Inflammatory Response

Acute inflammation is a transient initial phase of injury repair that lasts approximately 5 to 7 days. Directly after trauma, platelets migrate to the injury site and release specific growth factors and chemical mediators, which stimulate homeostasis and initiate the repair process. A fibrin scaffold structure is formed within the trauma bed, creating a matrix that allows for platelet aggregation and adherence to the injury site. This process of platelet activation stimulates synthesis of thrombin, fibrin, and the random organization of clot formation. Platelet plug formation is essentially a four-step process: (1) adhesion, (2) aggregation, (3) secretion, and (4) procoagulant activity (Fig. 7-1).

Adhesion of platelets is the deposition of these cells on the subendothelial matrix. Platelets have a surface receptor glycoprotein that binds to a sticky protein substance referred to as von Willebrand factor (vWF) found in the subendothelial matrix. Endothelial cells synthesize vWF, which is released into the circulating plasma, then deposited in the subendothelial matrix in response to exposure from injury.1,3 Aggregation is simply platelet-to-platelet cohesion via the surface fibrinogen receptor complex of the platelets. Secretion is the release of a number of platelet-derived growth factors (PDGFs) by stimulated platelets. The aggregating stimulators of serotonin, thrombospondin, and thromboxane also are secreted. Procoagulant activity refers to the process of thrombin formation and ensures that coagulation occurs at the site of the platelet plug.

Inflammation

Initially triggered by the release of histamine from mast cells in the surrounding tissue, the inflammatory response is amplified by cytokines (signaling molecules) such as interleukin-1 (IL-1) and tumor necrosis factors (TNFs) that are activated by leukocytes (white blood cells). These leukocytes are involved with phagocytosis, or the engulfment, of cellular debris. Cytokines stimulate vascular permeability allowing mononuclear cells to mobilize, proliferate, and differentiate into monocytes at the injury site. Other cytokines such as platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and transforming growth factor-β (TGF-β) help organize the specific sequence of migration of neutrophils, macrophages, and then fibroblasts to the injury site.

Fibroplasia

Several days (5 to 7) after the injury, the relative population of fibroblasts increases, whereas inflammatory cells and proinflammatory factors decrease. At this stage there is a proliferation of reparative cells. Fibroblasts stimulate PDGF and TGF-β among others to synthesize and deposit extracellular matrix constituents of fibronectin, laminin, collagen, and glycosaminoglycans.1,3,4

This phase also includes angiogenesis, the neovascular budding that helps reestablish oxygen-rich and growth factor–rich blood to new, fragile healing tissue. Angiogenic growth factors involved with the stimulation of this neovascularization are fibroblast growth factor (FGF), tumor necrosis factor-β (TNF-β), and wound angiogenesis factor (WAF). Endothelial cells from intact vascular membranes are mobilized to form new tissue from the secretion of specific enzymes and collagens. The end stages of angiogenesis signal vascular capillary and network tube formation, creating new vascular basement membranes that directly communicate with the injury site.1,3,4

Remodeling and Tissue Maturation

The remodeling phase of injury repair is essentially a balance between enzymatic (proteolytic) degradation of excess collagen and the deposition, organization, modification, and maturation of collagen, as well as a systematic regression of inflammatory cells (Fig. 7-2).

Collagenases are enzymes of the metalloproteinase family that act to fragment collagen. The regulation of the rate of collagen synthesis and degradation (turnover) is mediated by specific growth factors such as PDGF, IL, TGF-β, and TNF-β.⁴

Cummings and Reynolds² describe remodeling as “the process by which the architecture of connective tissue alters in response to stress.” Collagen fibers align parallel to the direction of applied stress, which increases the strength of the scar tissue union. Remodeling also includes absorption of collagen fibers that lie in opposing directions. If controlled properly, the effect of the remodeling phase is that the fibrous union becomes stronger and more supple with fewer adhesions.

Because of the vascularity, dense cell population of myofibroblasts, and nature of its small, fragile collagen fibers, new scar tissue is able to remodel quickly. Collagen fibers are initially small and disorganized, and they form a spongy randomly oriented tissue. As remodeling continues, larger, more parallel fibers replace the smaller fibers and orient across the wound, which forms a stronger yet supple repair (Fig. 7-3).²

Cellular Structure

Organelles define various component structures within cells. Smooth endoplasmic reticulum as a cell organ is involved with the synthesis of steroid hormones, particularly cholesterol. Rough endoplasmic reticulum organelles are actively involved with protein synthesis and secretion. Within cells the Golgi apparatus functions primarily as a storage and modification organ for various proteins that have been synthesized in the rough endoplasmic reticulum. Lysosomes and peroxisomes are cellular compartment structures that are involved with enzymatic degradation and oxidation of protein and fatty acids. Mitochondria, the powerhouses of the cell, are actively involved with adenosine triphosphate (ATP) production and protein synthesis.1,3

