When the body has a soft-tissue injury, during which phase does the body regenerate tissue?

1.1. INTRODUCTION AND OVERVIEW

The inevitable response to any implant is wound healing comprised of hemostasis, inflammation, repair, and remodeling. For nondegradable smooth-surfaced implants, repair and remodeling leads to isolation of the implant through tissue encapsulation. The nature of the encapsulation tissue and the cellular participants in the immune reaction leading to this outcome varies depending on the site of implantation and the type of tissue that hosts the implant (not to mention the skill of the surgeon).

It is now well-established that the wound healing process has substantial deleterious effects on the fidelity and reliability of implanted sensors and electrodes. A number of reviews [1–6] and a multitude of primary articles address this issue for implanted sensors, primarily glucose electrodes; however, there has been only one review [7] and a limited number of primary articles regarding electrodes implanted in central nervous system (CNS) tissue [8–11]. This chapter will provide a summary of the wound healing process in CNS tissue and compare it to the wound healing process in the peripheral nervous system (PNS), bone, and skin.

Recent reports have shown that implanted electrodes can monitor neural signals in the CNS and therefore can be used for the control of prosthetics in patients suffering from full or partial paralysis [12,13]. While initial studies are promising, chronically implanted sensors and electrodes frequently and unpredictably experience component failure or complications arising from the wound healing response [7]. The wound healing process begins as soon as the electrodes are inserted into the brain, inevitably disrupting the blood–brain barrier [14]. Breaching of the blood–brain barrier leads to hemostasis and the initial stages of inflammation in CNS tissue that are typical of those seen in all vascularized tissue. However, distinct differences arise in CNS tissue during the later stages of inflammation, repair, and remodeling (Figure 1.1).

Figure 1.1

Summary of wound healing response in the brain versus nonneural tissue.

The first difference is the blood–brain barrier. The CNS is highly vascularized; however, maintaining a barrier between capillaries and the surrounding tissue is important for regulating the concentration of ions and other molecules in both the vascular and extravascular space of the CNS [15]. Endothelial cells forming capillaries in the CNS differ from those in the rest of the body in two respects. First, they are able to form tight junctions, restricting paracellular flux, and, second, they have very few endocytotic vesicles, limiting transcellular solute movement from the blood to the brain interstices (reviewed by Rubin and Staddon [16]). As a result, the blood–brain barrier impedes entry of virtually all blood molecules, except those that are small and lipophilic, such as steroids. However, some larger molecules such as glucose and certain amino acids are still able to move into brain tissue through specific transporter proteins [16]. A dense basement membrane and astrocyte processes, termed end-feet, surround capillary endothelial cells, further contributing to the blood–brain barrier. Under normal nonpathological conditions the CNS vascular endothelium with its tight junctions completely separates brain tissue from circulating blood, isolating the brain’s unique cell types while protecting neural tissue from potentially harmful immune responses occurring outside of the neural tissue in the body [17]. This is important because unlike most peripheral tissues, the CNS functions through a network of neurons that is generally incapable of proliferation and cannot be replaced when impaired.

CNS tissue is distinguished from non-CNS tissue by the presence of cell types that are unique to the CNS. Neurons, which consist of a cell body and its axon, are the functional unit of brain tissue even though they account for fewer than 25% of the total cell population [7]. About half of the total CNS volume is occupied by the three types of supporting connective tissue glial cells: oligodendrocytes, astrocytes, and microglia. Each glial cell type performs functions that ensure the health of neurons in the CNS. Oligodendrocytes wrap around axons to provide support and produce a lipid-rich myelin sheath to protect neural processes; astrocytes have many neural support functions but are notable for forming the glial boundary between CNS and non-CNS structures to create the blood–brain barrier; microglia share many phenotypical and functional characteristics with blood-derived macrophages, existing in a resting state within brain tissue until activated to marshal the innate immune responses of the brain [7,18]. In contrast to the CNS, axons in the PNS have a continuous myelin sheath produced by Schwann cells. The continuous myelin sheath increases the propagation velocity of the nerve impulse, which is important for the axons that extend long distances through the PNS.

Subsequent sections of this chapter highlight the characteristics of each stage of the wound healing response (i.e., hemostasis, inflammation, repair, remodeling) while identifying the similarities and differences in the main tissue types. However, it is important to note that there are no defined boundaries between stages as the wound healing response “transitions” into the next stage of healing.

1.2. HEMOSTASIS (SECONDS TO HOURS)

Common to all tissue types, the wound healing process begins immediately after injury as tissue is disrupted and blood vessels are severed, releasing blood plasma and peripheral blood cells into the wound site. The earliest signals of tissue injury are the release of molecules such as ATP and the exposure of collagen on the blood vessel wall [19]. In the CNS, traumatic brain injury ruptures the blood–brain barrier as serum and blood cells are released into the wound site [14]. In all tissues, a clot is formed that acts as a temporary barrier that prevents excess bleeding and limits the spread of pathogens into the blood stream. Primary hemostasis occurs as platelets adhere to collagen fibers exposed in the damaged endothelium using specific collagen receptor glycoproteins (GPIb/IX/V) to form the primary hemostatic plug [19]. As platelets attach to the lesion site, they rapidly upregulate the high-affinity platelet integrin αIIbβ3, which mediates platelet aggregation [19]. Once platelets bind, they activate and degranulate, releasing their contents into the plasma to stimulate local activation of plasma coagulation factors (Figure 1.2b). These factors trigger the generation of a fibrin clot (Figure 1.2c) [20]. Exposure of blood plasma to tissue factor, produced by subendothelial cells not normally exposed to blood, such as smooth muscle cells and fibroblasts [21], or to foreign surfaces, such as implants, initiates an accelerated cascade of activated proteins that leads to fibrin formation. The cleaving of fibrinogen to fibrin monomers and its polymerization and cross linking forms an intertwined gelatin-like platelet plug, producing a stable clot [22].

Figure 1.2

Hemostasis in damaged blood vessel. (a) An injury to the vessel wall causes the release of blood-borne cells, proteins, and platelets into the periphery. Platelets are activated upon coming into contact with the surrounding collagen outside of the blood (more...)

Platelet activation also causes the release of a number of signaling molecules such as platelet-derived growth factor (PDGF) [23], transforming growth factor-β (TGF-β) [24], and vascular endothelial growth factor (VEGF) [25] from their cytoplasmic granules. Inflammatory and reparative cells are chemotactically attracted to the “reservoir” of molecules stored within the clot that gives rise to inflammation, the next step in the sequence of healing.

