Histology-guided protein digestion/extraction from formalin-fixed and paraffin-embedded pressure ulcer biopsies.

Herein we present a simple, reproducible and versatile approach for in situ protein digestion and identification on formalin-fixed and paraffin-embedded (FFPE) tissues. This adaptation is based on the use of an enzyme delivery platform (hydrogel discs) that can be positioned on the surface of a tissue section. By simultaneous deposition of multiple hydrogels over select regions of interest within the same tissue section, multiple peptide extracts can be obtained from discrete histological areas. After enzymatic digestion, the hydrogel extracts are submitted for LC-MS/MS analysis followed by database inquiry for protein identification. Further, imaging mass spectrometry (IMS) is used to reveal the spatial distribution of the identified peptides within a serial tissue section. Optimization was achieved using cutaneous tissue from surgically excised pressure ulcers that were subdivided into two prime regions of interest: the wound bed and the adjacent dermal area. The robust display of tryptic peptides within these spectral analyses of histologically defined tissue regions suggests that LC-MS/MS in combination with IMS can serve as useful exploratory tools.

Inhibition of Wnt/ß-catenin pathway promotes regenerative repair of cutaneous and cartilage injury.

Wound healing in mammals is a fibrotic process. The mechanisms driving fibrotic (as opposed to regenerative) repair are poorly understood. Herein we report that therapeutic Wnt inhibition with topical application of small-molecule Wnt inhibitors can reduce fibrosis and promote regenerative cutaneous wound repair. In the naturally stented model of ear punch injury, we found that Wnt/ß-catenin pathway is activated most notably in the dermis of the wound bed early (d 2) after injury and subsides to baseline levels by d10. Topical application of either of 2 mechanistically distinct small-molecule Wnt pathway inhibitors (a tankyrase inhibitor, XAV-939, and the U.S. Food and Drug Administration-approved casein kinase activator, pyrvinium) in C57Bl/6J mice resulted in significantly increased rates of wound closure (72.3 ± 14.7% with XAV-939; and 52.1 ± 20.9% with pyrvinium) compared with contralateral controls (38.1 ± 23.0 and 40.4.± 16.7%, respectively). Histologically, Wnt inhibition reduced fibrosis as measured by α-smooth muscle actin positive myofibroblasts and collagen type I α1 synthesis. Wnt inhibition also restored skin architecture including adnexal structures in ear wounds and dermal-epidermal junction with rete pegs in excisional wounds. Additionally, in ear punch injury Wnt inhibitor treatment enabled regeneration of auricular cartilage. Our study shows that pharmacologic Wnt inhibition holds therapeutic utility for regenerative repair of cutaneous wounds.

Imaging mass spectrometry for accessing molecular changes during burn wound healing.

The spatiotemporal analysis of the proteomic profile during human wound healing is a critical investigative step that can establish the complex interplay of molecular events that comprise the local response to burn injury. Partial-thickness wound samples with adjacent "normal" skin were collected from twenty-one patients with burn wounds and examined across a time spectrum ranging from the acute injury period at 3, 6, 11 days to the later hypertrophic scar period at 7 and 15 months. The techniques used for histology-directed tissue analyses highlighted inflammatory protein markers at the early time points after injury with diminished expression as burn wounds progressed into the proliferative phase. The datasets show the usefulness of MALDI MS and imaging mass spectrometry as discovery approaches to identify and map the cutaneous molecular sequence that is activated in response to the unique systemic inflammatory response following burn trauma. This information has the potential to define the unique factors that predispose human burn victims to disfiguring hypertrophic scar formation.

Wound samples: moving towards a standardised method of collection and analysis.

Chronic wounds, including diabetic foot ulcers, pressure ulcers and venous leg ulcers, impact the lives of millions of people worldwide. These types of wounds represent a significant physical, social and financial burden to both patients and health care systems. Wound care has made great progress in recent years as a result of the critical research performed in academic, clinical and industrial settings. However, there has been relatively little translation of basic research discoveries into novel and effective treatments. One underlying reason for this paucity may be inconsistency in the methods of wound analysis and sample collection, resulting in the inability of researchers to accurately characterise the healing process and compare results from different studies. This review examines the various types of analytical methods being used in wound research today with emphasis on sampling techniques, processing and storage, and the findings call forth the wound care research community to standardise its approach to wound analysis in order to yield more robust and comparable data sets.

