HISTOLOGY OF WOUND HEALING

Skin histology
Keratinocytes make up 95% of the epidermis, and the stratum basale is the source of all
replicating keratinocytes. It is these keratinocytes in the basallayer that are primarily responsible for the
epidermal response in wound healing.(126) As keratino cytesreplicate they push older cells towardthe
surface, and these cells progressively lose their nucleus and take on a more flattened ovoidshape. This
stratified squamous keratinized epithelium undergoes constant turnover, essentially regenerating fully
every 48 days. The stratum basales ends down finger-like projections that interdigitate with similar
structures reaching up fromthe dermis. This process forms the rete ridges that are often seen in cross
section. Freshly healing wounds as well as skin grafts lack these reteridges initially, which makes them
susceptible toshear trauma early on.The epidermis also contains essential appendages including hair
follicles with associated sebaceousglands (pilosebaceous unit), eccrine sweatglands, and apocrine glands.
The pilosebaceousun it, as well as rete ridges, contains epithelialstem cells that are able to regenerate and
differentiate into basal keratinocytes and are essential to the reepithelialization process. These stem cells
are critical, as they are relatively undifferentiated,have a large proliferation potential,and have a high
capacity for self-renewal(127). Healing difficulties are seen when these stem cells are destroyed by insults
such as burns and even iatrogenicsources such as dermabrasion. When dermabrasion or resurfacing is
taken too deep it can destroy the apocrine glands and pilosebaceousunit, leading to improper healing and
eventual scarring. Retinoid treatments such as isotretinoin(Accutane) cause atrophy of the
sebaceousglands, which is the source of their effectivenessto treat acne. Isotretinoin leads to obvious
problems with wound healing, directly reducing the cells needed for reepithelialization. Most surgeons
would agree that patients should be off isotretinoin for at least 1 year prior to any surgical resurfacing
procedure(128).The dermis is the next layer down, which receives the major blood supply for the skin and
contains most of the dermal appendages of the skin including the apocrine glands, eccrine glands,and hair
follicles. This layer itself is separated intoa superficial or papillary dermis and the deeper reticular dermis.
As a general rule, any damage that extends into this deeper reticular layer willinvariably cause scarring
and may require repairwith full-thickness grafts or flaps to assure properhealing.
FIG 18: Histologic section of the epidermis showing the strata from superficial to deep: corneum,
lucidum,granulosum, spinosum, and basale

HEMOSTASIS

Hemostasis is the initial phase that occurs with in seconds to minutes after the initial insult. Initially
hemorrhage into the wound exposes platelets to the thrombogenic subendothelium. Platelets are
integral to this phase and the entire healing pathway, as they not only serve to provide initial hemostasis
but also release multiple cytokines,hormones, and chemokines to set off the remaining phases of healing.
Vasoactive substancessuch as catecholamines and serotonin act viaspecialized receptors on the
endothelium to cause vasoconstriction of the surrounding blood vessels.Smaller vessels are signaled to
vasodilate to allow the influx of leukocytes, red blood cells,and plasma proteins. Platelets interact with
the GpIIb-IIIa receptor on the collagen of the damaged subendothelium to become activated and form
aninitial clot. Activated platelets release their granules and ignite the extrinsic and intrinsic coagulation
cascades. Fibrin polymerization helps forma mature clot and serves as scaffolding for the infiltratingcells
(leukocytes, keratinocytes, and fibroblasts)that are key to subsequent phases ofhealing. Platelets release a
myriad of chemokinesand cytokines to attract these inflammatory cellsto the area. Within minutes, there
is an influx of inflammatory cells (predominantly neutrophils and macrophages), leading to the next phase
of healing.

INFLAMMATION

The inflammatory phase is heralded by the influx of neutrophils, macrophages, and lymphocytes to the
site of injury. Neutrophils are the first leukocytes on site, arriving en masse within the first 24 hours.
Neutrophils are soon followed by macrophages, which are attracted by the byproductsof neutrophil
apoptosis. Phagocytic cells such as macrophages and other lymphocytes appear in the wound to begin to
clear debris and bacteria from the wound. These macrophages
infiltrate at approximately 48 hours post injury and stay until the conclusion of the inflammatory phase.
Macrophages have long been thought tobe the key cell to the wound-healing process,and they seem to
orchestrate the most important phases of healing. Although they are integral to proper healing, recent
research has also looked at their role in improper healing and scarring. Studies of macrophage function
have revealed that these key cells are intricate in reepithelialization,
granulation tissue formation, angiogenesis, wound cytokine production, and wound contracture.
Inflammation is a necessary step of the healingprocess, and inhibition of this key phase (via anti-
(129)

inflammatory medications) can result in improper healing. This phase of healing is important to combat
infection.(130) If disrupted or prolonged (ie,longer than 3 weeks), this inflammation can leadto a chronic
wound, impaired healing, and eventually more scarring. Important factors that can
abnormally lengthen this phase of healing include high bacterial load (greater than 105
microorganismsper gram of tissue), repeated trauma, and persistent foreign material in the wound. (126)
Once the wound has been debrided, the repair or proliferative phase begins.
FIG 19:The inflammatory phase heralded by the influxof neutrophils and macrophages into the wound