1.3. INFLAMMATION (HOURS TO DAYS)

1.3.1. Initial Events

The process of inflammation contains, neutralizes, or dilutes the injury-causing agent or lesion, regardless of tissue type [26]. In both the CNS and non-CNS, the tissue environment in which inflammation begins is a mixture of injured tissue, components of the clot (platelets, erythrocytes, and fibrin), extravasated serum proteins, and foreign material introduced at the time of the injury [27]. Inflammation is initiated by the release of signaling molecules from the wound site during hemostasis [28]. Chemoattractant molecules released by platelets also increase vasodilation and vascular permeability, subsequently enhancing leukocyte recruitment. Incoming leukocytes recognize plasma proteins such as fibronectin, vitronectin, and thrombospondin, which are passively absorbed by the clot. The initial process of inflammation is common to all tissue types because any physical trauma will usually result in the formation of the platelet plug and the recruitment of blood-borne inflammatory cells to the injury site. In CNS tissue, injury also results in the activation of microglia.

Neutrophils are the first inflammatory cells recruited to the wound, and they arrive within 24 hours after injury. They migrate into the wound by responding to chemoattractants released by platelets as well as chemokines presented on the endothelial cell surface [29]. The leukocyte receptor PSGL-1 binds to P-selectin expressed on both platelets and endothelial cells [30]. The low-affinity selectin binding to PSGL-1 on the neutrophil membrane causes flowing neutrophils to roll and briefly tether to endothelial cells. During rolling, neutrophils are activated further by interleukin-8 (IL-8) and macrophage inflammatory protein (MIP-1β) released from the endothelial cells. Neutrophil receptor binding to chemoattracants activates their β2 integrins, which then firmly attach to endothelial cell intracellular adhesion molecules (ICAM)-1 [31]. Neutrophils subsequently extravasate through the vessel wall into the wound site, where they release proteolytic enzymes for the digestion of foreign debris and the killing of bacteria through phagocytosis and superoxide and hydrogen peroxide production [32]. Neutrophils undergo apoptosis after 24 to 48 hours if wound decontamination is complete. They are replaced by extravasating monocytes and macrophages brought to the site of injury by further release of MIP-1α and MIP-1β, monocyte chemoattractant protein-1 (MCP-1), and a chemotactic cytokine called RANTES, all of which are produced by activated endothelial cells [33].

1.3.2. Later Events (Central Nervous System [CNS] versus Non-CNS)

Within 2 to 3 days after injury, inflammation continues as monocytes are recruited from the blood and differentiate into macrophages. Once in the tissue, macrophages release additional proinflammatory cytokines such as IL-1, TGF-β, and tumor necrosis factor-α (TNF-α) [26]. Macrophages remove foreign debris and can remain present for as long as a few months, depending on the extent of injury and the amount of foreign and necrotic debris to be cleared [26]. Macrophages are believed to be more important than neutrophils for successful inflammation resolution. In studies where neutrophils were depleted, wound repair was not disturbed [34], but when macrophages were removed instead, there was limited clearance of necrotic debris at the wound site, resulting in a modified wound healing process [32,25]. Mast cells within the tissue contribute to the inflammatory response by releasing histamine and serotonin to enhance blood vessel permeability and promote macrophage migration.

Once the blood–brain barrier is reestablished through the patching of damaged blood vessels during the later stages of hemostasis, the inflammatory responses of the CNS and non-CNS tissues begin to diverge (Figure 1.1). Specifically, the selectins expressed on vascular endothelial cells that attract leukocytes are less prevalent in CNS tissue, thus limiting recruitment [36–38]. Consequently, axon and myelin debris from damaged neurons can take months to clear in the CNS while only taking days in the PNS [39]. Reduced leukocyte activity and infiltration can be seen as a method to protect the brain from undue inflammatory damage, with the decreased levels of leukocyte recruitment replaced by the resident macrophage-like microglia cells.

Microglia, the only resident phagocytoic cells in the CNS, are normally found in a highly branched resting or “ramified state” and only become activated if debris needs to be cleared or if the blood–brain barrier is compromised [7]. Upon activation, microglia withdraw their processes and change from their resting ramified state to a more compact rod-like shape [40,41]. Microglia act in concert with macrophages that have penetrated the blood–brain barrier to phagocytose degenerating axons and myelin at the site of injury [42]. At this point, microglia can be distinguished from blood-derived macrophages through flow cytometric analyses in vitro by differences in CD45 expression (microglia: CD45 low; macrophages: CD45 high) [43,44].

Microglia, like macrophages, express surface receptors involved in the innate immune response after exposure to various pathogens or pathogen products such as lipopolysaccharide (LPS) [45]. Activated microglia also contribute aspects of adaptive immunity by upregulating major histocompatibility complex class II and costimulatory molecules to present antigens to incoming T-cells [45,46]. Microglia have been shown to appear stimulated within 1 hour after an initial tissue injury [10,47]. The number of activated microglia peaks at 3 days after an inflammatory insult, although microglia can remain activated up to 1 or 2 weeks after injury [48].

Microglia at the site of injury produce a variety of proinflammatory and neurotoxic factors such as MCP-1 [49], TNF-α [50], IL-1β [51], IL-6 [52], nitric oxide [53], and superoxide [54]. Paradoxically, microglia have also been reported to increase neuronal survival through the release of antiinflammatory factors, such as IL-10 [55,56], and neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) [57,58] (reviewed in [58]).

Astrocytes, which become activated within hours after injury, are the second type of effector cells in the immune response that reside in the CNS. Astrocytes also perform a wide variety of functions outside of inflammation such as providing growth cues and mechanical support for neurons during CNS development, creating and maintaining the blood–brain barrier, and helping control the surrounding chemical environment through the buffering of neurotransmitters and ions released during neuronal signaling [7]. Activated astrocytes are identified during inflammation by upregulation of glial fibrillary acid protein (GFAP), increased proliferation, and hypertrophic cytoplasm and nuclei. GFAP is upregulated as early as 1 hour post injury and is thus a sensitive early marker of reactive astrocytes [59]. However, GFAP upregulation is not solely indicative of astrocyte activation since microglia can engulf necrotic astrocytic GFAP fragments and thereby express the astroglial marker [60]. Therefore, cellular hypertrophy is an important co-indicator of astrogliosis. Astrocytes have been shown to help mediate inflammation through the release of both proinflammatory and antiinflammatory factors such as: (a) nitric oxide, which contributes to the innate immune response while also causing neurodegeneration; (b) expression of both class I and class II MHC molecules that aid in the secondary immune response; and (c) regulation of blood vessel diameter through release of vasoconstrictors [60].

Three days into an inflammatory response in the CNS, activated astrocytes are observed at the periphery of injury while macrophages and microglia are colocalized at the center of the wound [48] (Figure 1.3A). Microglia are likely the major players in the response since macrophages are largely excluded from the injury by the blood–brain barrier [61]. Nevertheless, macrophages and lymphocytes from nonneural tissue can also enter into the brain late in the inflammatory stage to further amplify the body’s wound healing response by secreting a variety of cytokines. However, only lymphocytes that are in an activated state are able to pass through the blood-brain barrier and enter the CNS (for review see [62]).

