Immediately after injury, the coagulation products fibrin, fibrinopeptides, fibrin split products, and complement components begin to attract inflammatory cells — particularly macrophages — into the wound. Platelets activated by thrombin release IGF-1, TGF, TGF, and PDGF, then attract leukocytes and fibroblasts into the wound. Damaged endothelial cells respond to a signal cascade involving complement product C5a, TNF, IL-1, and IL-8 and express receptors for integrin molecules on the cell membranes of leukocytes. This allows circulating leukocytes to adhere to the endothelium and then migrate into the wounded tissue. Their interleukins and other inflammatory components, such as histamine, serotonin, and bradykinin, cause vessels first to constrict in aid of hemostasis, and later dilate, becoming so porous that blood plasma and leukocytes can move freely into the area.
The newly arrived inflammatory cells increase metabolic demand. Since the local microvasculature has been damaged, a local energy sink results, and PaO2 falls while CO2 accumulates. Lactate in particular plays a critical role, but its source is mainly aerobic, from activated and oxygenated levels.
These conditions persist throughout repair, and together with other stimulants such as fibrin, foreign bodies, bacteria — they direct leukocytes, particularly macrophages, to release a variety of cytokines, chemoattractants, and growth factors. New information leads to a picture of wound healing as a reaction to oxidative stress. These events trigger reparative processes and ensure their continuation, because macrophages, which secrete most of the growth-promoting products, assume the control of repair as the flood of coagulation-mediated growth substances begins to ebb. Furthermore, macrophages, stimulated by fibrin, release large quantities of lactate. Though hypoxia may contribute, this process continues even in the presence of oxygen, thereby maintaining the environment of injury. The environment causes them to release growth promoters and more lactate. Lactate acts as a “surrogate” of hypoxia, and by itself stimulates angiogenesis and collagen deposition through instigation of growth factors.
By the third or fourth day after injury, the reparative cells become arranged in a characteristic spatial relationship. Wound cells proceed in orderly single file — leaders first, followers next. Unless the wound becomes infected, its granulocyte population, which dominated during the first days, diminishes. Macrophages now cover the cut surface. Immature fibroblasts, the product of growth signals, lie just beneath, mixed with buds of new vessels. More mature fibroblasts are scattered behind. The spatial relations of cells with respect to oxygen and lactate concentrations define zones of reparative activities. Recently, it has become apparent that stem cells contribute fibroblasts, but the extent of this contribution is unknown.
These spatial relationships show how macrophages condition an acidic, hypoxic, high-lactate growth-promoting environment that acts as a “growth center” synthesizing and depositing new collagen in an area still rich in lactate but better oxygenated as is required for collagen synthesis and deposition.
The fibroblasts secrete the collagen, proteoglycans of the connective tissue matrix that weld wound edges together. They assume high-molecular-weight polymeric forms and become the physical basis of wound strength.
Collagen synthesis is not a constitutive property of fibroblasts but must be stimulated. The mechanisms that regulate the stimulation and synthesis of collagen are not actively clear, except that they are multifactorial and include both growth factors and metabolic inputs such as lactate. The collagen gene promoter has binding sites to corticoids, TGF, and retinoids, which control collagen gene expression, whereas other growth factors regulate glycosaminoglycans, tissue inhibitors of metalloproteinase (TIMP), and fibronectin synthesis. A more basic observation, however, is that mere accumulation of lactate in the extracellular environment directly stimulates transcription of collagen genes as well as posttranslational processing of collagen peptide. This mechanism, which is not pH-dependent, rests on the size of the intracellular pool of adenosine diphosphoribose (ADPR), a key regulatory substance that normally inhibits collagen mRNA synthesis and other vital steps that facilitate collagen export from fibroblasts. ADPR results from the nicotinamide moiety being removed from NAD+. Accumulation of lactate converts NAD+ to NADH. As a consequence, less NAD+ is available to be converted to ADPR. When ADPR in the nucleus is depleted for any reason — as it is when the NAD+ pool is diminished — this inhibitory control is released, and more collagen mRNA is formed. The cause of decreased NAD+ can be hypoxia, accumulation of lactate, excessive conversion of NAD+ to ADPR, and probably others.
