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Notes: Background.  Although a series of vaccines against Helicobacter pylori have emerged in the past 10 years, the mechanism involved in their protective effect is yet to be elucidated, and more effective vaccine adjuvants remain to be developed. In this study, CpG-oligodeoxynucleotide (CpG-ODN) was investigated as a new candidate for a H. pylori vaccine adjuvant. Furthermore, the role of T helper 1 (Th1) type response and interferon (IFN)-γ in the protective immunity was explored.Methods.  C57BL/6 mice and IFN-γ knockout mice were intranasally or orally immunized with H. pylori whole cell sonicate (WCS)/CpG-ODN and challenged with different doses [5 × 108 and 5 × 106 colony-forming units (CFU)] of H. pylori. The protective effect was assessed as the percentage of noninfected mice. The responsive antibodies and cytokines were analyzed using an enzyme-linked immunosorbent assay (ELISA) and flow cytometry.Results.  The prevention rates against H. pylori infection in mice intranasally immunized with WCS plus CpG-ODN were dramatically higher than those in sham-immunized mice (70% vs. 0%, challenged with 5 × 108 CFU H. pylori; 90% vs. 20%, challenged with 5 × 106 CFU H. pylori). Significantly higher levels of immunoglobulin G2a (IgG2a) and IFN-γ were detected in the mice immunized with WCS/CpG than in sham-immunized controls. However, vaccination failed to effectively protect IFN-γ knockout mice challenged with H. pylori.Conclusions.  CpG-ODN given intranasally is a potent adjuvant for development of a H. pylori vaccine. Th1-type response and IFN-γ are involved in the protection.

Notes: The detection of Helicobacter species by genus-specific polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) was compared with that by species-specific PCR in murine intestinal samples. Results suggest that, in samples containing multiple Helicobacter species, genus-specific PCR-DGGE may fail to detect all Helicobacter species present and that this relates to the initial template DNA ratio.

Notes: The efficacy of established Helicobacter pylori regimes needs to be reviewed. In view of drug resistance, side effects, and compliance and expense of therapy, treatment failure is increasing and second-line treatment strategies need to be developed. A simulation model suggested by the Cochrane review group showed that H. pylori eradication is cost-effective for duodenal and gastric ulcer long-term. The duration of eradication therapy continues to be controversial. In Europe and other parts of the world, 7-day triple regimes are used, whereas guidelines from the United States recommend 10–14 days of therapy. Antibiotic resistance is a major factor affecting the outcome of eradication therapy. New modified eradication regimes involve substitution of antibiotics used in conjunction with other drugs. The newer generation fluoroquinolones have shown some promise as part of an eradication regimen. Quadruple therapy (bismuth, proton pump inhibitor [PPI] and two antibiotics and sequential treatment [PPI with three antibiotics]) are promising first-line treatments. Novel agents have been tried, but with disappointing results. New drugs and administration forms have been reported but their efficacy needs confirmation.

Notes: Pro-inflammatory cytokines such as IL-18, IFN-γ, and IL-1β play a significant role in the inflammation induced by Helicobacter. During the recent years of H. pylori research, the main focus has been on development of vaccines for therapeutic or prophylactic use against the infection. Both bacterial components of H. pylori as well as engineered vaccines have been tested as well as different forms of administration including systemic and oral/intranasal pathways.

Notes: A large number of studies on diagnostic tests have been published this year. New tests were proposed for the detection of Helicobacter pylori antigens in stools and new molecular methods (real-time polymerase chain reaction) to look for antimicrobial susceptibility. The other standard tests have been applied in different situations to improve the diagnosis of the infection.

Notes: Background.  In this study, we have aimed to show the possible relation between atrophic gastritis and premature atherosclerosis via hyperhomocysteinemia.Materials and Methods.  Thirty-four patients with atrophic gastritis were enrolled to the study. The control group consisted of 35 patients with non-atrophic gastritis. Classical cardiovascular disease risk factors did not significantly differ between atrophic gastritis and control subjects. The presence and degree of atrophic gastritis were assessed histologically and Helicobacter pylori infection was determined by both histologic and serologic methods. Body mass index was measured by standard technique blood fasting glucose, serum creatinine, total cholesterol, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, vitamin B12, folic acid, and homocysteine levels were measured by biochemical methods. Carotid intima-media thickness was measured by B-mode ultrasonography to examine the premature atherosclerosis.Results.  Plasma vitamin B12 levels were significantly lower (p = .00) and homocysteine levels were significantly higher (p = .01) in the atrophic gastritis group. There was no statistically significant difference in plasma folic acid levels between the two groups (p = .728). Carotid intima-media thickness was higher in the atrophic gastritis group than in the control group (0.516 mm versus 0.465 mm), but this difference did not show any statistical significance (p = .062).Conclusion.  Our results showed that atrophic gastritis may cause hyperhomocysteinemia, which is an independent risk factor for atherosclerosis and cardiovascular diseases. However, when compared with controls, carotid intima-media thickness of the atrophic gastritis patients was found to be higher but did not reach statistically significant levels.