Figure 1.3

Wound repair of different tissue types. (A) In the CNS, microglia and macrophages migrate into the lesion, secreting various cytokines and growth factors while also removing necrotic tissue. Activated astrocytes form a physical barrier around the area (more...)

The mechanism of lymphocyte extravasation during inflammation is modified in the CNS. First, there is no lymphocyte rolling along the epithelium within CNS, and, second, an activated lymphocyte can extravasate directly through an endothelial cell, leaving the neighboring tight junctions intact to preserve the blood–brain barrier [63]. This process can take several hours, whereas diapedesis through blood vessels into lymph nodes outside the CNS can occur within minutes [64]. The lymphocyte entry level varies within tissues of the CNS, with the highest entry in the spinal cord and the lowest entry in the cerebrum [36]. However, T cells find the CNS environment hostile and die rapidly via apoptosis or leave the CNS via the cerebrospinal fluid [36,65]. The reduced lymphocyte level results in significantly lower levels of immune surveillance in the CNS than in other tissues and is a reason why the CNS is often referred to as a site of “immunological privilege” [36]. A consequence of the inflammatory response in the brain is neuronal damage, as evidenced by the absence of neurofilaments around the lesion only a few days after injection of a proinflammatory agent [48].

The inflammatory phase is resolved by a mixture of antiinflammatory cytokines, although the specific individual cytokines that are involved and the mechanisms of their actions are largely unknown [66]. TGF-β secreted by macrophages is considered one of the main attenuators of inflammation in both CNS and non-CNS tissue [66]. TGF-β is involved in promoting the recruitment and proliferation of fibroblasts to push the wound healing response toward the repair phase. Macrophages are also known to produce other prowound healing factors such as VEGF to initiate angiogenesis within the hypoxic tissue environment [67].

1.4. REPAIR (DAYS TO WEEKS)

During inflammation the overall tissue strength of a wound is minimal, since tissues do not regain their normal functional strength until inflammation transitions into repair. This transition is mediated by macrophages and their antiinflammatory cytokines and generally occurs one week after the initial injury. The end result of the repair process is vitally important to wound healing because it establishes the scaffolding necessary to support and rebuild the damaged tissue.

Tissue repair is characterized by increased cell proliferation, capillary budding, and the synthesis of extracellular matrix (ECM) to fill in the damaged tissue that has been cleared during inflammation. The matrix material is initially made up of fibrinogen and fibronectin [68]. Thereafter, proteoglycans, large macromolecules with a core protein and one or more covalently attached glycosaminoglycan molecules, are synthesized by cells to make up the ground substance of the ECM. ECM-producing cells that produce the support matrix necessary to regain structural integrity include fibroblasts in connective tissue, chondrocytes in cartilage, osteoblasts in bone, and Schwann cells in the PNS.

The body’s reparative response to injury differs between soft collagenous cutaneous tissue, bone, the PNS, and the CNS (Figure 1.1). While complete repair of an injured tissue is ideal, this response can only consistently occur in mineralized tissue or bone, as re-establishing complete structural integrity is critical for functional recovery. In unmineralized connective tissue, specifically skin wounds, the depth of tissue loss dictates the repair response. While the epidermis can regenerate, deep tissue wounds in which the dermis is lost undergo secondary healing that requires excess ECM production, leading to the formation of fibrous scar tissue. In the right conditions, regeneration can occur in the PNS as injured axons can regrow through a Schwann cell scaffold. In the CNS, lost axons are not replaced, and a glial scar is formed by reactive astrocytes acting as a physical and molecular barrier that inhibits axon regeneration.

1.4.1. Non-CNS Tissue

The first stage of tissue repair is stabilization of the discontinuity created by the injury. Traditionally, there are two broad classifications of healing. Tissue that has little to no gap separating the wound boundaries will undergo “primary healing” from the apposed edges of the tissue. Tissue that is unstable with a large gap or discontinuity injury will undergo “secondary healing,” where excess ECM is produced to secure and fill the lesion. The ECM of secondary healing, which subsequently becomes vascularized, is referred to as granulation tissue—a term arising from its appearance. In general, the amount of granulation tissue formed is proportional to the eventual level of scarring.

1.4.1.1. Primary Repair (No Extracellular matrix [ECM] Production)

1.4.1.1.1. Partial-Thickness Skin Repair

Skin is composed of two distinct layers, the epidermis and the dermis. In partial thickness epidermal wounds, only the epidermis is damaged, leaving the basement membrane intact along with hair follicles and sweat and sebaceous glands. Because only the epidermal surface needs to be replaced and epithelial progenitor cells remain intact below the wound, the synthesis and deposition of collagen is not required. To repair a surface lesion such as a “paper cut,” the site must only be reepithelialized by migration of keratinocytes from below the wound and at the wound edge (Figure 1.3B). The degree of reepithelialization depends on the amount of tissue loss and the depth and width of the wound.

Reepithelialization begins within 24 to 48 hours as uninjured keratinocytes detach from the basal lamina to bore through or underneath the fibrin clot, crawling into and across the wound—a process termed lamellopoidial crawling (for review see [69]). Migration starts as keratinocytes at the wound edge upregulate their production of matrix metalloproteinases (MMPs), releasing the cells from their tethers to the basal lamina [70]. Keratinocytes resting on the basal lamina migrate across the wound site at a rate of 1 to 2 mm/day [71], attaching to fibronectin and vitronectin contained within the clot by upregulating their expression of α5β1 and αVβ6 integrins [69,72]. While moving through the dense fibrin clot, keratinocytes dissolve the dense fibrin matrix through the upregulation of several proteases such as tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), activating the fibrinolytic enzyme plasmin [73].

Keratinocyte locomotion occurs through the contraction of actinomyosin filaments of the cytoskeleton [69]. Because of changes in integrin expression and actinomyosin filament assembly as keratinocytes attach to the clot’s provisional matrix, there is a delay of several hours before the migration of basal keratinocytes is observed [74]. Once migration begins, keratinocytes move one by one over the wound site until the wound is covered by a complete layer of keratinocytes [72]. After migration is complete, the cells behind the wound edge undergo a proliferative burst to replace keratinocytes lost resulting from injury while also forming additional keratinocyte layers over the basal layer [75]. It is believed that epidermal growth factor (EGF), keratinocyte growth factor (KGF), and transforming growth factor (TGF-α) may drive cell proliferation and wound closure [69]. Migration and proliferation continue until keratinocytes receive a stop signal, likely due to contact inhibition [28]. At this time, MMP expression is interrupted, and a new basement membrane is produced whereby new cell-matrix adhesions are established [28].

