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Pressure Ulcer Tissue Histology: An Appraisal of Current Knowledge
A pressure ulcer is a wound caused by unrelieved pressure that damages underlying tissue.1 Risk factors for pressure ulcer development include reduced activity, poor nutrition, immobility, incontinence, low body weight, spinal cord injury, lengthy surgery, and stroke.2,3 Once a pressure ulcer is present, the microstructural changes initiated with pressure in the tissue are difficult to ascertain and assessment is limited to the surrounding tissue. Both in vitro and in vivo studies have been conducted to examine the changes present in tissue in response to pressure and with a pressure ulcer present. In vivo animal studies have allowed researchers to document the changes that occur in tissue as a response to pressure. In vivo studies in humans have primarily evaluated the tissue surrounding the pressure ulcer, the adjacent normal tissue, and in some cases, the tissue layers in the wound.
Difficult ethical issues need to be considered when conducting studies that may require punch biopsies from the intact skin of subjects with possible ulcers developing or from subjects with healing ulcers. As a result, few histological studies evaluating the effects of pressure on tissue in vivo have been performed in humans. The purpose of this literature review is to appraise the histology of pressure ulcer tissue. 
In Vitro Studies
In the author’s laboratory, in vitro studies utilized a bench-scale loading system to apply either static or cyclic pressures to tissue in a symmetrical fashion (see Figure 1). The system was designed to simulate the loading situation at the human heel using simplified geometry; study details have been described elsewhere.4 Agar was used to simulate the compliant tissue above the bone that was simulated by a hard spherical section (watch glass). External pressure was applied to tissue mounted over the agar layer using another identical watch glass. Stained tissue was histologically evaluated in different pressure conditions and compared to control tissue. In sections stained with hematoxylin and eosin, nuclei stained blue, cartilage and calcium deposits stained dark blue, cytoplasm and other constituents stained red shades, and blood stained bright red. Masson’s trichrome stained nuclei black; cytoplasm, keratin, and muscle fibers stained red; collagen and muscin stained blue. Verhoeff’s stain stained elastic fibers blue-black to black, nuclei stained blue to black, and collagen stained red. Other tissue elements stained yellow. Figure 2a-d show surface irregularities, abundant vascular component, and the multidirectional fiber array present in the tissue before pressure application. 
Tissue subjected to 50 mm Hg for 4 hours maintained multidirectional fiber bundles and capillaries of varied caliber (see Figure 3a,b). After being subjected to 170 mm Hg for 4 hours, the tissue was markedly different. The surface was flattened and keratin was fragmented. Most importantly, the fiber bundles were parallel to the surface and no longer exhibited the multidirectional appearance present in the control tissue (Figures 4 a,b). The pressures used for these trials were based on prior experiments using human subjects resting on support surfaces.
Although these are findings from healthy neonatal tissue in an in vitro model, the significant changes in the alignment of fiber bundles in the dermis may have serious implications with regard to tissue’s ability to sustain mechanical loads (pressure). In fact, tissue subjected to pressure before uniaxial tensile testing was found to be less stiff than control tissue.5
Changes observed occurred after 4 hours of pressure. If the tissue was subjected to pressure for longer periods of time, as would be the case in a clinical situation, the changes already seen may be critical in pressure ulcer formation.
In Vivo and Human Studies
Stage I pressure ulcers. As defined by the National Pressure Ulcer Advisory Panel (NPUAP),6 a Stage I pressure ulcer is intact skin with non-blanchable redness of a localized area usually over a bony prominence. The area may be painful, firm, soft, and warmer or cooler as compared to adjacent tissue. In darkly pigmented skin, visible blanching may not be present, but color may differ from the surrounding area.
Witkowski and Parish7 evaluated samples from 59 patients (age range 24 to 99 years) obtained from a variety of locations and from different stage pressure ulcers. Tissue samples from Stage I pressure ulcers had both diffuse and focal eosinophilia, as well as superficial crust and erosions. Subepidermal separation, necrosis of the epidermis, and subepidermal blisters and bulla were present in some sections. Some sections also had degeneration of follicular structures, as well as hemorrhage into the follicles in the dermis (see Figure 5a-e). The histology of the samples of tissue from Stage I pressure ulcers indicates changes in both the epidermis and dermis of the tissue.