Proteoglycans and Glycoproteins

Proteoglycans are macromolecules consisting of a protein core and bound polysaccharide units referred to as glycosaminoglycans (GAGs). GAGs are major components of ground substance within the extracellular matrix of various connective tissues, such as ligament, capsule, muscle, tendon, cartilage, nerve, vessel, and bone. GAGs are synthesized within the endoplasmic reticulum and Golgi apparatus of the fibroblast cells in connective tissues. Several GAGs are identified in musculoskeletal tissues, which significantly contribute to structure, composition, and function of the extracellular matrix of tissues: (1) chondroitin sulfate, (2) dermatan sulfate, (3) keratan sulfate, (4) hyaluronate, (5) aggregen, (6) decorin, and (7) biglycan.1,3

Generally, GAGs carry a high negative charge that renders GAGs hydrophilic. Within articular cartilage, GAGs are continuously synthesized, assembled, degraded, and secreted by the extracellular matrix to establish a relative homeostatic environment. Enzymatic degradation of GAGs occurs within lysosome organelles of the chondrocytes.1,3,4

Glycoproteins are molecules that organize and maintain the structure of the extracellular matrix. In articular cartilage these are fibronectin, laminin, anchorin, and tenascin.

Polymorphonuclear Leukocytes

Granulocytes, or polymorphonuclear leukocytes, differentiate into neutrophils, basophils, and eosinophils when stimulated by cytokine growth factors and colony-stimulating factors. Neutrophils, the most abundant granulocytes, are leukocytes that migrate to damaged tissue after mediation and activation of cytokines, platelet factor 4 (PF4), and TGF-β that are released by platelets. Primary function of neutrophils includes initial phagocytosis of foreign matter, bacteria, and cellular debris from the damaged tissue site. Neutrophils also contribute to the immune system inflammation process by stimulation, vasodilation, and vascular permeability assisting with transportation and migration of molecules and cytokine growth factor–PDGF propagating events to further minimize intense inflammatory reactions.

Mononuclear Leukocytes

Mononuclear leukocytes are single-nucleus cells that are derived from pluripotent hematopoietic stem cells, which differentiate into monocytes and lymphocytes. Circulating monocytes are mobilized to damaged tissue, where they are activated and become macrophages. Noted as scavenger cells, macrophages are leukocytes that display three essential functions during the inflammatory process: (1) phagocytosis, (2) antigen presentation, and (3) production of cytokine growth factors.1,3,4

Macrophages synthesize and secrete numerous cytokines, such as TGF-β, PDGF, transforming growth factor-α (TGF-α), and IL-1. The release of these cytokines stimulates proliferation of fibroblasts and collagen deposition and degradation of collagen by secreting enzymes (collagenases) also, which denatures collagen during the inflammatory and remodeling phases.1,3,4

Fibroblasts

Fibroblasts, important and highly specialized cells, are actively involved with collagen production of various stages of injury repair. Fibroblastic cell proliferation is activated by cytokines released by platelets and leukocytes. PDGF is responsible for the stimulation of fibroblast proliferation. Fibroblasts serve a critical function in wound healing mechanics. Fibronectin is a glycoprotein produced by fibroblasts, which acts to bind collagen within the extracellular matrix of healing tissue. Myofibroblasts are specialized contractile cell types that are important during the later stages of wound repair, contracting the edges of the wound.

Prostaglandins, Thromboxanes, and Leukotrienes

Prostaglandins and thromboxanes are lipid-derived powerful and important mediators of inflammatory reactions (Fig. 7-4), which are metabolized from arachidonic acids within the cells. Arachidonic acid metabolism is initiated by the degradation of cell membrane phospholipids by phospholipase enzymes. This cell membrane degradation releases arachidonic acid–synthesized cyclooxygenase enzymes, such as COX-1 and COX-2, which results in metabolic conversion to prostaglandins and thromboxanes.

