Protective and Damaging Aspects of Healing: A Review

Trauma, caused by mechanical, surgical, biological, or chemical means, generates a wound and activates a complex cascade of closely synchronized molecular events that initiate and complete the healing process.¹ Although normal healing may slightly differ from tissue to tissue, the overall process has a similar protective role throughout the body.² The damaging effects of abnormal healing caused by reduced or excessive healing activity may either lead to healing failure; acute, chronic, or over healing; and fibroproliferative scarring. The adaptation of trauma-activated healing response highlights the critical role of extracellular matrix (ECM) accumulation in directing the healing process toward protective or damaging outcomes (Figure 1). In addition to reviewing the most common protective and damaging aspects of healing, the authors propose a model of the healing process based on ECM homeostasis (Figure 1) and functional staging of the process in biological time (Figure 2), which may support the design of practical applications in wound management.

Biology of Healing

Wound healing is a complex insult-initiated biologic process that involves coordinate recruitment of multiple cellular and molecular events and affects growth factor-mediated ECM homeostasis. An extracellular matrix is a dynamic superstructure of self-aggregating macromolecules, including fibronectin, collagen, and proteoglycans, to which cells attach by means of surface receptors called integrins, forming a 3-dimensional supporting scaffold that isolates tissue compartments, mediates cell attachment, and determines tissue architecture.^3,4 Figure 2 illustrates the biology of healing, which consists of several distinct but overlapping phases including inflammation, mitosis, angiogenesis (migratory phase), synthesis (proliferative phase), wound contraction, and ECM remodeling. Their time course and approximate duration are also indicated. An ideogram of the biological time course followed by the functional stages of the normal healing sequence of a hypothetical individual in physical time scale is also included in this figure.
At the cellular level, wound healing is viewed primarily as an entity of dividing fibroblasts and other cells migrating into the wound. Cell division requires the release of growth factors from platelets at certain threshold concentrations necessary to activate the mitogenic potential of quiescent (Go) cells. Platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) are competence growth factors that are necessary to initiate a cellular response to additional progressive factors of the healing process, such as epidermal growth factor (EGF) and insulin-like growth factor (IGF).^5,6 If growth factor titers are below the threshold level, competence is not achieved, and the cells remain quiescent in the Go phase of the cell cycle. All harmful effects of healing could be assigned to discrete cell cycling abnormalities.⁷

Protective Effects of Healing

Healing is a primal life-protective property. Normal healing repairs damaged tissue architecture and protects vital organ integrity and the organism as a whole. From the general surgeon’s viewpoint, the alimentary tract provides the most representative clinical examples of the protective effects of healing, which are particularly evident in peptic ulceration, ischemia, anastomotic healing, and other analogous situations.²
Ulceration. Ulceration caused by various insults triggers a fibrotic repair process that may eliminate the ulcerated area and lead to the development of scar tissue. The resultant scar may resorb completely or persist permanently. Permanent scars may generate local deposition of fibrotic material in the underlying wall (muscularis propria), leading to the constriction of hollow viscera, such as in the pyloric stenosis of duodenal ulcers or the strictures in the small intestine as seen in Crohn’s disease. Repair response and scar fate appear to depend on the depth and duration of mucosal injury. For these reasons, all therapeutic interventions aim to reduce the extent of mucosal injury by avoiding over-development of fibrotic tissue.
Ischemia. Ischemia causes secondary insults leading to coagulative necrosis. The mucosal layer is most sensitive to ischemia. By analogy to peptic ulceration, tissue recovery following ischemic insult is determined by the severity and duration of the injury. Insults confined to the mucosa undergo spontaneous regeneration of the epithelium. However, once the submucosa has been affected, healing leads to fibrosis and scar development.⁸ Indeed, ischemic strictures are frequently the result of clinical conditions, such as necrotizing enterocolitis, where healing leads to the development of fibrotic tissue in the affected viscera. However, when ischemia profoundly affects all layers of the gut, transmural infarction results in death of that section of the bowel. Under these circumstances, spontaneous healing is not possible. It is evident that the protective effects of healing in both ulceration and ischemia depend upon the magnitude and duration of the insult. Insults that exceed a certain threshold unconditionally drive the healing process toward damaging effects.
Anastomotic healing. Incisional or anastomotic healing in the gastrointestinal tract differs from the previously described injuries in that a controlled, full-thickness injury of limited duration is applied. The incised ends are then held in apposition to reconstitute luminal integrity artificially (supported wound) while biologic healing takes place. The full-thickness injury elicits a fibrotic response with the participation of inflammation, proliferation, and connective tissue deposition. The result is a fibrotic scar at the site of repair. Skin healing and intestinal healing differ in that collagen production originates from fibroblasts and intestinal smooth muscle cells, respectively. Platelet-derived growth factor and transforming growth factor-beta (TGF-β) stimulate cell proliferation and collagen production in skin fibroblasts but have no effect on intestinal smooth muscle cells. These cells are stimulated primarily by interleukin-1 (IL-1) to proliferate, up-regulate collagen, and down-regulate collagenase biosynthesis.⁹ Complications of anastomotic healing are not rare and result either by healing failure (eg, anastomotic leak, fistulas) or excessive healing (eg, stricture formation). Various anti-inflammatory and anti-neoplastic agents have a negative effect on intestinal healing.^10–13