The increase in collagen mRNA leads to an increased procollagen peptide. This, however, is not sufficient to increase collagen deposition because procollagen peptide cannot be transported from the cell to the extracellular space until, in a posttranslational step, some of its prolines are hydroxylated. In this reaction, performed by the dioxygenase prolyl hydroxylase, an oxygen atom derived from dissolved O2 is inserted (as a hydroxyl group) into selected collagen prolines in the presence of ascorbic acid, iron, and ketoglutarate. The activity of this enzyme is normally suppressed by cytoplasmic ADPR. Thus, accumulation of lactate — or any other process that decreases the NAD+ pool—leads to production of collagen mRNAs, increased collagen peptide synthesis, and (provided enough ascorbate and oxygen is present) increased posttranslational modification and secretion of collagen monomers into the extracellular space.
Another dioxygenase, lysyl hydroxylase, hydroxylates many of the procollagen lysines and sets the stage for later lysyl to lysyl link between collagen molecules and fibers, thereby endowing them with considerable strength. It, too, requires adequate amounts of ascorbate and oxygen. These oxygenase reactions (and therefore collagen deposition) proceed only as fast as the local concentration of oxygen (PaO2) will allow. The rates are half-maximal at about 20 mm Hg and maximal at about 200 mm Hg. They can be “forced” to supernormal rates by tissue hyperoxia. Collagen deposition, wound strength, and angiogenesis rates can vary clinically as much as threefold as tissue PO2 is elevated.
Angiogenesis is necessary for all but the most minor wound healing. It becomes visible about 4 days after injury but begins 2 or 3 days earlier when new capillaries sprout out of preexisting venules and grow toward the injury in response to chemoattractants released by platelets and macrophages. In primarily closed wounds, sprouting vessels soon meet and connect with counterparts migrating from the other side of the wound, thus establishing blood flow across the wound. In unclosed wounds or those not well closed, the new capillaries fuse only with neighbors migrating in the same direction, and granulation tissue is formed instead.
The regulation of wound angiogenesis is inducible in unwounded tissue by the addition of chemoattractants to endothelial cells. Examples found in wounds include PDGF (homodimer BB), FGF, TNF, TGF, and VEGF.
Maintenance of the NAD+ pool severely inhibits the process. Thus, either unmet metabolic needs or the appearance of them lead through a growth factor mechanism involving VEGF to an anatomic response probably of general biologic significance.
Numerous growth factors, cytokines are said to stimulate angiogenesis, but animal experiments indicate that the dominant angiogenic stimulants in wounds are derived first from platelets in response to coagulation and then from macrophages in response to hypoxia or high lactate, fibrin, etc. Given the aerobic origin of wound lactate, it alone can stimulate VEGF in the presence of oxygen. Added oxygen then supports collagen deposition.
Epithelial cells respond to many of the same stimuli as do fibroblasts and endothelial cells. A variety of growth factors regulate their replication. TGF, for instance, tends to keep epithelial cells from differentiating and thus may potentiate and perpetuate mitogenesis, though it is itself not a mitogen for these cells. TGF and KGF are epithelial cell mitogens.
Mitoses appear in epithelium a few cells away from the wound edge. The new cells migrate over the cells at the edge and into the unhealed area and anchor to the first unepithelialized place that they encounter. The PaO2 on the underside of the cell at the anchor point is likely to be low. Low PaO2 stimulates squamous epithelial cells to produce TGF, presumably hindering terminal differentiation and favoring mitosis. This process repeats itself until the wound is closed.
Squamous epithelialization and differentiation proceed maximally when the local PaO2 approaches about 700 mm Hg and when surface wounds are kept moist.