Stage II pressure ulcers. Stage II pressure ulcers are defined as partial-thickness loss of dermis presenting as a shallow open ulcer with a red pink wound bed, without slough. The ulcer also may present as an intact or open/ruptured serum-filled blister.6 Arao et al8 evaluated tissue from a Stage II pressure ulcer from the sacrum of an 87-year-old woman post mortem. The tissue was collected from the interior of the ulcer (damaged), the edge of the ulcer (border), and healthy tissues. The tissue was evaluated using light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The latter two evaluative tools utilize electron beams to image the specimen: TEM passes the beam through the specimen, highly magnifying the image created by the transmitted beam, and SEM highly magnifies the surface of a specimen. Comparing damaged tissue, border, and healthy tissue revealed profound changes in the morphology of the dermal papillae present in this stage ulcer. This study clearly elucidates the transition in tissue from healthy to damaged regions in this ulcer and is unique in the literature. 
The variations in the dermal papillae seen between normal and damaged tissue are evident (see Figure 6). Changes in the collagen fibers are present at the surface of the dermis; in the damaged tissue, the collagen fibers are densely packed and visibly different from healthy tissue sections. Scanning electron micrographs looking down on the dermal papillae of healthy, border, and damaged tissue clearly show changes in the number and shape of the papillae. In healthy tissue, the papillae appear as regularly spaced, similarly sized appendages. In the border region, the number of the papillae decreases and the shape becomes irregular. In the damaged tissue, the difference is profound – no papillae are present (see Figure 7). At greater magnification, the finger-like projections appear to be missing their tops in some border area sections and are atrophic in others. Again, the damaged areas have no papillae present (see Figures 8a,b).
The reticular fibrils covering the papillary layer appear as a wide network in Figure 9a in healthy tissue but in Figure 9b this network appears to be more densely packed in border areas, similar to the changes seen in the tissue of Stage I pressure ulcers in Witkowski’s study.7 In the damaged tissue, this network is missing (see Figures 9b and 10). Witkowski and Parish7 also evaluated tissue from Stage II pressure ulcers and noted necrotic changes in appendages, as well as inflammatory cells in the dermis and fat in the tissue samples. Both the necrotic changes and fat were seen in all specimens (see Figure 11).
The changes seen in the dermal papillae and collagen fibers at the surface of the dermis are noteworthy. Damage to the dermal papillae in the border tissue and the disappearance of these structures in damaged tissue would disrupt or destroy the potential for viable epidermis. Hagisawa et al9 have suggested that the network of collagen and elastin fibers in the papillary and reticular layers may play a significant role in preventing pressures from extending from the surface of the skin to deeper tissues.
Stage IV pressure ulcers. The NPUAP defines Stage IV pressure ulcers as full-thickness tissue loss with exposed bone, tendon, or muscle. Slough or eschar may be present on some parts of the wound bed and the wounds often include undermining and tunneling. Undermining and sinus tracts also may be associated with Stage IV pressure ulcers.6
The author’s laboratory evaluated the microstructural changes in surgically debrided tissue from sites adjacent to Stage IV pressure ulcers and normal tissue.10 The tissue samples of the normal (control) group were obtained from the leg and breast area of a different patient group than the pressure ulcer tissue samples. The normal tissue exhibited a multidirectional fiber array similar to that seen in the in vitro studies with human foreskin tissue control samples see (Figure 12).4,10 Tissue was evaluated for number of fibers, fiber direction, fiber width, and wavy or straight fibers. The number of straight and wavy fibers was significantly greater in the control than in the pressure ulcer tissue. Additionally, the pressure ulcer tissue had longer and wider straight and wavy fibers compared to the control tissue (see Figure 13). Also of interest, fat was present in the dermal tissue. Van de Berg and Randolph11 evaluated tissue adjacent to pressure ulcers obtained from 20 patients and noted dense collagen and fat deposits present in the samples, as well as a decrease in vascularity (see Figure 14). Thicker fiber bundles, similar to the author’s and Aroa et al’s8 findings (see Figure 15), also were observed.
The changes seen in the fiber bundles of these tissues are impressive with respect to the tissue’s mechanical properties. The mechanical (load-bearing) properties of skin are a characteristic of the collagen and elastin fibers. Thus, changes in the tissue microstructure are assumed to result in change in the tissue’s mechanical properties. In fact, when tissue from pressure ulcers was tested in tension, mechanical properties were found to be altered substantially.10 The changes in the tissue structure impact the tissue’s response to load.