1.4.1.1.2. Stabilized Bone Repair

When motion at the fracture site is prevented and bone fractured ends are rigidly held in place immediately after injury, primary mineralized tissue repair occurs. In primary bone repair there is no need for ECM-rich structural support because the tissue is already stabilized. Two types of primary mineralized tissue healing can occur: gap repair, where there is a less than 0.5-mm breach separating stabilized bone fragments, and contact repair, where the fractured ends are held in direct apposition [76].

In gap repair, healing begins as blood vessels and loose connective tissue fill the wound. After 2 weeks pluripotent mesenchymal cells derived from the bone marrow arrive at the site of injury and differentiate into bone-producing cells called osteoblasts. Osteoblasts fill any gaps in the tissue by secreting layers of unmineralized bone matrix called osteoid at a rate of 1 to 2 μm per day, which becomes mineralized after approximately 10 days [77]. The new bone formed in this stage is perpendicular to the long axis of the bone (Figure 1.4A). As osteoid becomes mineralized, some osteoblasts remain embedded in the matrix and become entrapped as the tissue is mineralized. These osteoblasts become osteocytes and are responsible for mechanotransduction within the tissue. Osteoblasts are likely activated by proteins belonging to the TGF-β family such as bone morphogenic proteins (BMPs) [78]. The new bone fills the gap but does not completely unite the fracture ends. At this point, the interface between the new and original bone is the weakest link in the union [79]. This newly formed bone acts as a scaffold for future remodeling by osteoclasts and osteoblasts.

Figure 1.4

Primary versus secondary mineralized tissue repair. (A) When a small fracture gap occurs and the bone remains stabilized, osteoblasts deposit bone perpendicular to the bone’s long axis, filling the gap and serving as a scaffold for future remodeling. (more...)

Contact repair occurs when there is direct contact between bone ends. This allows organized lamellar bone to form directly across the fracture line. However, before new bone can be deposited to repair the wound, necrotic bone must be removed. Osteoclasts, large multinucleated cells that derive from hemopoietic progenitor cells in the bone marrow and are closely related to macrophages, tunnel across the fracture line and remove old bone and necrotic tissue through the release of proteases and lysosomal enzymes (Figure 1.4B). They attach to the tissue matrix through β3 integrins, creating a ruffled cell border between the cell and the bone surface [80,81]. Hydrogen ions are pumped toward the osteoclast membrane to create an acidic microenvironment to digest the bone’s mineral component. The bone’s organic matrix is degraded by lysosomal proteases and is then endocytosed by osteoclasts [82]. The rate of bone degradation by osteoclasts depends on the orientation of the tissue. Osteoclasts are capable of cutting bone at a speed of 20 to 40 μm per day parallel and 5 to 10 μm per day perpendicular to the bone’s long axis [83]. Capillaries form within these “cutting cones,” providing nutrients to the tissue being repaired [84]. Behind the cutting cone, rows of osteoblasts line the resorptive channel, depositing layers of osteoid. This region is referred to as the “closing cone,” where osteoblasts attempt to replace approximately as much bone as had been removed [83] (Figure 1.4B). Layers of osteoid are initially made up of collagen types I and IV, gradually becoming mineralized through the deposition of hydroxyapatite crystals to form lamellar bone. Bone formed by this method, in which there is no cartilage formation as an intermediate step, is called intramembranous ossification.

1.4.1.1.3. Peripheral Nervous System (PNS) Repair

A nerve is made up of a collection of several neurons and their supporting cells called Schwann cells (Figure 1.5). Schwann cells act as supporting cells, surrounding the signaling processes of neurons, called axons, and isolating them from the ECM by producing myelin, which increases signal propagation velocity. Each axon and its Schwann cells are encircled by a connective tissue matrix called the endoneurium, subsequently grouped into bundles, and bounded by another thin connective tissue matrix called the perineurium. A number of these bundles along with vasculature make up a nerve with an outer sheath, termed epineurium [85,86].

Figure 1.5

Cross-sectional anatomy of the peripheral nerve. Inset at left shows an unmyelinated fiber. Inset at bottom shows a myelinated fiber. (©2000 American Academy of Orthopaedic Surgeons. Reprinted from J. Am. Acad. Orthop. Surg., 8(4), 243, 2000. (more...)

The possibility of repair in the PNS depends on the extent of injury. The most severe injury capable of repair is classified as a third-degree injury, where the axon and the endoneurium are damaged or severed while the perineurium and epineurium remain intact [87]. In more traumatic injuries where there is disruption of not only axons but the entire epineurium or perineurium, the blood–nerve barrier is ruptured, requiring extensive fibrous ECM to physically repair the damaged tissue. This dense ECM prevents regeneration, as the cut axon is incapable of finding its innervated target [87].

No matter the extent of injury, if repair in the PNS is to be successful, the cell body of the neuron must first survive the injury to its extending axon [39]. The neuron cell body is required for metabolism in the neuron’s axonal processes; therefore, any extending axon that is severed from its body quickly degenerates downstream from where the injury occurred. Within minutes to hours after injury to a neuron and its surrounding environment, degeneration begins as Schwann cells surrounding the axon stop making myelin proteins. Within the axon, enzymatic proteases are activated in response to a calcium influx that causes axon fragmentation and causes the surrounding myelin to form droplet-shaped particulate, a process termed Wallerian degeneration [39]. By 3 to 4 days, impulse conduction is no longer possible, as the extending axon is destroyed. Axonal and myelin debris is cleared during the later inflammatory stages and during the beginning of the repair phase by the influx of macrophages over a matter of weeks, in contrast to that of the CNS, which proceeds over several months [61].

In injuries where the neuron survives and the greatest extent of injury to the extending nerve is crushing or severing of the axon and the endoneurium, repair begins as Schwann cells proximal to the injury proliferate (Figure 1.3C). Schwann cells help remove the degenerated axonal and myelin debris and fill the area previously occupied by the extending axon and the myelin sheath (Figure 1.3C) [88]. The proliferating Schwann cells form interconnected cellular tubes that act as conduits for axonal regeneration [42]. Within the conduit, Schwann cells increase their production of growth factors, such as NGF and brain-derived growth factor (BDGF), while synthesizing ECM proteins such as laminin and fibronectin [39]. Axons begin to regenerate within 3 to 24 hours, sprouting multiple new small-diameter axons at the site of injury called the “growth cone,” and attempt to grow through the Schwann cell conduit (Figure 1.3C) [39]. The regenerating axonal sprouts move across the lesion 0.5 to 5.0 mm per day, stimulated by contact and chemotactic guidance provided by Schwann cells [89,90]. Each axonal sprout contains filopodia that bind through β1 integrins to ECM molecules such as laminin and fibronectin that are produced by Schwann cells [91]. The newly regenerated axons are initially unmyelinated but are mitogenic, inducing adjacent Schwann cells to proliferate further and produce myelin around the axon as it regenerates [39]. Axonal sprouts continue to regrow in the attempt to reinnervate the tissue that was deinnervated by injury-induced axonal degeneration.