Imaging mass spectrometry for assessing cutaneous wound healing: analysis of pressure ulcers.

Imaging mass spectrometry (IMS) was employed for the analysis of frozen skin biopsies to investigate the differences between stage IV pressure ulcers that remain stalled, stagnant, and unhealed versus those exhibiting clinical and histological signs of improvement. Our data reveal a rich diversity of proteins that are dynamically modulated, and we selectively highlight a family of calcium binding proteins (S-100 molecules) including calcyclin (S100-A6), calgranulins A (S100-A8) and B (S100-A9), and calgizzarin (S100-A11). IMS allowed us to target three discrete regions of interest: the wound bed, adjacent dermis, and hypertrophic epidermis. Plots derived using unsupervised principal component analysis of the global protein signatures within these three spatial niches indicate that these data from wound signatures have potential as a prognostic tool since they appear to delineate wounds that are favorably responding to therapeutic interventions versus those that remain stagnant or intractable in their healing status. Our discovery-based approach with IMS augments current knowledge of the molecular signatures within pressure ulcers while providing a rationale for a focused examination of the role of calcium modulators within the context of impaired wound healing.

Adjuvant neurotrophic factors in peripheral nerve repair with chondroitin sulfate proteoglycan-reduced acellular nerve allografts.

BACKGROUND: Acellular nerve allografts are now standard tools in peripheral nerve repair because of decreased donor site morbidity and operative time savings. Preparation of nerve allografts involves several steps of decellularization and modification of extracellular matrix to remove chondroitin sulfate proteoglycans (CSPGs), which have been shown to inhibit neurite outgrowth through a poorly understood mechanism involving RhoA and extracellular matrix-integrin interactions. Chondroitinase ABC (ChABC) is an enzyme that degrades CSPG molecules and has been shown to promote neurite outgrowth after injury of the central and peripheral nervous systems. Variable results after ChABC treatment make it difficult to predict the effects of this drug in human nerve allografts, especially in the presence of native extracellular signaling molecules. Several studies have shown cross-talk between neurotrophic factor and CSPG signaling pathways, but their interaction remains poorly understood. In this study, we examined the adjuvant effects of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth postinjury in CSPG-reduced substrates and acellular nerve allografts. MATERIALS AND METHODS: E12 chicken DRG explants were cultured in medium containing ChABC, ChABC + NGF, ChABC + GDNF, or control media. Explants were imaged at 3 d and neurite outgrowths measured. The rat sciatic nerve injury model involved a 1-cm sciatic nerve gap that was microsurgically repaired with ChABC-pretreated acellular nerve allografts. Before implantation, nerve allografts were incubated in NGF, GDNF, or sterile water. Nerve histology was evaluated at 5 d and 8 wk postinjury. RESULTS: The addition of GDNF in vitro produced significant increase in sensory neurite length at 3 d compared with ChABC alone (P < 0.01), whereas NGF was not significantly different from control. In vivo adjuvant NGF produced increases in total myelinated axon count (P < 0.005) and motor axon count (P < 0.01), whereas significantly reducing IB4+ nociceptor axon count (P < 0.01). There were no significant differences produced by in vivo adjuvant GDNF. CONCLUSIONS: This study provides initial evidence that CSPG-reduced nerve grafts may disinhibit the prosurvival effects of NGF in vivo, promoting motor axon outgrowth and reducing regeneration of specific nociceptive neurons. Our results support further investigation of adjuvant NGF therapy in CSPG-reduced acellular nerve grafts.

4.7-T diffusion tensor imaging of acute traumatic peripheral nerve injury.

Diagnosis and management of peripheral nerve injury is complicated by the inability to assess microstructural features of injured nerve fibers via clinical examination and electrophysiology. Diffusion tensor imaging (DTI) has been shown to accurately detect nerve injury and regeneration in crush models of peripheral nerve injury, but no prior studies have been conducted on nerve transection, a surgical emergency that can lead to permanent weakness or paralysis. Acute sciatic nerve injuries were performed microsurgically to produce multiple grades of nerve transection in rats that were harvested 1 hour after surgery. High-resolution diffusion tensor images from ex vivo sciatic nerves were obtained using diffusion-weighted spin-echo acquisitions at 4.7 T. Fractional anisotropy was significantly reduced at the injury sites of transected rats compared with sham rats. Additionally, minor eigenvalues and radial diffusivity were profoundly elevated at all injury sites and were negatively correlated to the degree of injury. Diffusion tensor tractography showed discontinuities at all injury sites and significantly reduced continuous tract counts. These findings demonstrate that high-resolution DTI is a promising tool for acute diagnosis and grading of traumatic peripheral nerve injuries.