Notes: Abstract:  Idiopathic granulomatous mastitis (IGM) is a rare benign inflammatory disease of the breast that mimics carcinoma of the breast. Its etiology and treatment remain unclear. A retrospective review of nine women with histopathologic diagnosis of IGM was performed. The women had a mean follow-up of 18.7 months and a mean age of 45.7 years (range 32–83 years). The main presentation was breast mass (100%). Clinically and radiologically, 55.6% of the women were suspected to have malignancy. One patient was treated with lumpectomy without recurrence. Eight patients were treated with expectant management with close regular surveillance. No surgery was performed and no medications were given. Fifty percent of the patients had spontaneous complete resolution of disease after a mean interval of 14.5 months. These four patients had no recurrence. Fifty percent of patients had static disease. In conclusion, it is important to differentiate IGM from carcinoma of the breast. Tissue biopsy remains the gold standard to confirm the diagnosis. Expectant management with close regular surveillance is the treatment of choice.

Notes: Abstract:  A prospective audit of 221 breast biopsies was carried out to assess the pain/discomfort experienced during image-guided breast biopsies. The only significant factor in pain scores was the size of the needle used. Fine-needle aspiration cytology using a 21-gauge needle was found to cause the most discomfort.

Notes: sAbstract:  Duct ectasia consists of dilation of the mammary ducts and is clinically manifested as nipple discharge, which is more commonly multiductal, bilateral, and colored. To identify clinical factors that might be related to duct ectasia. A case-control study was carried out on a population of 150 patients divided into two groups. Group 1 (the experimental group) comprised 100 patients with multiductal, bilateral, and colored nipple discharge, clinically representing the nipple secretion of duct ectasia. Group 2 (the control group) was composed of 50 patients without nipple discharge. The odds ratio of duct ectasia was three times higher for current smokers (p = 0.04). Likewise, smokers from the duct ectasia group had smoked for a longer time (median 25 months) compared to smokers from the control group (median 15 months) (p = 0.02). Parity, history of abortion or termination, breast-feeding, hormonal contraceptive use, and history of breast abscess did not increase the risk for duct ectasia. The group of women with duct ectasia was associated with current tobacco smoking.

Notes: Abstract:  Our objective was to describe the characteristics of subareolar breast abscesses and to analyze the results of surgical treatment in relation to the prevention of recurrences. Almost 70% of patients smoked more than 10 cigarettes a day. The recurrence rate after excision of the lactiferous ducts was 28% and after management without excision of the lactiferous ducts was 79% (p 〈 0.001). Gram-positive bacteria were isolated more frequently in primary subareolar breast abscesses (not significant). Anaerobic microorganisms were more frequently cultured in recurring subareolar breast abscesses (p = 0.02). Definitive treatment of subareolar breast abscesses should consist of excision of the affected lactiferous ducts.

Notes: Abstract:  The primary objective of this study was to evaluate the relative prevalence of estrogen receptor-negative contralateral breast cancer to the first primary cancer and to assess the correlation between the relative overexpression of HER-2/neu in the first primary cancer and contralateral breast cancer. A total of 144 women diagnosed with cancers in contralateral breasts were identified from the Henry Ford Health System tumor registry. Data were retrieved from electronic databases and medical records. Women were dichotomized into users and nonusers of tamoxifen. Hormone receptors were scored as positive or negative. HER-2/neu overexpression, assessed by immunohistochemistry, was scored as 0, 1+, 2+, or 3+. Concordance between hormone receptors of the two cancers was low (κ = 0.27, p = 0.06). Stratification of women by tamoxifen therapy yielded an almost fivefold increase in the proportion of estrogen receptor-negative cancers among the users, while the proportion of cancers expressing no estrogen receptor remained the same among the nonusers (39.6% versus 40.6%). Matched, archived, paraffin-embedded specimens of the first and contralateral breast cancers were available for 57 women. The correlation between the relative overexpression of HER-2/neu between the first primary and the contralateral breast cancer was 0.4 (p = 0.002). The higher prevalence of estrogen receptor-negative contralateral breast cancer among tamoxifen users concurs with previous reports. The biological mechanism for this observation is not understood; however, it has been proposed that tamoxifen inhibits the proliferation of estrogen receptor-positive breast cancer cells, while estrogen receptor-negative cells may continue to grow because of selective pressure. The correlation between HER-2/neu overexpression in the matched first primary and contralateral breast cancers was statistically significant, suggesting that the diagnosis of HER-2/neu overexpression in contralateral breast cancer is associated with HER-2/neu overexpression in the first primary cancer.