In Vivo Animal Studies
In vivo animal studies provide an important window into the changes occurring in tissue as a result of the pressure applied. The results from human in vivo studies have been limited by the fact that samples were collected only after ulcers were identified. The histological and microstructural characterization is of tissue already damaged or bordering damaged tissue. Studies performed by Kosiak12,13 on dogs and rats have shown how quickly load leads to changes in the tissue. Muscular necrosis, hyaline degeneration, and venous thrombosis were seen in tissue samples from the trochanteric region of dogs subjected to greater than 60 mm Hg for >1 hour12 (note: 60 mm Hg is not considered an extreme load clinically but it is greater than the arbitrary 32 mm Hg, often targeted as the highest acceptable pressure). In rat tissue subjected to pressures >115 mm Hg for >3 hours, more than 10% of the muscle fibers had signs of inflammation.13
Husain14 found significant changes (muscular necrosis, subcutaneous edema, and fiber degeneration) in the tissue of rats subjected to pressure of 100 mm Hg for 2 hours. In tissue subjected to the same load for 6 hours, compressed muscle with a larger number of inflammatory cells was noted. The Kosiak and Husain studies are noteworthy because of the degree and duration of the load on the tissue, which caused damage that extended into the muscle. These results illuminate how great the changes in human tissue may be as a result of modest pressure on the tissue.
Salcido et al15 subjected rats to 145.3 mm Hg for 6 hours. The 6-hour treatments were repeated on successive days for up to 5 days. After one treatment, hyperemia was noted; after two treatments, necrosis in the deep gluteal muscles and panniculus carnosus muscle was noted. Interestingly, 3 days after the fifth daily treatment, the site could be located by noting the microstructural changes apparent in the necrotic panniculus carnosus muscle, as well as the occurrence of epidermal thickening and loss of adipose tissue. Of the sites receiving five pressure sessions, 10% did not have ulceration of the skin and 23% had redness (Stage I). The others had yellow discoloration with a red border and were classified as Stage II pressure ulcers. Dermal ulcerations were rare; only 6% of “loaded” sites had ulceration, with most of the large changes occurring in deeper layers of the tissue. These findings are of particular interest because the findings in Witkowski and Parish’s study7 evaluating tissue from Stage I pressure ulcers in humans did not extend beyond the subcutaneous fat.
Stekelenburg et al16 used two protocols to apply either uniaxial or ischemic loading to rats. The tibialis anterior region of the rats was imaged using contrast-enhanced MRI. The changes seen in the tissue as a response to the ischemic (inflatable tourniquet 140kPa) were reversible; whereas, the compressive loading (indentation of 4.5 mm) for 2 hours led to irreversible muscle tissue damage. The damage included necrotic regions of the muscle compressed and disorganization of the necrotic fibers.
Applying the Research
Researchers face the difficult challenge of trying to determine how the changes seen in human in vivo studies, which examine tissue post pressure ulcer formation, compare to the results seen immediately following pressure application in animal models. The changes seen in the tissue of the animals in the studies reveal the effects of pressure on tissue. What is still unknown is human tissue’s tolerance for pressure. Tissue changes were observed as a result of loading in animals, but such changes in response to the load also may be the beginning of adaptive remodeling. Animal models differ because both dogs and rats have fur and loose skin with minimal subcutaneous tissue, which may create pressure distribution different from that seen in humans.
Also, in humans the studies published evaluate the tissue present at the site of pressure ulcers. However, as new studies evaluating tissue changes in patients at risk for developing pressure ulcers are published, the understanding of these visible changes as a precursor to ulcer development versus remodeling in response to load may become clearer. Studies that utilize ultrasound, MRI, or other imaging techniques to evaluate visible changes in tissue that may serve as precursors to ulcer formation are critical to understanding ulcer formation.
Conclusion
Pressure ulcers are classified in stages defined by the visible layers of tissue damaged from the surface toward the bone. The literature clearly demonstrates that bottom-to-top pathogenesis is occurring. In many cases, the changes visible at the surface of the tissue are minor compared to the damage seen at the deepest layers of tissue. Certainly, a number of factors are involved in the development of pressure ulcers, including the type of load (eg, presence of shear) and changes in the biochemistry of the tissue surrounding the load as a result of reperfusion or tissue damage. It is apparent from the in vitro and in vivo studies reviewed that further studies evaluating the changes occurring in deeper layers of the tissue in humans in response to loading are necessary to better understand the effects of pressure on tissue and the development of pressure ulcers. Additionally, understanding the response of the tissue to pressure as either the precursor to ulcer development or as an adaptation to load must be improved.
Future research utilizing imaging techniques to identify areas of risk of pressure ulcer development and the earliest microstructural precursors to pressure ulcers is needed. The identification of deep tissue injuries provides an additional opportunity to study quickly evolving tissue changes in response to severe loading conditions. Each individual’s response to loading on tissue has a biochemical component; as the biochemistry of these loading situations is better understood, the link between load and tissue tolerance will be further elucidated.
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