Human acellular dermal wound matrix: evidence and experience.

A chronic wound fails to complete an orderly and timely reparative process and places patients at increased risk for wound complications that negatively impact quality of life and require greater health care expenditure. The role of extracellular matrix (ECM) is critical in normal and chronic wound repair. Not only is ECM the largest component of the dermal skin layer, but also ECM proteins provide structure and cell signalling that are necessary for successful tissue repair. Chronic wounds are characterised by their inflammatory and proteolytic environment, which degrades the ECM. Human acellular dermal matrices, which provide an ECM scaffold, therefore, are being used to treat chronic wounds. The ideal human acellular dermal wound matrix (HADWM) would support regenerative healing, providing a structure that could be repopulated by the body's cells. Experienced wound care investigators and clinicians discussed the function of ECM, the evidence related to a specific HADWM (Graftjacket(®) regenerative tissue matrix, Wright Medical Technology, Inc., licensed by KCI USA, Inc., San Antonio, TX), and their clinical experience with this scaffold. This article distills these discussions into an evidence-based and practical overview for treating chronic lower extremity wounds with this HADWM.

A physiological role for connective tissue growth factor in early wound healing.

Mesenchymal stem cells (MSCs) that overexpress secreted frizzled-related protein 2 (sFRP2) exhibit an enhanced reparative phenotype. The secretomes of sFRP2-overexpressing MSCs and vector control-MSCs were compared through liquid chromatography tandem mass spectrometry. Proteomic profiling revealed that connective tissue growth factor (CTGF; CCN2) was overrepresented in the conditioned media of sFRP2-overexpressing MSCs and MSC-derived CTGF could thus be an important paracrine effector. Subcutaneously implanted, MSC-loaded polyvinyl alcohol (PVA) sponges and stented excisional wounds were used as wound models to study the dynamics of CTGF expression. Granulation tissue generated within the sponges and full-thickness skin wounds showed transient upregulation of CTGF expression by MSCs and fibroblasts, implying a role for this molecule in early tissue repair. Although collagen and COL1A2 mRNA were not increased when recombinant CTGF was administered to sponges during the early phase (day 1-6) of tissue repair, prolonged administration (>15 days) of exogenous CTGF into PVA sponges resulted in fibroblast proliferation and increased deposition of collagen within the experimental granulation tissue. In support of its physiological role, CTGF immunoinhibition during early repair (days 0-7) reduced the quantity, organizational quality and vascularity of experimental granulation tissue in the sponge model. However, CTGF haploinsufficiency was not enough to reduce collagen deposition in excisional wounds. Similar to acute murine wound models, CTGF was transiently present in the early phase of human acute burn wound healing. Together, these results further support a physiological role for CTGF in wound repair and demonstrate that when CTGF expression is confined to early tissue repair, it serves a pro-reparative role. These data also further illustrate the potential of MSC-derived paracrine modulators to enhance tissue repair.

Blocking the P2X7 receptor improves outcomes after axonal fusion.

BACKGROUND: Activation of the P2X7 receptor on peripheral neurons causes the formation of pannexin pores, which allows the influx of calcium across the cell membrane. Polyethylene glycol (PEG) and methylene blue have previously been shown to delay Wallerian degeneration if applied during microsuture repair of the severed nerve. Our hypothesis is that by modulating calcium influx via the P2X7 receptor pathway, we could improve PEG-based axonal repair. The P2X7 receptor can be stimulated or inhibited using bz adenosine triphosphate (bzATP) or brilliant blue (FCF), respectively. METHODS: A single incision rat sciatic nerve injury model was used. The defect was repaired using a previously described PEG methylene blue fusion protocol. Experimental animals were treated with 100 µL of 100 µM FCF solution (n = 8) or 100 µL of a 30 µM bzATP solution (n = 6). Control animals received no FCF, bzATP, or PEG. Compound action potentials were recorded prior to transection (baseline), immediately after repair, and 21 d postoperatively. Animals underwent behavioral testing 3, 7, 14, and 21 d postoperatively. After sacrifice, nerves were fixed, sectioned, and immunostained to allow for counting of total axons. RESULTS: Rats treated with FCF showed an improvement compared with control at all time points (n = 8) (P = 0.047, 0.044, 0.014, and 0.0059, respectively). A statistical difference was also shown between FCF and bzATP at d 7 (P < 0.05), but not shown with d 3, 14, and 21 (P > 0.05). CONCLUSIONS: Blocking the P2X7 receptor improves functional outcomes after PEG-mediated axonal fusion.