Notes: Abstract:  Distal obstruction of the lymphatics by tumor and extensive tumor infiltration of the draining lymph nodes may prevent migration of the tracer to the sentinel lymph node (SLN), adversely affecting SLN identification. Rerouting of lymphatic drainage may divert flow to internal mammary nodes and cause an alternative nonsentinel node to become “sentinel,” increasing the risk of a false-negative result. A total of 618 breast cancer patients underwent SLN biopsy using 99mTc albumin colloid and patent blue V injected peritumorally. This was followed by standard axillary node clearance in all patients at the same operation. The overall SLN identification and false-negative rates were 96% (593/618) and 7.6% (17/223), respectively. There was no difference in the SLN identification rate and the false-negative rate with increasing axillary tumor burden (as determined by the total number of positive nodes in the axilla). Further detailed analyses are based on the 64 patients from one center (Cardiff) who had at least one positive SLN and proceeded to axillary clearance. A total of 83 positive SLNs were removed from 64 patients. Tumor burden in the positive SLN was assessed by measuring the size of the metastasis and percentage replacement of the SLN by tumor, and by documenting extranodal invasion. Increasing tumor burden in the SLN (as determined by percentage replacement of SLN by tumor and presence of extranodal invasion) was associated with decreased radioisotope uptake (p = 0.005 and p 〈 0.0001, respectively). There was no correlation between radioisotope uptake and the size of the metastasis in the SLN. There was no correlation between blue dye uptake, internal mammary drainage on lymphoscintiscan, and tumor burden in the positive SLN. In conclusion, increased axillary lymphatic tumor burden is not associated with failure to identify a SLN or false-negative results when both blue dye and radioisotope are used for SLN biopsy. In an individual SLN, the percentage replacement by tumor, but not the absolute size of the metastatic deposit is associated with reduced radioisotope uptake. Extranodal invasion in the SLN is a marker of lymphatic obstruction and is significantly associated with reduced radioisotope uptake. The lymphatic tumor burden does not seem to affect blue dye uptake or internal mammary drainage.

Notes: Abstract:  Over 7 years, 57 women with breast cancer underwent lumpectomy and bilateral mammoreduction. Physical complaints about large or lax breast shape were the predominate rationale. Two patients were immediately lost to follow-up, 55 patients remained and were followed every 3 months for an average of 1.6 years. This is the largest series traceable by computer and literature search. Chart review and patient examination in this retrospective review were utilized as the basis for data within the article. Collated notes from patients’ doctors were assessed, as well as documented patient responses to the procedure. Pictures without head/face for identifiers were taken of the patients. Chart data were collected by clinicians, but were reviewed blindly by a statistician. The overall control and cosmesis rates as well as alleviation of heavy breast problems were noted. Only 6% of women had fair to poor cosmetic results; the majority (82%) had excellent to good results. Women with very large breasts or markedly relaxed breast tissue of concern to the patients proved optimal candidates for lumpectomy of cancer and bilateral mammoreduction in the conservative treatment of these cancers. There was a significant reduction in the physical complaints of the patients as well. For women with very pendulous or extremely large breasts, lumpectomy and bilateral mammoreduction may prove to be the optimal course of action.

Notes: Abstract:  Increasing experience with positron emission tomography (PET) scanning in breast cancer patients is revealing a significant role for this imaging modality. This report summarizes the experience of 2-[F18]fluoro-2-deoxy-D-glucose (FDG) PET scanning in 165 breast cancer patients from the BC Cancer Agency, British Columbia, Canada, and reviews the literature on this topic. Using the database at PETSCAN Vancouver, we identified imaged patients with a diagnosis of breast cancer. We then conducted a retrospective review of these patients’ BC Cancer Agency charts to extract demographic and follow-up information. Between November 2000 and March 2003 we identified 165 patients with histologically confirmed breast cancer who had undergone PET scanning, were registered at the BC Cancer Agency, and had follow-up information. The median patient age was 52 years. The sensitivity of PET in detecting axillary metastases was 28%, and the specificity was 86%. At diagnosis, 5% of patients were diagnosed with distant metastases. In patients undergoing PET scanning because of suspected recurrence, the sensitivity and specificity for detecting recurrence were 89% and 88%, respectively. Distant metastases were demonstrated in 30% of patients who were thought only to have local-regional recurrence. The results suggest that there are two clinical situations in which PET appears to be particularly valuable. The first is in the evaluation of patients who are suspected of having a tumor recurrence. The other is in identifying patients with multifocal or distant sites of malignancy who otherwise appear to have an isolated, potentially curable, local-regional recurrence.

