Salim A, Inaba K. A prospective look at the current state of open abdomens.

Open Abdomen: The Surgeons' Challenge http://dx.doi.org/10.5772/intechopen.81428 201

[8] Coccolini F, Roberts D, Ansaloni L, Ivatury R, Gamberini E, Kluger Y, et al. The open abdomen in trauma and non-trauma patients: WSES guidelines. World Journal of Emergency

[9] Demetriades D. Total management of the open abdomen. Surgical Clinics of North

[10] Moore LJ, Moore FA. Epidemiology of sepsis in surgical patients. Surgical Clinics of

[11] Coccolini F, Montori G, Ceresoli M, Catena F, Moore EE, Ivatury R, et al. The role of open abdomen in non-trauma patient: WSES consensus paper. World Journal of Emergency

[12] Plantefeve G, Hellmann R, Pajot O, Thirion M, Bleichner G, Mentec H. Abdominal compartment syndrome and intraabdominal sepsis: Two of the same kind? Acta Clinica

[13] Sartelli M et al. The role of the open abdomen procedure in managing severe abdominal sepsis: WSES position paper. World Journal of Emergency Surgery. 2015;**10**(1):1-11. DOI:

[14] Sartelli M, Catena F, Ansaloni L, Coccolini F, Corbella D, Moore EE, et al. Complicated intra-abdominal infections worldwide: The definitive data of the CIAOW study. World

[15] Bradley M, Dubose J, Scalea T, et al. Independent predictors of enteric fistula and abdominal sepsis after damage control laparotomy: Results from the pro- spective AAST

[16] Kirkpatrick AW, Roberts DJ, Ball CG, Regli A, Amours SD. Intra-abdominal hypertension and the abdominal compartment syndrome: Updated consensus definitions and clinical practice guidelines from the World Society of the Abdominal Compartment Syndrome World Society of the Abdominal. Intensive Care Medicine. Jul 2013;**39**(7):1190-1206 [17] Kaplan M, Banwell P, Orgill D. Guidelines for the management of the open abdomen.

[18] Papavramidis T et al. Abdominal compartment syndrome–Intra-abdominal hypertension: Defining, diagnosing, and managing. Journal of Emergencies, Trauma, and Shock.

[19] Malbrain M et al. Results from the international conference of experts on intra-abdominal hypertension and abdominal compartment syndrome. I. definitions. Intensive Care

[20] Howdieshell T, Proctor C, Sternberg E, Cue J, Mondy J, Hawkins M. Temporary abdominal closure followed by definitive abdominal wall reconstruction of the open abdomen.

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### **Author details**

Juan José Santivañez Palomino<sup>1</sup> \*, Arturo Vergara<sup>2</sup> and Manuel Cadena<sup>2</sup>

\*Address all correspondence to: juan.santivanez@urosario.edu.co

1 Department of Surgery, Fundación Santa Fe de Bogotá, Colombia

2 Department of Metabolic Support and General Surgery, Fundación Santa Fe de Bogota, FACS, Universidad Los Andes, Colombia

### **References**


[7] Teixeira P, Salim A, Inaba K. A prospective look at the current state of open abdomens. The American Surgeon. 2008;**74**(10):891-897

**6.1. Key points**

200 Wound Healing - Current Perspectives

**Author details**

**References**

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hensive care of intestinal failure.

Juan José Santivañez Palomino<sup>1</sup>

FACS, Universidad Los Andes, Colombia

America. 2014;**94**(1):131-153

1997;**77**:783-800

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fistula to a high-output enteroatmospheric fistula.

• Open abdomen is associated with significant postsurgical complications, including wound infection, fluid and protein loss, a catabolic state, loss of abdominal wall domain, and

• Enteroatmospheric fistula can range from an easily controlled low-output colocutaneous

• Enteric fistula management is comprised of three phases: recognition and stabilization of the patient, anatomical definition and decision-making, and finally the definitive operation.