Prostaglandins are generally of three forms1,3: (1) prostaglandin E₂ (PGE₂), which stimulates smooth muscle relaxation and vasodilation; (2) prostaglandin I₂ (PGI₂), which is synthesized in endothelial cells and incites vascular dilation and inhibition of platelet adhesion; and (3) prostaglandin F₂ (PGF₂), which is a potent vasoconstrictor and stimulates smooth muscle contractions. These molecules are capable of producing pain and stimulating synthesis of pain-producing chemicals.^1,3 Thromboxanes are synthesized by platelets and are products of the COX pathway along with prostaglandins. These cell-signaling molecules are potent vasoconstrictors and smooth muscle contractors. Leukotrienes are products of an alternate arachidonic acid metabolic pathway. In this pathway, lipoxygenase is converted to leukotrienes, which act as smooth muscle contractors; lipoxygenase also stimulates bronchoconstriction and is a strong mediator of other various inflammatory chemicals (chemotactic).^1,3

Various pharmacologic agents involved with antiinflammatory action, namely corticosteroids and nonsteroidal antiinflammatory drugs (NSAIDs), target arachidonic acid for the inhibition of prostaglandins, thromboxanes, and leukotrienes. Specifically, corticosteroids inhibit production of arachidonic acid metabolites (cyclooxygenase and lipoxygenase) by inhibiting the conversion of cell membrane phospholipids to arachidonic acid. Conversely, NSAIDs are post–arachidonic acid inhibitors of the specific cyclooxygenase (COX-1, COX-2) metabolic pathway.

Cytokines

Cytokines, including many growth factors, are a large and complex group (more than 100 identified) of protein-soluble peptide signaling molecules that are synthesized and secreted by all musculoskeletal tissues. They are used for cellular communication and stimulate cell proliferation, differentiation, and regulation of normal growth, homeostasis, injury, disease, and repair (Table 7-1). Generally referred to as mitogenic, cytokines are powerful and important immunologic mediators that coordinate and amplify various repair processes of injured musculoskeletal tissues.^1,3

Cytokines are named for either the biological effects they perform or the tissue on which they exert action. Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine synthesized and secreted by macrophages, lymphocytes, and monocytes. The biological effects and anatomic target tissues are varied and diverse. General target tissues include stimulation of leukocytes, mononuclear phagocytes, vascular endothelial cells, fibroblasts, chondrocytes, and synovial macrophages. TNF-α activates granulocytes and stimulates other proinflammatory cytokines, which are also important regulators of bone resorption. IL-1 is also a proinflammatory cytokine that is synthesized by various cells. Tissue targets include monocytes, synovial macrophages, fibroblasts, chondrocytes, and endothelial cells. Biologically mediated effects include inhibition of extracellular matrix synthesis within chondrocytes, stimulation of fibroblast proliferation, proliferation of T cells, and stimulation of other proinflammatory cytokine synthesis. Interleukin-7 is also a proinflammatory growth factor responsible for additional cytokine secretion and stimulation of prostaglandins in epithelial, endothelial, and fibroblastic cells.

Transforming growth factor-β is a potent immunosuppressive cytokine with strong anabolic activity in cartilage. TGF-β also reduces enzymatic degradation activity specifically within cartilage. In addition, TGF-β promotes wound healing, bone formation, and neovascular activity.1,3,4

Insulin-like growth factor (IGF) regulates many musculoskeletal functions. Generally, IGF stimulates proteoglycan synthesis, chondrocyte proliferation, and osteoblast matrix synthesis. Target cells for neovascularization include platelets and endothelial cells.

Platelet-derived growth factor contributes significant stimulation toward the repair process of musculoskeletal and vascular tissue. Cell types activated by PDGF include platelets, neutrophils, macrophages, fibroblasts, and endothelial cells. Biological activity activated by PDGF includes homeostasis, initiation of wound repair cascade phagocytic activity, synthesis, and deposition of extracellular matrix constituents and angiogenesis activity.

Vascular endothelial growth factor (VEGF) is an important angiogenic cytokine that stimulates endothelial cell proliferation. VEGF significantly contributes to neovascularization. In addition, VEGF has been identified in the hypertrophic zone of calcified cartilage in the epiphyseal plate. VEGF stimulates endothelial cell ingrowth in this area, possibly enhancing cartilage conversion to bone. VEGF also is responsible for the release of degradative enzymes in the extracellular matrix.

Fibroblast growth factor (FGF) stimulates cell proliferation of cartilage matrix and bone tissue. FGF is also an effective angiogenic stimulant for revascularization after injury. FGF promotes epithelial cell activity during remodeling and neovascular growth.

Overall, tissue disruption and subsequent repair processes initiate and propagate complex cellular events mediated by cytokines. Cytokines regulate, stimulate, and express other growth factors to synthesize, proliferate, excrete, and mobilize numerous molecules involved with extracellular matrix deposition, cartilage synthesis, vascular growth, enzymatic degradation, and modulation of inflammatory immune reactions during injury and repair of musculoskeletal and neurovascular repair (Fig. 7-5).^1,3