http://surgerybook.net/

13. Knittle JL, Timmers K, Ginsberg-Fellner F, et al. The growth of adipose tissue in children and

adolescents. Cross-sectional and longitudinal studies of adipose cell number and size. J Clin Invest

1979;63:239–246.

14. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha:

direct role in obesity-linked insulin resistance. Science 1993;259(5091):87–91.

15. Lord G, Matarese G, Howard J, et al. Leptin modulates T-cell immune response and reverses

starvation-induced immunosuppression. Nature 1998;394:897–901.

16. O’Rourke RW, White AE, Metcalf MD, et al. Hypoxia-induced inflammatory cytokine secretion in

human adipose tissue stromovascular cells. Diabetologia 2011;54(6):1480–1490.

17. Shin MK, Drager LF, Yao Q, et al. Metabolic consequences of high-fat diet are attenuated by

suppression of HIF-1α. PLoS One 2012;7(10):e46562.

18. Lumeng CN, Bodzin JL, Saltiel AR. Obesity induces a phenotypic switch in adipose tissue

macrophage polarization. J Clin Invest 2007;117:89–93.

19. Khan T, Muise ES, Iyengar P, et al. Metabolic dysregulation and adipose tissue fibrosis: role of

collagen VI. Mol Cell Biol 2009;29(6):1575–1591.

20. Virtanen KA, Lidell ME, Orava J, et al. Functional brown adipose tissue in healthy adults. N Engl J

Med 2009;360:1518–1525.

21. Boström P, Wu J, Jedrychowski MP, et al. A PGC1-α-dependent myokine that drives brown-fat-like

development of white fat and thermogenesis. Nature 2012;481:463–468.

22. Shi H, Strader AD, Woods SC, et al. The effect of fat removal on glucose tolerance is depot specific

in male and female mice. Am J Physiol Endocrinol Metab 2007;293(4):E1012–E1020.

23. Tran TT, Kahn CR. Transplantation of adipose tissue and stem cells: role in metabolism and disease.

Nat Rev Endocrinol 2010;6:195–213.

24. Donnelly KL, Smith CI, Schwarzenberg SJ, et al. Sources of fatty acids stored in liver and secreted

via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest 2005;115(5):1343–

1351.

25. Barbuio R, Milanski M, Bertolo MB, et al. Infliximab reverses steatosis and improves insulin signal

transduction in liver of rats fed a high-fat diet. J Endocrinol 2007;194:539–550.

26. Tilg H, Jalan R, Kaser A, et al. Anti-tumor necrosis factor-alpha monoclonal antibody therapy in

severe alcoholic hepatitis. J Hepatol 2003;38:419–425.

27. Calle EE, Rodriguez C, Walker-Thurmond K, et al. Overweight, obesity, and mortality from cancer

in a prospectively studied cohort of U.S. adults. N Engl J Med 2003;348(17):1625–1638.

119

http://surgerybook.net/

Chapter 5

Wound Healing

Rajiv Chandawarkar and Michael J. Miller

Key Points

1 Nonhealing wounds affect about 3 to 6 million people in the United States, with persons 65 years and

older accounting for 85% of these events. The annual cost of this problem in the United States is

estimated to be as high as $25 billion for hospital admissions, antibiotics, and local wound care. The

development of new data regarding the normal and pathologic wound healing responses both at the

cellular levels and the biological markers associated with them will help us develop new strategies

to treat these difficult expensive clinical problems.

2 Normal wound healing is achieved through four highly integrated and overlapping biophysiological

phases: hemostasis, inflammation, proliferation, and tissue remodeling or resolution. Each phase initiates a

cascading set of processes critical to the final aim of a healed wound.

3 Wound healing is a complex biological process that consists of hemostasis, inflammation,

proliferation, and remodeling. Large numbers of cell types – including neutrophils, macrophages,

lymphocytes, keratinocytes, fibroblasts, and endothelial cells – are involved in this process. Multiple

factors can cause impaired wound healing by affecting one or more phases of the process and are

categorized into local and systemic factors.