PROLIFERATION

The proliferative (repair) phase begins early on in the form of reepithelialization. The repair phase also
involves capillary budding and extracellular matrix production to fill in the defects leftbehind from
debridement of the wound. Epithelialization is marked by the proliferation and influx of keratinocytes
near the leading edge of the wound.As discussed previously, stem cells within the bulbs of the hair
follicles and apocrine glandsbegin to differentiate into keratinocytes and repopulatethe stratum basale,
and also begin to migrate over the edge of the wound. Once they encounter the mesenchyme of the
extracellular matrix (ECM), they attach near the inner wound edge and begin to lay down a new base
mentmembrane. Following this, another row of keratinocytes migrates over the newly laid epithelialcells
to fill in the defect. These cells migrate and digest the ECM using proteases until they are in physical
contact and they stop migrating, signaledby contact inhibition from neighboring keratinocytes. (131) This re
epithelialization protects the wound from infection and desiccation. During this processa layer of
uninfected exudates lies over the wound, which provides an important moisture layer and contains growth
factors essential to healing. Any improper wound dressings that destroy this healthy layer will result in
delayed healing. Underneath this reepithelialization process the ECM continues to be laid down. Wounds
healing by secondary intention fill in with granulation tissue. Under the influence of vascular endothelial
growth factor (VEGF), angiogenesis begins as the vessels begin to bud from the blood vessels
surrounding the wound. Granulation tissue consists of these fibroblasts, new budding vessels,and
immature collagen (collagen type III). Some fibroblasts will also begin to differentiate in this phase into
myofibroblasts that have contractile function to bring gaping wound edges together. (131)

FIG 20:Reepithelialization takes place as keratinocytesdifferentiate from the stem cells in the basal
stratum
and migrate over the wound edge to fill in the defect.Migration stops, signaled by contact inhibition as
thewound defect fills in.

ANGIOGENESIS

Angiogenesis follows a typical pattern of sprouting,looping, and pruning signaled by a complex gradient
of cytokines. These small and delicate sprouting vessels repopulate the dermis, and any trauma to this
area will lead to destruction of these vessels and delayed healing. Shearing of these new vessels is often a
problem in skin grafting, where by the graft is solely supplied by initial imbibitionfor 24 hours followed
by growth of these vessels into the tissue. It is the role of the bolster placed over these delicate graft sites
to prevent hematoma that would limit diffusion of nutrients,but more importantly to prevent shearing of
these delicate immature vessels. Bolsters are usually lefton for 5 to 7 days to allow these vessels to
become more robust and mature, and to resist these shearing forces. Angiogenesis is also a key process
affected by primary versus secondary closures. Angiogenesis is greatly accelerated by primary closure,
due to the proximity of the budding vessels. In healing through secondary intention, this process takes
place through formation of the afore mentioned granulation tissue induced by hypoxia, elevated lactate,
and various growth factors. Healing by secondary intention involves epithelialization over this
granulation and then extensive remodeling. Any medications that interfere with new blood vessel
formation (ie, the antiangiogenic drug bevacizumab [Avastin]) can be detrimental to wound healing.

REMODELING PHASE

The remodeling phase begins as the provisional ECM and type III collagen is replaced with type I
collagen and the remaining cell types of the previous phases undergo apoptosis. Withthe laying down of
type I collagen, the tensile strength of the wound dramatically increases.Granulation tissue begins to
involute and excess blood vessels retract. This phase lasts the longe stand results in the final appearance
of the wound following healing. A successful remodeling phase involves a delicate balance requiring
synthesis more than lysis. Synthesis is greatly energy dependent,and any depletion of nutrients will push
thebalance toward lysis and affect the healing process. Excess fibrosis at this stage results in hypertrophic
scarring (with the scar limited to thewound area) or keloid formation (with the scar extending beyond
wound edge).

FIG 21:The maturation phase is longest of the 4 phasesand results in the final appearance of the wound