Hydrophilic polymers enhance early functional outcomes after nerve autografting.

BACKGROUND: Approximately 12% of operations for traumatic neuropathy are for patients with segmental nerve loss, and less than 50% of these injuries obtain meaningful functional recovery. Polyethylene glycol (PEG) therapy has been shown to improve functional outcomes after nerve severance, and we hypothesized this therapy could also benefit nerve autografting. METHODS: We used a segmental rat sciatic nerve injury model in which we repaired a 0.5-cm defect with an autograft using microsurgery. We treated experimental animals with solutions containing methylene blue (MB) and PEG; control animals did not receive PEG. We recorded compound action potentials (CAPs) before nerve transection, after solution therapy, and at 72 h postoperatively. The animals underwent behavioral testing at 24 and 72 h postoperatively. After we euthanized the animals, we fixed the nerves, sectioned and immunostained them to allow for quantitative morphometric analysis. RESULTS: The introduction of hydrophilic polymers greatly improved morphological and functional recovery of rat sciatic axons at 1-3 d after nerve autografting. Polyethylene glycol therapy restored CAPs in all animals, and CAPs were still present 72 h postoperatively. No CAPS were detectable in control animals. Foot Fault asymmetry scores and sciatic functional index scores were significantly improved for PEG therapy group at all time points (P < 0.05 and P < 0.001; P < 0.001 and P < 0.01). Sensory and motor axon counts were increased distally in nerves treated with PEG compared with control (P = 0.019 and P = 0.003). CONCLUSIONS: Polyethylene glycol therapy improves early physiologic function, behavioral outcomes, and distal axonal density after nerve autografting.

The impact of reduction mammaplasty on breast sensation: an analysis of multiple surgical techniques.

Our prospective clinical trial collected sensory data using a computerized pressure-specified sensory device comparing 4 procedures for reduction mammaplasty. A total of 48 patients were assessed at baseline, 6 weeks (n = 42), 6 months (n = 15), and 1 year (n = 24) postoperatively. The findings of our study showed pressure sensitivity for women <43 years of age improved by pressure-specified sensory device assessment; whereas, outcome data merely indicated return to baseline in pressure sensitivity for women ≥ 43 years of age. Improved sensitivities for moving and static pressures were found in patients receiving vertical or inferior pedicle reduction mammaplasties. Reductions based on superior pedicles exhibited sensory loss as compared with baseline measurements while those receiving free nipple grafts showed negligible change. Moving and static sensation showed differential return after breast reduction irrespective of the specific surgical approach but sensation was uniquely conserved for the nipple. In the total cohort, the type of breast reduction procedure did not produce significant differences in breast sensation.

Spatial mapping by imaging mass spectrometry offers advancements for rapid definition of human skin proteomic signatures.

Investigations into the human skin proteome by classical analytical procedures have not addressed spatial molecular distributions in whole-skin biopsies. The aim of this study was to develop methods for the detection of protein signatures and their spatial disposition in human skin using advanced molecular imaging technology based on mass spectrometry technologies. This technology allows for the generation of protein images at specific molecular weight values without the use of antibody while maintaining tissue architecture. Two experimental approaches were employed: MALDI-MS profiling, where mass spectra were taken from discrete locations based on histology, and MALDI-IMS imaging, where complete molecular images were obtained at various MW values. In addition, proteins were identified by in situ tryptic digestion, sequence analysis of the fragment peptides and protein database searching. We have detected patterns of protein differences that exist between epidermis and dermis as well as subtle regional differences between the papillary and reticular dermis. Furthermore, we were able to detect proteins that are constitutive features of human skin as well as those associated with unique markers of individual variability.

Multiplexed molecular descriptors of pressure ulcers defined by imaging mass spectrometry.