Damaging Effects of Healing

Abnormal healing alters normal tissue architecture, disrupts vital organ integrity, and harms the organism as a whole, leading to notable deviations from normalcy. These harmful developments or damaging effects are the result of either insufficient healing (healing failure) or over healing that leads to proliferative scarring. Healing failure may be acute or chronic. Some clinical examples of excessive collagen synthesis in over healing are the development of keloids and hypertrophic scars in the skin, adhesions in the peritoneal cavity, and progressive fibrosis of healing tissues and organs (fibroproliferative disorders) as a response to an inflammatory insult. Both healing failure and over healing may lead to debilitating disease, unconditional suffering, and/or death.
Healing failure. Acute healing failure occurs when the load placed across the wound exceeds the resistive capacity of the suture line and provisional matrix. This failure usually occurs when there is abnormal progression through the integrated phases of acute tissue repair. Acute wounds are completely dependent on suture until breaking strengths capable of offsetting the increased loads placed across an acute wound by a recovering patient are achieved.
In contrast to acute healing failure, acute wounds usually occur secondarily to surgery or trauma in healthy individuals healing quickly and completely and have no underlying healing defect. Successful acute wound healing depends on timely, effective, and regulated hemostasis, inflammation, proliferation, and remodeling. Certain patient characteristics, such as age > 65, wound infection, pulmonary disease, hemodynamic instability, ostomy within the incision, hypoproteinemia, sepsis, obesity, uremia, malignancy, ascites, use of steroids, and hypertension, are used to predict acute healing failure risk.^14,15
The 2 parameters that determine the fate of acute healing failure are wound breaking strength and healing zone. The metabolically active region of the wound—the healing zone—is the gradient zone of active collagenolysis and matrix degradation around the wound, which extends approximately 0.75 cm from each edge of the wound.¹⁶ Most often, wound failure results from suture pulling through the healing zone and not suture fracture or knot slippage. Tissue failure occurs in the biochemically active zone adjacent to the acute wound edge, where proteases activated during normal tissue repair result in a loss of native tissue integrity in the zone where sutures are placed.
Chronic healing failure is defined as a loss in tissue integrity produced by insult or injury that is of extended duration or frequent recurrence.^1,17 At the molecular level, chronic healing failure may result either from deficient supply or functional inhibition of growth factors. Growth factor deficiency may be due to increased protease levels that degrade growth factors and ECM components at the wound site.¹⁷
Because fibroblasts are the most common cell type in the dermis and the most influential in producing collagen and other ECM components, their role in the wound repair process becomes fundamental in understanding and evaluating growth factor therapies in chronic wounds. Wounds that contain a significant number of nondividing fibroblasts due to senescence, damaged DNA, or enduring quiescence do not heal. The wound is more likely to achieve closure as the arrested population of cells decreases and more dividing cells populate the wound. Senescence is irreversible, and senescent cells are refractory to growth factor therapies. These therapies may prove valuable in the treatment of chronic wounds.^17–23
A basic difference between acute and chronic wounds is the amount and type of proteases and their inhibitors present in the wound fluid. Acute wound fluid is stimulatory to cells in culture, whereas chronic wound fluid appears to inhibit cell proliferation. It may, therefore, be possible to induce a healing response in chronic healing failure by adding exogenous growth factors, by inhibiting protease activity at the wound site, or by recreating conditions of initial wound response to trauma by programmed corrective surgery (Figure 1). A variety of exogenous insult-related factors as well as endogenous local and systemic factors may convert acute into chronic healing failure conditions, primarily in skin ulcers, pressure ulcers, diabetic ulcers, lower leg ulcers, post-operative open wounds, and enterocutaneous fistulae. Local factors include infection, foreign bodies, tissue hypoxia, venous insufficiency, local toxins, mechanical trauma, irradiation, cigarette smoking, and alcoholism. Systemic factors include malnutrition, cancer, diabetes mellitus, uremia, jaundice, old age, corticosteroids, chemotherapeutic agents, cigarette smoking, and alcoholism.^24–26