• High-output fistulas and EAFs are best managed in medical centers that provide compre-

and Manuel Cadena<sup>2</sup>

\*, Arturo Vergara<sup>2</sup>

2 Department of Metabolic Support and General Surgery, Fundación Santa Fe de Bogota,

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[2] Demetriades D, Salim A. Management of the open abdomen. Surgical Clinics of North

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[4] Coccolini F et al. The open abdomen, indications, management and definitive closure. World Journal of Emergency Surgery. 2015;**10**(1):1-10. DOI: 10.1186/s13017-015-0026-5 [5] Ivatury R, Diebel L, Porter J. Damage control surgery: Intra-abdominal hypertension and the abdominal compartment syndrome. The Surgical Clinics of North America.

[6] Lee JC, Peitzman AB. Damage control laparotomy. Current Opinion in Critical Care.

\*Address all correspondence to: juan.santivanez@urosario.edu.co 1 Department of Surgery, Fundación Santa Fe de Bogotá, Colombia


[21] Quyn A et al. The open abdomen and temporary abdominal closure systems–Historical evolution and systematic review. Colorectal Disease. Aug 2012;**14**(8):e429-438

**Chapter 12**

Provisional chapter

**Facilitation of Wound Healing Following Laparoscopic**

DOI: 10.5772/intechopen.82614

Facilitation of Wound Healing Following Laparoscopic

**and Conventional Abdominal Surgery with Dressings,**

Wounds due to surgical incisions and due to injuries often do not heal and can result in complications like slow healing and infections. Several approaches to facilitate wound healing are constantly being developed. Here we discuss various wounds and multiple ways to treat wounds especially those resulting from abdominal surgery either due to conventional surgery or due to laparoscopic surgery. In future, there are various possibilities in the pipeline that could result in accelerated wound healing as well as tissue

An estimated 313 million surgical procedures are performed worldwide annually [1]. The quality of incision selection and postoperative wound healing play significant critical roles in patient recovery and rehabilitation. Mortality and morbidity rates are affected by surgical wound complications. The importance of surgical incisions and wound care has been documented throughout history to primarily prevent wound infection has been from the time Alexander the Great was treated with saffron for injuries from a piercing spear. Over the years, several new ways of performing surgery and treating wound have advanced wound care. In the last century, endoscopic surgery has significantly reduced incision damage, and antibiotics were introduced to control infections and facilitate healing. Neosporin with its triple antibiotics has been commonly used, can be purchased over the counter, and can be used for minor

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and Conventional Abdominal Surgery with Dressings,

**Patches, Antibiotics, etc.**

Patches, Antibiotics, etc.

http://dx.doi.org/10.5772/intechopen.82614

Abstract

regeneration.

1. Introduction

Rebekah Amarini, Sufan Chien and Girish J. Kotwal

Keywords: wound, healing, surgery, laparoscopic, infection

Rebekah Amarini, Sufan Chien and Girish J. Kotwal

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter


#### **Facilitation of Wound Healing Following Laparoscopic and Conventional Abdominal Surgery with Dressings, Patches, Antibiotics, etc.** Facilitation of Wound Healing Following Laparoscopic and Conventional Abdominal Surgery with Dressings, Patches, Antibiotics, etc.

DOI: 10.5772/intechopen.82614

Rebekah Amarini, Sufan Chien and Girish J. Kotwal Rebekah Amarini, Sufan Chien and Girish J. Kotwal

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.82614

#### Abstract

[21] Quyn A et al. The open abdomen and temporary abdominal closure systems–Historical evolution and systematic review. Colorectal Disease. Aug 2012;**14**(8):e429-438

[22] Popivanov G, Chiara O, Tugnoli G. What is the effectiveness of the negative pressure wound therapy (NPWT) in patients treated with open abdomen technique? A systematic review and meta-analysis. The Journal of Trauma and Acute Care Surgery. Sep 2016;**81**(3):575-584

[23] Frazee RC, Abernathy SW, Jupiter DC, Hendricks JC, Davis M, Regner JL, et al. Are commercial negative pressure systems wortzh the cost in open abdomen management? Journal of the American College of Surgeons. 2013;**216**(4):730-735. DOI: 10.1016/j.

[24] Delgado A, Sammons A. In vitro pressure manifolding distribution evaluation of ABThera™ active abdominal therapy system, V.A.C. ® abdominal dressing system, and Barker's vacuum packing technique conducted under dynamic conditions. SAGE Open

[25] Chiara O, Cimbanassi S, Biffl W, Leppaniemi A, Henry S, Scalea TM, et al. International consensus conference on open abdomen in trauma. The Journal of Trauma and Acute

[26] Davis KG, Johnson EK. Controversies in the care of the enterocutaneous fistula. The

[27] Schecter WP, Hirshberg A, Chang DS, Harris HW, Napolitano LM, Wexner SD, et al. Enteric fistulas: Principles of management. Journal of the American College of Surgeons.

