What is the correct order of events in tissue repair?

Following tissue injury via an incision, the initial response is usually bleeding. The cascade of vasoconstriction and coagulation commences with clotted blood immediately impregnating the wound, leading to hemostasis, and with dehydration, a scab forms. An influx of inflammatory cells follows, with the release of cellular substances and mediators. Angiogenesis and re-epithelialization occur and the deposition of new cellular and extracellular components ensues.

The initial injury results in an outflow of blood and lymphatic fluid. This is also the process during which the initial reparative coagulum is created. Both the intrinsic and extrinsic clotting mechanisms are activated. The intrinsic mechanism is enjoined from the thrombocytes and the extrinsic mechanism from the injured tissues. Following vasoconstriction, platelets adhere to damaged endothelium and discharge adenosine diphosphate (ADP), promoting thrombocyte clumping, which dams the wound. With the short-lived vasoconstriction complete, the vessels dilate allowing the influx of more thrombocytes and other blood cells.

At this stage, one can think of the commencement of the inflammatory phase. Although some speak of a separate inflammatory phase, it commences during the hemostasis phase, again providing evidence of the overlapping nature of the healing compendium. These thrombocytes, as well as the recruited white blood cells, release numerous factors to ramp up the healing process. Alpha-granules liberate platelet-derived growth factor (PDGF), platelet factor IV, and transforming growth factor (TGF)–β). The processes of inflammation, collagen degradation and collagenogenesis, myoblastic creation from transformed fibroblasts, growth of new blood vessels, and reepithelialization have all commenced.

These processes are mediated by a host of cytokines and growth factors. The interleukins strongly influence the inflammatory process. Vascular endothelial growth factor (VEGF) and other factors enhance blood vessel formation, and some have multiple roles such as fibroblast growth factor (FGF)–2, which affects not only the process of angiogenesis but also that of reepithelialization. Vasoactive amines such as histamine and serotonin are released from dense bodies found in thrombocytes. PDGF is chemotactic for fibroblasts and, along with TGF-β, is a potent modulator of fibroblastic mitosis, leading to prolific collagen fibril construction in later phases. Fibrinogen is cleaved into fibrin, and the framework for completion of the coagulation process is formed. Fibrin provides the structural support for cellular constituents of inflammation. This process starts immediately after the insult and may continue for a few days.

As the hemostasis phase can be construed to consist of an early and a late phase, the early phase being bleeding and hemostasis and the late phase being coagulation, so is it also with inflammation. While the inflammatory phase commences during the hemostasis phase, the early component of the inflammatory phase is predominated by the influx of the polymorphonuclear leukocytes (PMNs) and the later component predominated by monocytes/macrophages.

Within the first 6-8 hours, the next phase of the healing process is underway, PMNs engorging the wound. TGF-β facilitates PMN migration from surrounding blood vessels, where they extrude themselves from these vessels. These cells cleanse the wound, clearing it of debris. The PMNs attain their maximal numbers in 24-48 hours and commence their departure by hour 72. Other chemotactic agents are released, including FGF, TGF-β and TGF-α, PDGF, and plasma-activated complements C3a and C5a (anaphylactic toxins). They are sequestered by macrophages or interred within the scab or eschar. [12]

As the process continues, monocytes also exude from the vessels. These are termed macrophages once they leave the vessel. The macrophages continue the cleansing process and manufacture various growth factors during days 3-4. The macrophages orchestrate the multiplication of endothelial cells with the sprouting of new blood vessels, the duplication of smooth muscle cells, and the creation of the milieu created by the fibroblast. Many factors influencing the wound healing process are secreted by macrophages. These include TGFs, cytokines and interleukin 1 (IL-1), tumor necrosis factor (TNF), and PDGF.

This phase consists of different subphases. These subphases do not happen in discrete time frames but constitute an overall and ongoing process. The subphases are "fibroplasia, matrix deposition, angiogenesis and re-epithelialization". [11]

In days 5-7, fibroblasts have migrated into the wound, laying down new collagen of the subtypes I and III. Early in normal wound healing, type III collagen predominates but is later replaced by type I collagen.

Tropocollagen is the precursor of all collagen types and is transformed within the cell's rough endoplasmic reticulum, where proline and lysine are hydroxylated. Disulfide bonds are established, allowing 3 tropocollagen strands to form a triple left-handed triple helix, termed procollagen. As the procollagen is secreted into the extracellular space, peptidases in the cell wall cleave terminal peptide chains, creating true collagen fibrils.

The wound is suffused with GAGs and fibronectin produced by fibroblasts. These GAGs include heparan sulfate, hyaluronic acid, chondroitin sulfate, and keratan sulfate. Proteoglycans are GAGs that are bonded covalently to a protein core and contribute to matrix deposition.

Angiogenesis is the product of parent vessel offshoots. The formation of new vasculature requires extracellular matrix and basement membrane degradation followed by migration, mitosis, and maturation of endothelial cells. Basic FGF and vascular endothelial growth factor are believed to modulate angiogenesis.

Re-epithelization occurs with the migration of cells from the periphery of the wound and adnexal structures. This process commences with the spreading of cells within 24 hours. Division of peripheral cells occurs in hours 48-72, resulting in a thin epithelial cell layer, which bridges the wound. Epidermal growth factors are believed to play a key role in this aspect of wound healing.

This succession of subphases can last up to 4 weeks in the clean and uncontaminated wound.

After the third week, the wound undergoes constant alterations, known as remodeling, which can last for years after the initial injury occurred. Collagen is degraded and deposited in an equilibrium-producing fashion, resulting in no change in the amount of collagen present in the wound. The collagen deposition in normal wound healing reaches a peak by the third week after the wound is created. Contraction of the wound is an ongoing process resulting in part from the proliferation of the specialized fibroblasts termed myofibroblasts, which resemble contractile smooth muscle cells. Wound contraction occurs to a greater extent with secondary healing than with primary healing. Maximal tensile strength of the wound is achieved by the 12th week, and the ultimate resultant scar has only 80% of the tensile strength of the original skin that it has replaced.

Author: Dr. Christopher Leonard, DO, MHI

Dr. Leonard is the Chief Information Officer at Vohra Wound Physicians. His experience includes developing a niche-specific, ONC-certified, proprietary electronic medical records (EHR) system. His expertise also lies in managing the data flow spectrum, machine learning, and product design related to healthcare technology. His creative vision supports Vohra’s mission in the continuous improvement of its novel healthcare delivery model.

View all posts by Dr. Christopher Leonard, DO, MHI