Contrary to classic thought, even short periods of drying can impair the process. Wounds should be kept moist. The exudate from acute, uninfected superficial wounds also contains growth factors and lactate and therefore recapitulates the growth environment found internally.
Collagen Fiber Maturation, Lysis, & Contraction
Replacement of an extracellular matrix is a complex process. First, fibroblasts replace the provisional fibrin extracellular matrix with collagen monomers. Extracellular enzymes, some of which are PaO2-dependent, quickly polymerize these monomers but in a pattern that is much more random than normal, thus leaving young wounds weak and brittle. This brittleness is overcome when the first, hastily placed, matrix is replaced with a more mature one that contours larger, better organized, stronger, and more durable fibers.
Turnover and reorganization of new matrix is an important feature of healing, and fibroblasts and leukocytes secrete collagenases that ensure the lytic component. Turnover occurs rapidly at first and then more slowly. Even in simple wounds, increased turnover can be detected chemically for as long as 18 months. Healing is successful when a net excess of matrix is deposited despite concomitant lysis. Lysis, being destructive, is less dependent upon energy and nutrition. If synthesis is impaired, lysis weakens wounds. In some tissues (eg, colon), lysis unnecessarily weakens tissue structure, and inhibition of lysis with orally administrable agents leads to quicker gain of tensile strength. This is one of many features of healing that has not yet been clinically exploited.
During rapid turnover, wounds gain strength and durability but also are vulnerable to contraction and stretching. Fibroblasts exert the force for contraction. Fibroblasts attach to collagen and each other and pull the collagen network together when the cell membranes shorten as the fibroblasts migrate. The fibers are then fixed in the packed positions by a variety of cross-linking mechanisms. Both open and closed wounds tend to contract if not subjected to a superior counterforce. The phenomenon is best seen in surface wounds, which may close 90% or more by contraction alone in loose skin. For instance, the residual of a large open wound on the back of the neck may be only a small area of epithelialization. On the back, the buttock, or the neck, this is often a beneficial process, whereas in the face and about joints, the results may be disabling or disfiguring. This undesirable result is usually termed a contracture or a stricture. Skin grafts, especially thick ones, impede but do not totally stop the process. Dynamic splints, passive or active stretching, or insertion of flaps containing dermis and subdermis may be needed to counteract contraction. The force can be quite strong, but even severely contracted joints can usually be straightened with countertraction. Prevention of a stricture in a ureteric repair, for example, depends on ensuring that the opposing tissue edges are well perfused so that healing can proceed quickly to completion and contraction can stop. The longer the wound is open, the larger role that contraction plays in its final closure.
Healing wounds may also stretch during active turnover when tension overcomes contraction. This may account for the laxity of scars in ligaments of injured but unsplinted joints and the tendency for hernia formation in abdominal wounds of obese patients. If wounds are traumatized when passively stretched, contraction or weakness may continue for long periods and may become troublesome.
Completion of Healing
Healing and growth of malignancies are strikingly similar processes. As opposed to their role in oncogenesis, however, growth factors in wound healing are more obedient to the basic controls, and healing stops at an appropriate point. Normally, the final stimuli to release of growth factors, cytokines seem to be local hypoxia and lactic acidosis. When these stimuli disappear as the new microcirculation matures, healing should stop. However, the reason for cessation of healing is debated.
Keloids, that is, local overgrowths of connective tissue, and hypertrophic scars, which occur particularly in pigmented skin, probably represent a loss of normal control over the healing process, but few facts are known. Hypertrophic scars are generally self-limited, are related to residual inflammation, and may regress after a year or so. The last areas of a burn to heal are the most often hypertrophic through traction, reinjury, and tension. Immune mechanisms may also contribute to scar. Prolonged inflammatory reactions potentiate scar. Therapy is another intralesional injection of anti-inflammatory steroids, dressing with thick layers of Silastic that raise the level temporarily and thereby increase the activity of lytic enzymes, or both.