1.4.1.2. Secondary Repair (ECM Production)

1.4.1.2.2. Full-Thickness Cutaneous Tissue Repair

If the basement membrane is damaged in a full thickness skin wound and substantial dermis is lost, the wound cannot heal by reepithelialization alone. ECM-producing dermal fibroblasts adjacent to the wound site are activated and proliferate, migrating into the wound hematoma within 3 to 4 days [69].

In secondary repair, during approximately the same time period that keratinocytes attempt to re-epithelialize the wound (as previously illustrated in primary skin repair), fibroblasts migrate through the provisional matrix by contraction of their actinomyosin cytoskeleton. Migrating fibroblasts initially synthesize fibronectin [92], but in response to TGF-β1 released by macrophages, fibroblasts switch to a more fibrotoic phenotype, synthesizing collagen matrix that begins to provide structural support for the wound [93]. Keratinocytes utilize this newly produced ECM to migrate over and epithelialize the site of injury (Figure 1.3D). ECM acts also as a conduit where new capillaries are formed as endothelial cells migrate into the wound site, responding to growth factors such as fibroblast growth factor-2 (FGF-2) and VEGF [94]. Neovascularization delivers nutrients to the migrating fibroblasts at the site of injury, giving the replacement tissue a pink, granular appearance—hence the name granulation tissue. New blood vessels are formed as there is a shift in the balance between the relative amounts of molecules that induce versus the molecules that inhibit vascularization. Proangiogenic growth factors such as VEGF are secreted predominantly by keratinocytes on the wound edge while FGF-2 and TNF- α are released from damaged endothelial cells and macrophages [69]. Hyaluronan, an oligosaccharide component of the ECM, also promotes angiogenesis and aids in repair [95]. Revascularization of the wound site is critical, as angiogenic failure can result in chronic wounds such as venous ulcers that are unable to heal.

1.4.1.2.2. Bone Repair

For bone repair, secondary healing is more commonly seen than primary fracture healing bone because most bones are not rigidly supported after injury [96]. When injury results in the disruption of the bone’s external vascular covering (periosteum) as well as the surrounding soft tissue, the damaged tissue is usually left unsupported. Unstabilized mineralized tissue will undergo secondary repair where the first step is creating an ECM-rich bridge to support the fracture (Figure 1.4C).

Most compact bone surfaces that make up the outermost layer of mineralized tissue are covered with a lining of osteoblasts that become active and produce a small amount of intramembranous ossification as early as 24 hours after injury along either side of the fracture (Figure 1.4C) [96]. However, this limited early bone formation provides little stability [96]. The wound site does not begin to regain mechanical strength until 3 to 4 days after injury, when fibroblasts and undifferentiated mesenchymal cells arrive at the periosteum via the circulation [97]. In secondary healing osteoprogenitors arriving from the vasculature differentiate into chondrocytes, whereas in primary healing they differentiate into osteoblasts. Chondrocytes and fibroblasts team to produce collagenous and fibrous tissue that forms around the outside of the fracture at the periosteum and internally within the marrow to provide an internal splint (Figure 1.4C) [98]. This collagen-rich tissue is called soft callus and can be classified as a type of granulation tissue that is critical for providing vascularity and structural support to the fracture. Maximum callus is usually observed a week after injury (Figure 1.6, day 7) [99]. In general, the amount of soft callus formation is dependent on the relative stability of the fracture fragments. The greater the motion at a fracture site, the more callus is required to prevent this motion [100]. Growth factors such as PDGF, TGF-β, and FGF released from platelets immediately after injury are possible initiators of callus formation [100].

Figure 1.6

Histological analysis of secondary fracture healing in bone showing the progression of repair on days 1, 3, 14, 21, and 28. Fractured bone appears denser than the surrounding tissue. On day 7, extensive soft callus is seen forming around the injured bone. (more...)

At 2 weeks, the collagenous soft callus is gradually mineralized to form hard callus, increasing the stability of the fracture site (Figure 1.6, day 14) [99]. Collagen mineralization to form woven bone is different from osteoid mineralization to form lamellar bone. In collagen mineralization, chondrocytes stop their production of collagen, elongate, and release proteases [101]. Glycosaminoglycans within the collagen matrix inhibit mineralization and must be removed by chondrocyte proteoglycanases for mineralization to occur [101]. As the cartilage matrix is degraded, chondrocytes differentiate and secrete angiogenic factors such as VEGF to induce capillary ingrowth from adjacent tissue [102]. Osteoblasts lining the bone surface then secrete collagen-free organic matrixes such as osteonectin and osteopontin, providing nucleation sites for the initiation of nanocrystaline calcium phosphate mineralization. Bone formed by this method, where collagen is mineralized to form bone, is termed endochondral ossification.

By the third week, the majority of the cartilage has become bone and union is achieved (Figure 1.6, day 21) [99]. At this point, the healing bone is generally able to support loads. However, even after stabilization, the newly formed bone is still weaker than normal uninjured bone. Only after the remodeling phase in which woven bone becomes remodeled to more compact laminar bone, does the tissue achieve full strength [98].

1.4.2. CNS Repair (Gliosis)

The repair of a wound in the CNS is not followed by neuronal regeneration. Unlike the response seen in the PNS, where degenerated axons can regenerate, damaged axons of the CNS initially sprout, but regeneration is impeded as the growth cones collapse within a day. CNS tissue repair begins hours after injury as astrocytes outside the lesion core and the surrounding area are activated. This marked glial response, commonly called gliosis, is made up of a multilayered sheet of activated astrocytes that form a boundary around areas of tissue damage (Figure 1.3A). Like the response seen in unmineralized tissue repair, astrocytes alter their integrin expression, migrating toward the lesion while also secreting MMPs for ECM degradation. MMP expression by astrocytes and neurons 1 to 2 weeks after injury can also promote repair by stimulating VEGF production to initiate angiogenesis [103]. Once astrocytes arrive at the site of injury, their processes encircle the lesion and become tightly intertwined to give the glial barrier a highly disordered appearance, forming what is referred to as the glial scar. The overall magnitude of glial activation roughly correlates with the amount of blood–brain barrier disruption and tissue damage [104].