4 Clinically the process of wound healing is important to understand from several perspectives. These

include: development of precise, least-traumatic surgical technique; the clear understanding of how

newer developments in the field of biofilm and anti-infective therapies affect wound management;

factors that lead to the formation of chronic versus acute wounds, and importantly, the comorbid

conditions that affect wound healing. The role of age, gender, nutrition, obesity, and diabetes are

critical factors to incorporate into the therapeutic repertoire.

The role of disease states including diabetes, and cancer-related treatments including radiation,

chemotherapy and ways to mitigate these factors are as important to assimilate.

1 A fundamental understanding of wound healing is essential to surgical practice. Years of research

have yielded extraordinary details of the wound healing process. The complexity can lead the practicing

clinician to the conclusion that this topic is perhaps best reserved for the wound specialist. It is critical,

however, for the practicing surgeon to have a fundamental understanding of wound healing. Besides the

controlled injury that occurs in elective surgery, all invasive surgical procedures cause soft tissue

trauma regardless of the circumstances. All aspects of surgical care from patient selection, surgical

instruments and technique, and postoperative management are intended to optimize tissue healing and

avoid complications. Highly specialized neurosurgical or cardiac procedures can be fraught with

complications if sound principles of soft tissue wound healing are overlooked. In addition to surgical

procedures, there is the growing problem of people suffering from chronic wounds. Nonhealing wounds

affect about 3 to 6 million people in the United States, with persons 65 years and older accounting for

85% of these events. The annual cost of this problem in the United States is estimated to be as high a

$25 billion for hospital admissions, antibiotics, and local wound care.1 Minimizing wound complications

is essential in the current health care environment with an emphasis on quality and safety with publicly

reported outcomes such as surgical site infection and readmission rates. For the benefit of the patient

and to meet increasingly stringent scrutiny of surgical outcomes by the public, it is incumbent on the

surgeon to be more than a technician. Specifically, the surgeon needs to understand how surgery alters

tissues and the physiology of healing in order to obtain the best outcomes for their patients. This

understanding is essential to allow the surgeon to constantly improve personal technique and quality

outcomes. The development of new data regarding the normal and pathologic wound healing responses

both at the cellular levels and the biological markers associated with them will help develop new

strategies.

120

http://surgerybook.net/

NORMAL WOUND HEALING

The series of events associated with wound healing begins at the moment of injury. Although different

kinds of tissue (i.e., skin, fat, muscle, bone, nerve, parenchymal organs) have unique responses to

injury, each follows a similar process that involves four sequential overlapping periods known as the

hemostasis, inflammatory, proliferative, and remodeling phases of wound healing. The process is best

understood by considering a full-thickness injury of the skin, dermis, and subcutaneous tissue associated

with a simple surgical incision common to every surgical specialty.

2 Normal wound healing is achieved through four highly integrated and overlapping biophysiological

phases: hemostasis, inflammation, proliferation, and tissue remodeling or resolution.2 For a wound to heal

successfully, as shown in Figure 5-1, all four phases must occur in the proper sequence and time frame

(that takes almost 1 year to complete) and continue for a specific duration at an optimal intensity3

(Table 5-1).

Cellular components, the dominant processes as well as biochemical environments that elicit each of

the phases are markedly different in their functionality and work in concert to result in a normal healed

wound (Fig. 5-2). As shown in Figure 5-3, each phase initiates a cascading set of processes critical to the

final aim of a healed wound. In addition, the dominant cytokines for each phase are shown in Figure 5-

4.

Figure 5-1. Four phases of wound healing: plotted against “Time” on the X axis, the four phases shown in different colors occur

sequentially, and overlap. Overall the total time period for completion is 1 year.