The pathogenesis of impaired healing within pressure ulcers remains poorly characterized and rarely examined. We describe the results of a pilot study that applies matrix-assisted laser desorption/ionization imaging mass spectrometry technology for direct tissue analysis to evaluate proteomic signatures ranging from 2 to 20 kDa and phospholipids from 300-1,200 Da in focal regions within the wound microenvironment. Distinguishing molecular differences were apparent between upper vs. lower regions of ulcers and further contrasted against adjacent dermis and epidermal margins using protein profiles, ion density maps, principal component analysis and significant analysis of microarrays. Several proteins previously uncharacterized in pressure ulcers, the α-defensins (human neutrophil peptide [HNP]-1, -2, -3), are potential markers indicating whether the wound status is improving or being prolonged in a deleterious, chronic state. Thymosin ß4 appears to be a favorable protein marker showing higher relative levels in adjacent dermis and maturing areas of the wound bed. Lipidomic examination revealed the presence of major lipid classes: glycerophosphocholines, glycerophosphoglycerols, glycerophosphoinositols, and triacylglycerols. Our pilot data examined from either a global perspective using proteomic or lipidomic signatures or as individual distributions reveal that imaging mass spectrometry technology can be effectively used for discovery and spatial mapping of molecular disturbances within the microenvironment of chronic wounds.

Integrating Specialty-Specific Clinical Anatomy Education into the Post-Clerkship Curriculum.

Anatomy is a foundational science mainstay of undergraduate medical school education, particularly in the pre-clerkship curriculum. During the post-clerkship curriculum, students closer to graduate medical education may benefit from a focused concentration on human anatomy related to their specific clinical interests. Here, we describe a course for post-clerkship students that uniquely incorporates a multimodal approach of dissection, didactics, and clinical correlation to radiologic imaging, with the opportunity to personalize student learning on a specialty-specific anatomic region. The course increased students' confidence of anatomical knowledge and its clinical relevance. Other institutions may benefit from establishing a similar multimodal integrated post-clerkship anatomy curriculum.

Calreticulin enhances porcine wound repair by diverse biological effects.

Extracellular functions of the endoplasmic reticulum chaperone protein calreticulin (CRT) are emerging. Here we show novel roles for exogenous CRT in both cutaneous wound healing and diverse processes associated with repair. Compared with platelet-derived growth factor-BB-treated controls, topical application of CRT to porcine excisional wounds enhanced the rate of wound re-epithelialization. In both normal and steroid-impaired pigs, CRT increased granulation tissue formation. Immunohistochemical analyses of the wounds 5 and 10 days after injury revealed marked up-regulation of transforming growth factor-beta3 (a key regulator of wound healing), a threefold increase in macrophage influx, and an increase in the cellular proliferation of basal keratinocytes of the new epidermis and of cells of the neodermis. In vitro studies confirmed that CRT induced a greater than twofold increase in the cellular proliferation of primary human keratinocytes, fibroblasts, and microvascular endothelial cells (with 100 pg/ml, 100 ng/ml, and 1.0 pg/ml, respectively). Moreover, using a scratch plate assay, CRT maximally induced the cellular migration of keratinocytes and fibroblasts (with 10 pg/ml and 1 ng/ml, respectively). In addition, CRT induced concentration-dependent migration of keratinocytes, fibroblasts macrophages, and monocytes in chamber assays. These in vitro bioactivities provide mechanistic support for the positive biological effects of CRT observed on both the epidermis and dermis of wounds in vivo, underscoring a significant role for CRT in the repair of cutaneous wounds.

African Americans show increased risk for pressure ulcers: a retrospective analysis of acute care hospitals in America.

In an earlier study, we reported a significantly increased risk of pressure ulcer hospital discharge diagnoses in African Americans, higher age groups, and those with certain medical conditions. The objectives of the present study were to: (a) investigate the demographics associated with a higher odds ratio (OR) in African Americans and (b) determine whether African Americans have different rates of medical risk factors. The 2003 Nationwide Inpatient Sample database was queried. Patients with pressure ulcers were identified by discharge diagnoses using ICD-9 codes 707.0-707.09. Discharge diagnosis was examined using the agency for healthcare research and quality clinical classifications software (CCS). The present study used identified CCS discharge diagnoses present in at least 5% of all patients, with an OR>2. African Americans exhibited a higher incidence of an OR>2 for 28 identified CCS risk factors for pressure ulcers. The pressure ulcer diagnoses tended to occur at younger ages in African Americans. No significant differences were noted in African Americans with pressure ulcers when a subanalysis was conducted by zip code income quartile, region of the country, or teaching status of the hospital. Hospitalized African Americans exhibit an age-dependent, higher prevalence of pressure ulcers compared with Caucasians. Socioeconomic factors tracked within the Nationwide Inpatient Sample do not provide an explanation for this phenomenon.