Inside the lesion and surrounded by reactive astrocytes, microglia and macrophages persist in the attempt to remove potential pathogens and digest the fibrin clot [105] (Figure 1.3A). Because of the robust inflammatory response produced by microglia and macrophages, there are generally no neurofilaments at the wound site 3 days after injury [48,105]. Without neuronal viability, the CNS loses its functionality at the site of injury. From a structural standpoint, while smaller lesions in the CNS can be filled by reactive astrocytes [48], glial hypertrophy and proliferation cannot compensate for larger amounts of tissue loss. These large wounds remain cavities with reactive astrocytes forming a dense barrier around the lesion. Since the lesion is not filled with cells or ECM, surgeons can visualize past traumatic brain injuries during imaging because of missing tissue architecture (Figure 1.7A) [106]. In cases when the outer meningeal surface of the brain is penetrated, the lesion is filled with fibrosis tissue as meningeal fibroblasts lining the outside of the brain are able to migrate into the lesion [9,105,107]. Meningeal fibroblasts, like those in connective tissue repair, are capable of producing collagen (types I, III, IV) and ECM proteins (laminin, fibronectin) to fill the wound site [108,109]. Other nonglial cells such as vascular endothelial cells and mesenchymal cells are present in the glial scar. Endothelial cells attempt to form new blood vessels, whereas mesenchymal cells deposit basal lamina, which is known to inhibit axon regrowth [60,110].

Figure 1.7

Wound remodeling of different tissue types. (A) At the site of injury, the fibrin clot and necrotic tissue is removed by microglia and macrophages. Unlike nonneural tissue, lost tissue is not replaced, leaving a lesion with a cerebral spinal fluid– (more...)

1.4.2.1. Purpose of the Glial Scar

Spinal cord injury studies where astrocytes have been inactivated have led to a better understanding of the purpose of the glial scar. Selective removal of reactive astrocytes shows no glial scar formation at 2 weeks after stab injuries [109]. However, astrocyte removal resulted in the loss of injury containment as inflammatory cells spilled into the tissues surrounding the initial wound, increasing neuronal degeneration next to the injury [109]. The glial scar is believed to protect neuronal function following injury by repairing the blood–brain barrier and to subsequently limit inflammatory response to cells of the CNS [60,104].

Unlike the PNS, axons within the CNS are unable to regenerate. Within the glial scar environment two main groups of inhibitory molecules impede axonal regeneration: those associated with the glial scar and those associated with myelin [111]. Inside the glial scar, chondroitin sulfate proteoglycans (CSPGs) are upregulated by astrocytes, oligodendrocyte precursors, and meningeal fibroblasts. CPSGs are made up of a core protein to which a variable number of repeating disaccharide chondroitin sulfate chains attach [111]. CSPGs are secreted by reactive astrocytes within 24 hours after injury and can continue for months thereafter [104,112,113]. Proteoglycan expression is highest in the center of the lesion and diminishes outward [114].

Myelin within the CNS also contains growth inhibitory ligands that are released locally following axonal trauma. These inhibitory molecules are Nogo, myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp), Semaphorin 4D, and myelin associated CSPGs (for review see [111]). Both the glial scar and myelin-associated molecules prevent regrowth by repelling or collapsing growth cones though the Rho GTPase signaling pathway, blocking microtubule assembly [115].

The major difference between the PNS and CNS is that while the supporting Schwann cells and astrocytes both proliferate and activate after injury, Schwann cells of the PNS undergo changes to provide a supportive environment for regeneration, while astrocytes of the CNS undergo changes to produce inhibitory molecules that prevent neuronal regeneration from occurring. This has been demonstrated, as CNS axons are capable of regenerating through peripheral nerve grafts implanted into wounds outside of the CNS [116]. It has been proposed that the glial scar not only protects CNS tissue adjacent to injury from further damage but also prevents neurons from reforming inappropriate neuronal connections after injury [60,113,117]. Glial scarring has additionally been linked to the clearance of glutamate and the production antiinflammatory cytokines [110].

1.4.2.2. Glial Scar Induction

Cytokines such as IL-1, TGF-α, TGF-β, TNF-α, INF-α, and INF-γ have all been shown to be upregulated in scar tissue after injury, promote astrocyte proliferation in vitro, and augment glial scarring in vivo (for recent review see [104]) [110]. TGF-β1 and TGF-β2 expression has been shown to increase immediately after injury in the brain and spinal cord. TGF-β2 increased both proteoglycan production [118,119] as well as glial scarring by astrocytes [120]. INF-γ and FGF2 have also been shown to increase the extent of glial scarring and increase astrocyte proliferation in culture [104].

1.5. REMODELING (WEEKS TO MONTHS)

The ultimate endpoint following remodeling depends on the tissue type. In non-CNS tissue that undergoes primary healing, very little remodeling occurs because of the lack of ECM produced during repair. Secondary healing, in contrast, involves fiber alignment and contraction to reduce the wound size and to reestablish tissue strength. Complete recovery of original tissue strength is rarely obtained in secondary healing because repaired tissue remains less organized than noninjured tissue, which results in scar formation. Collagen-rich scars are characterized morphologically by a lack of specific organization of cellular and matrix elements that comprise the surrounding uninjured tissue. In CNS tissue where there is no repair or regeneration of injured neurons, there is also relatively little reestablishment of structural integrity in the region. Instead, during CNS remodeling, the glial scar around the lesion becomes denser as astrocytic processes become more intertwined and more or less isolates but does not repair the injured region.

1.5.1. Non-CNS Remodeling

1.5.1.1. Primary Remodeling

1.5.1.1.1. Partial-Thickness Cutaneous Tissue Remodeling

In superficial injuries, wounds can heal by epithelialization alone, with little or no additional ECM required to fill in the tissue. Because these primary tissue injuries can heal by keratinocyte regeneration and minimal ECM production, very little additional remodeling is required (Figure 1.7B). As a result, there is no scarring, and the repaired tissue is virtually indistinguishable from uninjured tissue.

In the clinical setting, when there is little contamination or necrotic debris, physicians use suturing to bring dermal edges in direct apposition. Upon careful alignment and elimination of tension, the epidermal and dermal layers can heal primarily by epithelialization within the epidermis and with limited ECM production in the dermis, leading to limited scarring [121]. Over a period of weeks to months, the wound gradually increases its tensile strength as the ECM is remodeled. During this process the injured tissue’s ECM becomes reoriented with respect to tensile force. Remodeling is more critical in secondary healing and hence will be expanded upon further in the section discussing full-thickness unmineralized tissue wounds.

1.5.1.1.2. Stabilized Bone Remodeling

Mineralized tissue remodeling is an active and dynamic process. Bone is unique in that remodeling occurs throughout the life of the tissue as mechanical stress induces bone to reorient itself and produce new bone to better handle the demands that are placed on it. According to Wolff’s law, the tissue adapts to the environment by becoming oriented along lines of maximum stress [112].

Remodeling after primary fracture repair where bone is stabilized is similar to the remodeling response that occurs over the life of the tissue, lasting up to several years before full preinjury strength is restored [76]. In primary gap healing, remodeling is important for restoring tissue strength. However, in primary contact healing, remodeling is coupled to the repair process. During contact remodeling, the cutting cones mature, depositing lamellar bone centripetally to form ring-shaped structures with a center blood vessel-containing canal (Figure 1.8B).