[28] Schecter WP. Management of enterocutaneous fistulas. The Surgical Clinics of North

Medicine. 2016;**4**:205031211562498. DOI: 10.1177/2050312115624988

jamcollsurg.2012.12.035

202 Wound Healing - Current Perspectives

Care Surgery. Jan 2016;**80**(1):173-183

America. 2011;**91**(3):481-491

Surgical Clinics of North America. 2013;**93**(1):231-250

2009;**209**(4):484-491. DOI: 10.1016/j.jamcollsurg.2009.05.025

Wounds due to surgical incisions and due to injuries often do not heal and can result in complications like slow healing and infections. Several approaches to facilitate wound healing are constantly being developed. Here we discuss various wounds and multiple ways to treat wounds especially those resulting from abdominal surgery either due to conventional surgery or due to laparoscopic surgery. In future, there are various possibilities in the pipeline that could result in accelerated wound healing as well as tissue regeneration.

Keywords: wound, healing, surgery, laparoscopic, infection

### 1. Introduction

An estimated 313 million surgical procedures are performed worldwide annually [1]. The quality of incision selection and postoperative wound healing play significant critical roles in patient recovery and rehabilitation. Mortality and morbidity rates are affected by surgical wound complications. The importance of surgical incisions and wound care has been documented throughout history to primarily prevent wound infection has been from the time Alexander the Great was treated with saffron for injuries from a piercing spear. Over the years, several new ways of performing surgery and treating wound have advanced wound care. In the last century, endoscopic surgery has significantly reduced incision damage, and antibiotics were introduced to control infections and facilitate healing. Neosporin with its triple antibiotics has been commonly used, can be purchased over the counter, and can be used for minor

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Table 1. Wound care table for different types of wounds.

cuts and wounds that do not require stitches. With antibiotic resistance manifesting itself in recent decades, other new ways have become essential. Current and future wound healing measures are in a constant state of flux, and when an agent proves useful, it can help many patients who have been affected. Carbohydrate-derived fulvic acid (CHD-FA) is a topical agent, which has been used to prevent drug-resistant bacterial and fungal infections, and contains anti-inflammatory properties for those who have suffered a traumatic wound [2]. Wounds can occur due to several different causes as summarized in Table 1. Several wound care options are currently available (Table 2) and could become available in the future (Table 3). A surgical incision is an aperture into the body to permit the work of the planned operation to proceed. The choice of incision for laparotomy depends on the area that needs to be exposed, the elective or emergency nature of the operation, and personal preference. There are two approaches used nowadays: traditional incision and minimally invasive. This book chapter provides a brief review of recent progress in surgical procedure and wound care of incisions during abdominal surgeries.

### 2. Traditional incision

Traditional abdominal surgery refers to operating through an open abdominal incision known as laparotomy. The goal is to provide adequate exposure for the anticipated procedure while taking into account the possibility that the planned procedure may change depending upon

Types of dressing

Hydrogels

Primary

•

Give the wound a moist envi-

•

Hydrates

•

Must have a

•

Change

when

other

dressings

are

changed

secondary

dressing

wound, liquefies necrotic

tissue on wound

•

Not meant for

significant

exudate

surface

• • •

Significantly

•

Must have a

•

1–2x a

week

secondary

dressing

•

Silver–

Weekly

absorbent

Soothing effect

Nonstick

ronment

• •

Autolytic

debridement

Used for shallow dry ulcers

(e.g., IntraSite Gel, generic

hydrogel)