Estrogen receptor beta expression in nevi during pregnancy.

Estrogen levels increase during pregnancy and clinical evidence has long suggested that melanocytes are estrogen-responsive. We hypothesized that nevi from pregnant patients would exhibit increased expression of estrogen receptor beta (ERbeta) and thus enhanced potential to respond to altered estrogen levels. Normal, dysplastic and congenital nevi (n = 212) were collected from pregnant and non-pregnant women ranging from 18 to 45 years of age. Immunohistochemical staining was performed on these nevi using antibodies specifically directed against estrogen receptor alpha (ERalpha) and ERbeta. ERalpha was not observed in any lesions; thus, ERbeta was the predominant estrogen receptor in melanocytic cells from all types of nevi. Enhanced positivity for ERbeta in normal nevi during pregnancy was noted, compared with non-pregnant controls including nevocytes residing in both the epidermal and dermal micro-environments (P = 0.005 and P = 0.001 respectively). Nevi with increasingly melanocytic atypia showed increased ERbeta in nevocytes nested within the epidermis. No additional increase in ERbeta in atypical nevi was observed during pregnancy. For normal and congenital nevi, regardless of pregnancy status, dermally associated nevocytes tended to have greater ERbeta immunoreactivity. Significant decreases in ERbeta immunoreactivity were observed in congenital nevi from pregnant women compared with normal and dysplastic nevi from pregnant women. Our data suggest that nevi possess the capacity to be estrogen-responsive. Factors such as pregnancy and degree of atypia are associated with enhanced ERbeta with the exception of congenital nevi where the melanocytes were unique in their response to pregnancy.

In-vivo optical imaging of hsp70 expression to assess collateral tissue damage associated with infrared laser ablation of skin.

Laser surgical ablation is achieved by selecting laser parameters that remove confined volumes of target tissue and cause minimal collateral damage. Previous studies have measured the effects of wavelength on ablation, but neglected to measure the cellular impact of ablation on cells outside the lethal zone. In this study, we use optical imaging in addition to conventional assessment techniques to evaluate lethal and sublethal collateral damage after ablative surgery with a free-electron laser (FEL). Heat shock protein (HSP) expression is used as a sensitive quantitative marker of sublethal damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and green fluorescent protein (GFP) expression (hsp70A1-L2G). To examine the wavelength dependence in the mid-IR, laser surgery is conducted on the hsp70A1-L2G mouse using wavelengths targeting water (OH stretch mode, 2.94 microm), protein (amide-II band, 6.45 microm), and both water and protein (amide-I band, 6.10 microm). For all wavelengths tested, the magnitude of hsp70 expression is dose-dependent and maximal 5 to 12 h after surgery. Tissues treated at 6.45 microm have approximately 4x higher hsp70 expression than 6.10 microm. Histology shows that under comparable fluences, tissue injury at the 2.94-microm wavelength was 2x and 3x deeper than 6.45 and 6.10 microm, respectively. The 6.10-microm wavelength generates the least amount of epidermal hyperplasia. Taken together, this data suggests that the 6.10-microm wavelength is a superior wavelength for laser ablation of skin.

Risk factors for pressure ulcers in acute care hospitals.

Selection of patients for preventive measures to protect against pressure ulcers relies on clinical scales and provider judgment, which vary widely. Our objectives were to: (a) identify risk factors by clinical classification and report demographic differences in pressure ulcer risk and (b) develop criteria for identification of high-risk patients. Patients with pressure ulcer as a discharge diagnosis were identified from the 2003 Nationwide Inpatient Sample (NIS). The effect of discharge diagnosis was examined using the Agency for Healthcare Research and Quality Clinical Classification Software (CCS). Multiple regression analysis for survey data was used to assess risk factors. The 2003 NIS listed 94,758 with a discharge diagnosis of pressure ulcer, identified as International Classification of Disease-9 code 707.0-707.09, for an overall incidence of 143 per 10,000. Forty-five CCS discharge diagnoses were present in at least 5% of these patients and 28 of these CCS diagnoses had odds ratios >2.0. African-American race and advanced age were identified as risk factors for pressure ulcer diagnosis. Disorders of skin integrity, organ system failure, and infection were found to be broad categories of risk factors as well. Using the NIS, risk factors for pressure ulcer including diagnoses and demographic factors have been identified.