Over healing (fibroproliferative disorders). In cases of proliferative healing, although the various dynamic and interrelated processes are the same as in normal healing, it seems that the degree of penetration of the individual processes may differ from that of normal healing. The processes of coagulation, inflammation, angiogenesis, fibroplasia, contraction, and remodeling may be different in wounds and may result in proliferating scarring. Transforming growth factor-beta is the most important cytokine that is released during inflammation and may trigger many of the cell-to-cell and cell-to-matrix interactions that lead to proliferative scarring.^27,28 The TGF-β growth factor may influence scar formation by the modulation of apoptosis or programmed cell death. It has been shown that the Bcl-2 oncogene, which is known to inhibit apoptosis, is markedly elevated in the blood of patients with proliferative burn scars.²⁸ Another observation suggests that programmed fibroblast cell death is markedly decreased in cases of proliferative scarring, which is mediated by TGF-β.²⁸ Extraction of tissue samples from hypertrophic and mature scars revealed increased water content, decreased collagen content, 4-fold reduction in decorin, and 6-fold increase in biglycan and versican levels relative to normal skin and normal scar.^29,30 The proteoglycans biglycan and versican are normally found in articular cartilage. Their hydrophilic properties lead to increased water content in ECM. Interestingly, TGF-β co-localizes with decorin, a finding consistent with one of the theoretical functions of decorin: resolution of fibrosis by its ability to bind and neutralize TGF-β in the ECM.³¹ Significant fibroproliferative disorders leading to various morbid conditions develop:
1. In the skin: Keloids and hypertrophic scars are unique human dermal fibroproliferative disorders that occur spontaneously following trauma, inflammation, surgery, and burns. Keloids usually extend beyond the margins of the original wounds. They occur most commonly in individuals with a familial predisposition and may rarely regress spontaneously. Hypertrophic scars, which are raised erythematous, pruritic, fibrous lesions that typically remain within the confines of the original wound, usually undergo at least partial spontaneous resolution over widely varying periods and are often associated with contractures of the healing tissues. These disorders represent aberrations in the fundamental phases of wound healing (Figure 2).
2. In other tissues: Adhesions remain a significant source of unfavorable consequences of surgery, and their prevention would significantly improve post-operative morbidity. All abdominal surgical procedures have the potential of creating adhesions. In the absence of surgery, abdominal and pelvic infections and therapy, such as peritoneal dialysis, may trigger the inflammatory cascade that eventually leads to the development of adhesions.
A primary consequence of infectious and autoimmune insults is the development of tissue and organ fibrosis. Several lines of evidence indicate that TGF-β initiates and terminates tissue repair. Its sustained production underlies the development of tissue fibrosis.³² Over-accumulation of ECM in tissues is the chief pathologic feature of fibrotic diseases. Transforming growth factor-beta enhances the deposition of ECM, which is continually degraded by proteases. To accomplish these roles, TGF-β acts simultaneously as a cellular stimulator to increase the synthesis of most matrix proteins by several fold, a cellular suppressor to decrease the production of inhibitors of certain collagen proteases, and a modulator of integrin expression in a manner that increases cellular adhesion to the matrix. These effects on ECM reflect the diverse biologic properties of TGF-β and may also be part of a negative feedback loop that normally regulates its own expression.³³ Increased levels of TGF-β and collagen are present in tissue sections from patients with a variety of clinical fibrotic disorders. The involvement of TGF-β in diseases of the organs, such as the kidney (glomerulonephritis), the liver (cirrhosis, veno-occlusive disease), and the lung (idiopathic fibrosis), has been thoroughly investigated.^34–38 There is supportive evidence that points to a causal relation between elevated production of TGF-β and the accumulation of ECM in the aforementioned organs. Glomerular immunostaining of kidney biopsy specimens from patients with mesangial proliferative glomerulonephritis for TGF-β was intense, and the intensity correlated with the amount of mesangial matrix.³⁴ Transforming growth factor-beta was detected by immunostaining in biopsies from patients with chronic liver disease but not in areas of inactive disease or normal liver.³⁵ Elevated plasma TGF-β concentrations were highly predictive of the development of hepatic fibrosis (veno-occlusive disease) in recipients of bone marrow transplants.³⁶ Transforming growth factor-beta was increased in alveolar walls at the sites of ECM accumulation in idiopathic pulmonary fibrosis.³⁷ Bronchoalveolar cells obtained by lavage from patients with autoimmune diseases and lung fibrosis contained 10 times more TGF-β m-RNA than similar cells obtained from normal subjects or patients with asthma.³⁸