Alginates

Primary

• •

Silver can be added to give

antibacterial

ulcers/presence

oxidized silver reduces viable

biofilm

•

High absorptive capacity can

be used in packing

> •

Form a gel like covering over

the wound keeping it moist

> •

•

Use with negative pressure

wound therapy

> •

Used for packing wounds with

dead space

> •

Semipermeable

waterproof

water vapor and oxygen

> •

Keep wound moisturized

preventing

nation

• •

Superficial/light

wounds

•

Often the preferred secondary

dressing

•

Used to prevent friction injuries

 exudative

Provides autolytic

debridement

gas permeable

> •

Keep moist

environment

 bacterial contami-

 while

 but permeable to

 membrane

•

Transparent

•

Skin damage

Every 7–10

days

if removed improperly

evaluation of wound without

removal of

•

Limited

absorptive

properties

http://dx.doi.org/10.5772/intechopen.82614

dressing

•

Waterproof

 and •

Shouldn't be

used for infected ulcers

> •

Roll up in coc-

cyx region 205

72 h of

antibacterial

 properties

•

Doesn't require

•

Doesn't eradi-

Every 72 h

> cate bacteria

in wound

Facilitation of Wound Healing Following Laparoscopic and Conventional Abdominal Surgery…

a secondary

dressing

•

Inexpensive

 of a biofilm-

effect-infected

•

Don't inhibit wound contrac-

•

May be too

drying if

wound isn't

exudative

tion

•

Can be used in

infected pres-

sure ulcers

•

Fiber residue

Ulcers with copious exudate

> •

Calcium alginate (AlgiSite,

Aquacel, Sorbsan)

> •

Calcium alginate with silver

(Aquacel Ag)

> •

Calcium-sodium

(Kaltostat)

•

Carboxymethyl

(CMC) silver oxysalt dressing

> •

Biosorb gelling fiber

Antimicrobial

• • •

Foam

Transparent

• • •

Opsite

Tegaderm

Generic transparent

 gauze

 film

Primary or secondary

Alginate

Gauze

 dressings

Primary

 cellulose

 alginate

Primary or secondary

Functions/indications

Pros

Cons

Frequency

dressing

change

 of

dressing


cuts and wounds that do not require stitches. With antibiotic resistance manifesting itself in recent decades, other new ways have become essential. Current and future wound healing measures are in a constant state of flux, and when an agent proves useful, it can help many patients who have been affected. Carbohydrate-derived fulvic acid (CHD-FA) is a topical agent, which has been used to prevent drug-resistant bacterial and fungal infections, and contains anti-inflammatory properties for those who have suffered a traumatic wound [2]. Wounds can occur due to several different causes as summarized in Table 1. Several wound care options are currently available (Table 2) and could become available in the future (Table 3). A surgical incision is an aperture into the body to permit the work of the planned operation to proceed. The choice of incision for laparotomy depends on the area that needs to be exposed, the elective or emergency nature of the operation, and personal preference. There are two approaches used nowadays: traditional incision and minimally invasive. This book chapter provides a brief review of recent progress in surgical procedure and wound care of

Diabetic wounds Types of dressings Process of cleaning and caring for wound

• Offload wound

• Diabetic footwear • Hyperbaric oxygen chamber

• Skin grafting [6]

• Antibiotic therapy if indicated • Control blood glucose levels

• Keep incision clean and dry • Prophylactic antibiotics

• Prophylactic antibiotics • Betadine prep

the cause of infection • Possibly need tetanus shot

• Antibiotics

• Debridement if necessary of nonviable tissue to promote accelerated wound healing

• Correction of peripheral arterial insufficiency

• Gauze pad or soft cloth to clean wound with normal saline or soapy water [7]

• Post-op dressing can be removed in 48 h

• Possible surgery to remove bullet or bone [8] • Crushing tissue or stretching of tissue

• If through the sole of a shoe Pseudomonas can be

• Daily saline

• Sterile dressing • Wet to dry dressing

• Transparent film

tourniquet

• Clean dressing

Gunshot wounds • Keep wound clean and dry

Table 1. Wound care table for different types of wounds.

• Nonadherent dressing gauze to absorb light drainage

• Stop bleeding with pressure/

• Keep wound clean and dry

• Dressings that provide a moist environment [5]

Traditional abdominal surgery refers to operating through an open abdominal incision known as laparotomy. The goal is to provide adequate exposure for the anticipated procedure while taking into account the possibility that the planned procedure may change depending upon

incisions during abdominal surgeries.

2. Traditional incision

General surgical incisions Stitches, skin glue, staples,

204 Wound Healing - Current Perspectives

Laparoscopic incisions Subcuticular stitch, staples,

Wounds from sharp objects (knives, blades)

steri-strips

skin glue