Hemostasis

The first phase of hemostasis begins almost instantaneously after wounding. A scalpel drawn across

intact skin injures cells in the epidermis and dermis and separates components of the extracellular

matrix (ECM) that support the normal three-dimensional framework of the tissue and preserves the

barrier function. The zone of injury is limited to the dimensions of the blade when using a conventional

metal scalpel. With electrosurgery, the injury extends some distance into the surrounding tissues

depending on the power settings on the device. As the surgeon deepens the incision, the blood vessels in

the sub-dermal plexus and deeper in the subcutaneous planes, are cut, and bleeding occurs. The tissue

response is mediated by factors from three sources: (1) blood leaking into the wound, (2) proteins

stored in the ECM, and (3) locally resident surviving cells.

Table 5-1 Cellular and Biological Events that Frame the Normal Wound Healing

Process

121

http://surgerybook.net/

Circulating factors that initiate the inflammatory phase of healing are platelets, plasma proteins, and

leukocytes. Platelets are unnucleated formed elements in the blood produced in the bone marrow by

megakaryocytes. The plasma membrane of each platelet contains specific receptors for collagen known

as the glycoprotein Ia/IIa complex. The platelet cytoplasm contains granules holding an array of factors

important for hemostasis and inflammation. When plasma membrane receptors come into contact with

collagen of the ECM (type I) and the basement membrane of the vascular endothelium (Type IV),

platelets bind and anchor to the site. Simultaneously, platelet activation occurs and the platelet changes

from a rounded amorphous shape to a flattened configuration and discharges the contents of stored

cytoplasmic granules in an event known as the platelet release reaction. These bioactive factors serve a

dual purpose in hemostasis and wound healing. Hemostasis is promoted by factors that cause

strengthened force of platelet binding, accelerated platelet aggregation, vasoconstriction, and activation

of the clotting cascade. Platelet anchoring is strengthened by von Willebrand factor (vWF) released by

damaged endothelium and activated platelets. Platelet binding is also characterized by rapid vascular

constriction and the formation of a stable fibrin clot. Platelets are the main cellular players of the first

phase (Fig. 5-5A and Fig. 5-5B). By their collective function, they prevent hemorrhage. Platelet-derived

functions that achieve this goal include: adhesion, aggregation, and formation of a procoagulant surface

that facilitates the generation of thrombin and results in a fibrin plug. In addition, platelets express and

release substances that promote tissue repair and influence processes such as angiogenesis,

inflammation, and the immune response. They contain large secretable pools of biologically active

proteins, including platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-B), and

vascular endothelial growth factor (VEGF) as well as cytokines while newly synthesized active

metabolites including proteins such as PF4 and CD40L are also released. Although anucleate, activated

platelets possess a spliceosome and can synthesize tissue factor and interleukin-1β. The binding of

secreted proteins within a developing fibrin mesh or to the ECM can create chemotactic gradients

favoring the recruitment of stem cells, stimulating cell migration and differentiation, and promoting

repair.4 The therapeutic use of platelets in a fibrin clot has a positive influence in clinical situations

requiring rapid healing. Dental implant surgery, orthopedic surgery, muscle and tendon repair, skin

ulcers, hole repair in eye surgery and cardiac surgery are situations where the use of autologous

platelets accelerates healing.

122

http://surgerybook.net/

Figure 5-2. The dominant cells, physiologic process as well as the temporal distribution of the four phases of wound healing.

Figure 5-3. Flow chart of the phases and the cascading steps that result from each phase.

123

http://surgerybook.net/

Figure 5-4. The patterns of cytokines and growth factors within each phase of wound healing.

Figure 5-5. A: Diagrammatic wound healing model. Platelets, the first cells to arrive at the wound are critical to create a clot and

establish hemostasis B: Phase 1 – Hemostasis.

The growing platelet plug inside the vessel is stabilized by fibrin polymerized from circulating

fibrinogen. The vascular response is mediated by local factors modulated by the systemic control of the

sympathetic nervous system. Vasoconstriction, mediated by catecholamines and prostaglandins

(thromboxane and PGF2a), normally is limited to the time required to achieve hemostasis. Vasodilation

and alteration in capillary permeability soon follow mediated by histamine, prostaglandins (PGE2 and

PGI2

), and VEGF released from resident interstitial mast cells and damaged endothelium. This causes

increased blood flow and controlled delivery of fluid, leukocytes, macrophages, and relevant plasma

proteins to the wound environment. Platelets release two factors with particular importance for wound

healing: PDGF and TGF-β. Which in turn stimulate chemotaxis and proliferation of inflammatory cells.