Figure 1.8

Primary versus secondary bone remodeling. (A) Lamellar bone deposited perpendicular to the bone’s long axis during primary repair is used as a scaffold for cutting cones. Osteoclasts create tunnels through which new blood vessels follow, stimulating (more...)

In primary gap remodeling, lamellar bone deposited perpendicular to the long axis within the injury during repair is used as a scaffold (Figure 1.8A). This process is commonly referred to as Haversian remodeling, where bone is remodeled in small packets of cells called basic multicellular units (BMUs) [123]. This is the same process that occurs during primary contact repair where osteoclasts form cutting cones allowing the influx of endothelial cells that form capillaries (Figure 1.4B). The budding capillaries bring in pluripotent mesenchymal cells that differentiate into osteoblasts after attaching to the surface of the internal cutting cone. Osteoblasts are activated and synthesize new lamellar bone concentrically, gradually closing the diameter of the tunnel (Figure 1.8A). After about four weeks, bone production stops as the tunnel is closed, leaving behind a vascularized cavity called an osteon that runs parallel to the long axis [96]. It is believed that the greater number of osteons that cross the site of injury, the greater the ultimate strength [79].

1.5.1.1.3. PNS Remodeling

In the PNS, once regenerating axons find their target, the tissue matures as axons gradually increase in thickness through neurofilament synthesis (Figure 1.7C). The axonal diameter and myelin sheath thickness of regenerated neurons are usually thinner and never reach normal preinjury levels [39]. Daughter axons that do not make contact with the target are cleaved off. During regeneration, a parent axon sprouts an average of 3 daughter axons, although up to 25 daughter axons have been observed [39]. Greater amounts of neural death generally lead to greater sprouting since there is less competition for access to the target [39]. The PNS is capable of maintaining a regenerative response at least 12 months after injury [124]. Schwann cell scaffolds that remain uninnervated slowly shrink in diameter, and if they do not receive a regenerating axon, they lose supporting ability as they are progressively filled with fibrous tissue [124].

Axonal repair and remodeling depends on the severity of the trauma. Most injuries in the PNS are stretch related, where the tensile force applied to the tissue exceeds the nerve’s ability to stretch. Generally in these injuries, the outer connective tissue layer of the nerve, the endoneurium, is intact, and the axon is able to regenerate as illustrated previously (Figure 1.3C) [124]. However, in more traumatic injuries such as lacerations, the endoneurium and the axon are severed and the tissue usually fails to regenerate. After traumatic injuries the fibroblasts contained within the endoneurium proliferate and produce a collagenous scar around the nerve trunk during the repair and remodeling phases. This collagenous scar misdirects or blocks axonal regeneration [124]. Sprouting axons that cannot find their target or contact with a Schwann cell conduit are either cleaved or grow into a disorganized mass, resulting in the formation of a neuroma. Meanwhile, the target muscle remains inactive, and neural cell bodies atrophy and eventually die. Even when the endoneurium is not severed, regeneration of the target site fails more often than not, and even in the best case, regeneration of a peripheral nerve does not fully restore the tissue back to its original status since there is inevitably inaccuracy during the attempted return of an axon to its original target [88].

Surgical insertion of an autologous nerve graft can be used to repair PNS lesions that are too large to be bridged by Schwann cells. The purpose of the graft is to reconnect damaged nerves end-to-end without causing tension. Suturing is used to connect the connective tissue sheath of the damaged nerve to the autologous graft. Freshly injured tissue sheaths do not hold sutures very well, so surgical repair is generally not performed until three weeks after injury, when the sheaths have had time to thicken. The major drawback of this method of repair is that harvest of the autologous nerve entails sacrificing one or more nerves. A number of groups are working to engineer synthetic nerve grafts for PNS repair [86,125].

1.5.1.2. Secondary Remodeling

1.5.1.2.1. Full-Thickness Cutaneous Tissue Remodeling

The major goal of secondary wound remodeling is to reduce the amount of excess ECM and align the ECM through contraction. If the extent of the wound is relatively small and the reorganization of the ECM is efficient, relatively little contracture occurs, and little or no scarring is observed. However, in larger injuries where there is more extensive tissue repair through ECM production, significant remodeling is required, which results in scarring. Remodeling occurs over a long time period. This phase can overlap with the repair phase, as it can begin as early as 1 week after injury and can last as long as 2 years, depending on the wound.

During repair, fibroblasts migrate into the site of injury and produce ECM to replace lost tissue. The tractional forces that fibroblasts create as they move through the wounded tissue generate mechanical tension, which promotes wound closure [126]. The remodeling phase officially begins when TGF-β and other cytokines released from platelets and activated macrophages cause fibroblasts to differentiate into a more contractile phenotype called myofibroblasts. Myofibroblasts are characterized by their expression of alpha smooth muscle actin and production of collagen I [28,127]. Once activated, they increase their cytoskeletal stress fibers and focal adhesions, providing constant tension to contract the wound bed (for review see [127]). Myofibroblast contraction is similar to that of smooth muscle cells, though the mode of activation is vastly different. Smooth muscle cells contract because of elevations of Ca2+. Since Ca2+ levels around cells can change rapidly, the contractility of smooth-muscle cells can change quickly [128]. Myofibroblasts are believed to be regulated by the Rho–Rho kinase pathway, which is less transient, and causes a longer-lasting contraction force. Contractures of 10 to 20 μm per day are possible without the need to generate large amounts of force because of the low basal tension level of skin [127]. Myofibroblasts continue to support loads in contracted tissue until ECM is produced and crosslinked, leading to stress shielding [127]. Collagen fibers gradually thicken and, along with myofibroblasts, they become oriented parallel to the wound bed along lines of stress, resulting in the appearance of striated scar tissue (Figure 1.7D). This is in direct contrast to the basket weave pattern seen in uninjured skin [129].

Once wound contraction occurs, stress relaxation causes myofibroblasts to return to a quiescent state. The cells then receive a signal to undergo apoptosis, transforming the wound from cell-rich granulation tissue to cell-poor scar tissue with an excess of ECM [130]. At the same time, the capillary density gradually diminishes and the wound loses its pink color, becoming progressively paler [27]. The ultimate end point of the remodeling process is the formation of acellular scar tissue that is poorly reorganized into dense parallel bundles, as opposed to the tightly woven meshwork of normal dermal tissue (Figure 1.7D) [72]. Dermal structures such as hair follicles, sweat glands, and sebaceous glands that are lost during injury are not regenerated [69]. After three months unmineralized tissue can have a maximum of 80% of the strength of unwounded tissue [68]. This is generally the highest level of strength that a healed tissue can achieve.