Wound Care Improvements: The Role of Wound Healing Assessment

Neutrophils, macrophages, fibroblasts, and lymphocytes migrate into the wound site in an orderly fashion during the first 2 weeks post trauma.¹ In theory, the type and relative number of cells recruited in the wound site may form the basis for effective healing progress assessment protocols. Other parameters, such as fibroblast-derived proteins, collagen, fibronectin, and proteoglycan, may be used as markers of ECM accumulation and may contribute to the functional evaluation of the healing process. In practice, however, experimental attempts to explain why some wounds heal and others do not by means of wound fluid activity and composition have been inconclusive.^39–41 This may be explained in part by the close dependence of biological time to subject age and other biological parameters and individualities,⁴² which appear to obviate a direct correlation between biological and physical time during healing and reduce the efficiency of proper therapeutic intervention. More work is needed in this area to establish a meaningful correlation between functional staging and effective enhancement of the protective aspects of the healing process by suitable stage-specific treatment.⁴³ Changes in nuclear chromatin conformation and cytoplasmic catecholamine-containing particle content44 are likely to play pivotal roles in assessing local cellular homeostasis in the wound site, which will in turn assist in the proper functional staging of the healing process.
The quiescent, senescent, or even apoptotic stage of wound repair cells in chronic wounds may help explain why delays occur in wound healing. Increased numbers of repair fibroblasts arrested at the G1 phase of the cell cycle have been measured in nonhealing wounds.⁷ Proliferating cell nuclear antigen (PCNA) is a classic marker of cellular ability for DNA synthesis and cell cycle progression, whereas p21 is a cell cycle inhibitor. Increased numbers of arrested fibroblasts expressing p21 and reduced numbers expressing PCNA have been observed in nonhealing ulcers and in a model of fascial wound failure leading to incisional hernia formation.^7,45–48

Conclusion

The protective and damaging aspects of healing summarized in Figure 1 suggest that wound management protocols should aim to activate the normal healing cascade and block the pathways that lead to healing deviations. Furthermore, the biological aspects of the healing sequence shown in Figure 2 underscore the importance of accurate healing progress assessment for the stage-specific administration of therapeutics, such as monoclonal antibodies TGF-β 1, 2, and 3, in order to achieve the greatest healing benefit.⁴⁹ The proposed model of wound healing, as summarized in Figures 1 and 2, may help identify potent activators of normal healing via functional staging and select the proper timing for their administration for optimal therapeutic benefit. The successful use of the ECM component fibulin-5 in functional staging⁵⁰ supports the authors’ proposed approach and suggests that the use of additional staging markers will decisively improve wound healing management practices.