Inflammatory Phase

The second phase of healing involves acute inflammation that begins at the moment of tissue disruption.

Inflammatory cells appearing during this phase of healing are polymorphonuclear leukocytes (PMNs)

and macrophages. PMNs are first to appear (Fig. 5-6). Their primary role is to clear devitalized tissue,

blood clot, foreign material, and bacteria from the wound. PMNs are part of natural host defenses that

destroy bacteria by phagocytosis and secreting oxygen free radicals. Migration of PMNs from the

intravascular compartment (the lumen) to the ECM and to the wound site is controlled by several

biochemical agents including selectins, cytokines, and integrins that act in series to activate, tether, and

facilitate extravascular escape (Fig. 5-7). PMNs work in concert with the immune system antibodies.

Their numbers and persistence depend on the initial wound conditions. A clean surgical wound

performed with atraumatic tissue handing will have low PMN activity, whereas a poorly performed

surgical incision or a traumatic wound may be characterized by a large amount of debris and require

124

http://surgerybook.net/

prolonged participation of PMNs to set the stage for appearance of macrophages, the most important

cellular mediator of the inflammatory phase of wound healing.

Figure 5-6. Phase 2 – Inflammation.

Macrophages appear in large numbers within 48 hours of wounding and play a central role in the

inflammatory phase. They are derived from circulating monocytes or resident interstitial cells that

migrate into the wound from adjacent tissues. Macrophages are multifunctional (Fig. 5-8) and complete

the cleanup activities initiated by the PMNs by phagocytizing remaining wound debris. Most

importantly, they secrete a vast array of cytokines and growth factors, which function in a rapidly

amplifying process that affects all aspects of healing during the inflammatory phase. These factors

induce recruitment and activation of additional macrophages, angiogenesis, proliferation of fibroblasts,

and ECM production. Matrix production is accelerated as the inflammatory phase of healing transitions

into the next phase of healing, the proliferative phase.

Proliferative Phase

The defining characteristic of the proliferative phase of healing is ECM production (Fig. 5-9). The

phases of wound healing are not discreet. While the inflammatory phase is still most active, the

proliferative phase begins with formation of a provisional ECM composed of fibrin and fibronectin

precipitated from blood extravasated into the wound at the time of the initial injury. The provisional

matrix is a protein scaffold that stabilizes the wound edges and provides a framework for migration of

PMNs, macrophages, fibroblasts, and other cells into the wound from surrounding tissues. As the

inflammatory phase slows the proliferative phase begins and becomes dominant. Fibroblasts replace

macrophages as the most numerous cell type. Like macrophages, fibroblasts are multifunctional. They

are responsible for new tissue formation, collagen production, and the laying down of the ECM (Fig. 5-

9). Angiogenesis occurs simultaneously as new capillaries form and blood vessels penetrate the

provisional matrix sprouting from vessels of the surrounding uninjured tissues. The processes that

actually coordinate formation of ECM and new tissue with angiogenesis are not well defined. Signaling

pathways that stimulate new tissue formation involve the role of low oxygen tension that increases the

expression of “hypoxia-inducible factor” (HIF) by vascular endothelial cells. HIF in turn binds to specific

sequences of DNA that regulate the expression of VEGF thus stimulating angiogenesis. This also has a

negative feedback loop – increased formation of new blood vessels normalizes the oxygen tension. In

response, oxygen binds to HIF and blocks its activity resulting in decreased synthesis of VEGF.

Epidermal growth factor and TGFa produced by activated wound macrophages, platelets, and

keratinocytes play an important role at the creation of a robust scaffold.

125