1.5.1.2.2. Unstabilized Bone Remodeling

The ultimate goal of secondary remodeling of mineralized tissue is full structural restoration with little to no scarring (Figure 1.5E). While remodeling can last up to 6 months, the endpoint is healed tissue that is remarkably similar to noninjured tissue in terms of robustness but may appear slightly less organized [78]. The only major difference between primary and secondary mineralized tissue remodeling is the extensive bone removal required to remove the excess callus produced during bone repair.

In secondary healing, osteoclasts arrive at the site in need of remodeling via the circulation. They recognize and attach to cell adhesion proteins such as osteopontin, osteocalcin, and osteonectin [83]. Osteoclasts are present not only to remove excess bone not needed for structural support, but also to digest woven bone synthesized from the soft callus during repair so it can be replaced with lamellar bone aligned in response to stress. Like primary remodeling, secondary remodeling occurs mainly through cutting cones. Within the cortex, woven and necrotic lamella bone is removed by osteoclasts. Osteoblasts follow closely and produce new compact lamellar bone, giving the tissue greater strength (Figure 1.8C). Each concentric 100-μm-thick lamellar layer is directed in a specific manner to buttress fracture fragments [96]. Secondary remodeling is altered from primary remodeling because of the large external callus formed during repair. The external callus is formed outside the cortex and is generally remodeled by osteoclasts without the formation of cutting cones and osteons. This is possible because osteoclasts have immediate access to woven bone of the external callus via the periosteum (Figure 1.8C).

Much of the bone removed during remodeling of the cortex is replaced by lamellar bone. During remodeling, mechanical loads applied to mineralized tissue are capable of generating signals at the cellular level to increase bone production (for review see [80]). As the most abundant cell in bone, osteocytes are enclosed within the bone matrix and are capable of communicating with neighboring cells through their network of processes connected by gap junctions. Mechanical stress within mineralized tissue causes fluid shear on osteocytes and causes an influx of extracellular calcium ions as well as ATP release leading to ion channel activation [80]. This in turn may stimulate lamellar bone synthesis by activating specific prostaglandins and increase nitric oxide production [80]. Osteoblast activity has been shown to increase in the presence of certain prostaglandins [131], while nitric oxide is known to inhibit bone resorption by osteoclasts [132].

1.5.2. CNS Remodeling

Remodeling in the CNS is limited. Because of the need to protect the CNS from the body’s robust inflammatory responses, reactive astrocytic processes become further intertwined, forming a dense sheath around the wound site (Figure 1.7A). During the repair stages, GFAP reactivity is seen approximately 400 μm outside the lesion. During the remodeling stages, GFAP reactivity is only observed less than 100 μm from the site of tissue damage [133]. Glial scar density increases as hypertrophic astrocytes become more condensed around the site of damage (Figure 1.9) [11].

Figure 1.9

Time course of glial scar formation at four time points as imaged by GFAP staining. At 2- and 4-week time points, the astrocytic processes fall back into the void left by the probe extraction before tissue processing. By 6 weeks, the processes have interwoven (more...)

Inside the lesion, removal of the fibrin clot and the necrotic neurons and supporting glial cells is completed by microglia and macrophages (Figure 1.7A) [105]. Once complete, microglia and macrophages undergo apoptosis, leaving a cerebral spinal fluid–filled cyst in the center of the lesion where the initial wound occurred [105]. The cyst is bordered by a thin, dense layer of reactive astrocytes that serve as a barrier between healthy and lost tissue and may help protect neurons outside the injury. The axons of neurons protected outside the glial scar cannot regrow into lost tissue because of inhibitory molecules within the scar, and thus tissue function is never regained (Figure 1.7A).

1.6. CONCLUSION

The cellular reaction after injury depends on the tissue type as well as the extent of the wound. In injury to CNS tissue that damages neurons and the supporting glial cells, the body’s response is unforgiving, as regeneration of lost neurons is not possible. Activated astrocytes wall off the lesion, creating a glial scar. These activated astrocytes may prevent further tissue damage, although neuron axonal regrowth is inhibited. In contrast, in non-CNS tissue, a single tissue type can have multiple responses depending on the magnitude of injury. For example, a superficial skin wound often has lower levels of inflammatory infiltrate and can undergo primary healing, while a deeper wound with more extensive tissue damage and cellular loss will undergo secondary healing. This chapter focused on the primary healing response seen after injuries in skin, bone, and the PNS where the general tissue architecture is maintained after injury, allowing the tissues to undergo repair predominantly by the regeneration of lost cells to restore the tissue’s normal structure. In secondary healing, the wounds are often deeper and the general tissue structure is compromised. The inflammatory response may be more intense and prolonged as well, recruiting fibroblasts and endothelial cells that produce granulation tissue. Within granulation tissue, the deposition of collagen by fibroblasts changes the structure and function of the tissue and eventually leads to scar formation upon the completion of the remodeling stage. Secondary healing in bone is the exception, as bone tissue is capable of removing the initial fibrous callus and replacing it with lamellar bone. Other examples of scarring after secondary healing occur in tissues that are not capable of undergoing regeneration such as the heart.

In summary, while the overall outcome of CNS and non-CNS tissue wound healing has been described, for therapeutic purposes in the CNS, it is important to consider the different junctures within the wound healing process at which the response can be modified to push the body’s wound healing response toward a desired outcome. These points of intervention are mainly within the stages hemostasis, inflammation, and repair. Within each stage there are points that may be useful for modulating the wound healing response. For instance, to limit extensive clot formation, it has been suggested that there are three main stages within clot formation for which therapeutics can be developed: initiation (platelet activation), propagation (coagulation cascade), and fibrin formation (thrombin) [134]. During inflammation, modulation strategies center around limiting immune cell activation and reducing inflammatory cell migration into the wound [67]. Since neurons within the CNS are especially sensitive to inflammatory damage [48], and because chronic inflammation is involved in many other diseases outside the CNS, there has been a great deal of effort to address mechanisms through which inflammation is dampened, with the hope of achieving a balance between infection prevention and resolution of inflammation [67]. Finally, to alter the CNS repair process for improving CNS function after injury, researchers have attempted to reduce astrocyte activity [135] and stimulate neural regeneration [111].

CNS wound healing interventions focusing on altering the glial scar and the inflammatory processes should be approached with caution. A decrease in glial scaring could result in unnecessary tissue damage due to the inability to reestablish normal homeostasis and repair of the blood–brain barrier [109,136]. One of the major concerns regarding antiinflammatory approaches is loss of defense against infection, although methods so far have not shown substantial increases in the susceptibility to infection, suggesting that it is difficult to completely shut off the inflammatory process because of redundancies in the pathway [67].

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What are the phases of soft tissue healing?

What are the stages of tissue regeneration?

Which is Phase 2 of tissue healing?

What is the three phase healing cycle in a soft tissue or wound injury?