Notes: Abstract:  The management of breast cancer in elderly women is controversial. Breast cancer in this age group tends to be biologically less aggressive and is highly responsive to hormonal intervention. The risk of dying of other causes often exceeds the risk of cancer recurrence. For these reasons, older patients tend to be treated less aggressively. One large study of elderly women with breast cancer found that half of the patients were undertreated. Four patients (mean age 72 years, range 61–95 years) underwent a unilateral total mastectomy for cancer under local anesthesia using the tumescent technique of infiltrating dilute lidocaine with epinephrine (25 ml of 1% lidocaine [250 mg] and 1 ml of 1:1000 epinephrine [1 mg] to 1 L of Ringers lactate) via an infusion pump. Three of the patients had estrogen receptor (ER)-negative tumors and one patient had tumor progression despite switching from tamoxifen to anastrozole. All four patients were class IV as defined by the American Society of Anesthesiology (ASA). There was no morbidity related to the surgery in the form of hematoma, wound infection, or skin flap necrosis. The patients were discharged 1–4 days after surgery. The anesthesia was adequate in all four cases and there was no deviation from the described technique. The mean operative time was 35 minutes (range 24–46 minutes). The tumescent technique is a safe, effective method for performing a total mastectomy in patients who would not be considered candidates for general anesthesia. 

Notes: Abstract:  The techniques for performing sentinel lymph node biopsy (SLNB) vary from institution to institution. Some advocate blue dye only, others radioisotope only, and many utilize a combination of both. The purpose of this study is to evaluate the additional benefit that blue dye provides when used in combination with a radioisotope. From October 2001 to June 2004, 102 SLNBs were attempted in 99 patients with breast cancer using a combination of blue dye and radioisotope. A lymph node was considered a sentinel lymph node (SLN) when it was stained with blue dye, had a blue lymphatic afferent, or had increased radioactivity. Ninety-eight patients had 101 successful identifications of SLNs, for an identification rate of 99%. Twenty-eight patients had positive SLNs. In three of those patients, although there were SLNs identified by both techniques, the positive SLNs were identified with only blue dye. Of the 102 SLNB procedures, there were two patients whose only SLN was identified by blue dye only. Although blue dye did not improve the identification rate, there was a definite benefit in improving the false-negative rate.

Notes: Abstract:  The objectives of this study were to evaluate quality of life (QOL) and identify its associated factors in climacteric women with a history of breast cancer. A cross-sectional study was performed including 75 breast cancer survivors age 45–65 years who had undergone complete oncologic treatment and nonusers of hormone therapy or tamoxifen in the last 6 months. Sociodemographic and clinical characteristics in addition to the prevalence of climacteric symptoms were evaluated. QOL was evaluated by the Medical Outcomes Study 36-item Short-Form Health Survey (SF-36) questionnaire, including eight components that can be condensed into two summaries: a physical component summary (physical functioning, role-physical, body pain, general health) and a mental component summary (vitality, social functioning, role-emotional, and mental health). Generalized linear models were used to analyze the data, allowing the identification of factors affecting QOL, adjusting for confounding variables. The mean age of the participants was 53.1 ± 5.9 years. Breast cancer survivors reported good QOL. The most prevalent symptoms were nervousness (69%) and hot flashes (56%). Factors associated with poorer QOL were dizziness, postmenopausal status, and breast-conserving therapy (physical component), as well as insomnia and being married (mental component). In conclusion, participants demonstrated good QOL. We identified factors that may influence QOL in women with breast cancer, highlighting being married, climacteric symptoms, postmenopausal status, and breast-conserving therapy. Given the impact of these factors, health professionals and patients must discuss choices for alleviating climacteric symptoms and explanations for the potential repercussions of breast cancer treatment.

Notes: Abstract:  The HER-2/neu gene is a proto-oncogene that is amplified in 10–30% of breast cancers. New drugs for targeted therapy, such as Herceptin, are effective for patients with HER-2/neu-positive tumors, making it necessary to have a noncostly and accurate method to assess HER-2/neu status. We studied the correlation of findings made by fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) staining and the possibility of combining IHC and other clinicopathologic characteristics of breast tumors to predict FISH-determined HER-2/neu status. The clinicopathologic characteristics analyzed were the size of the tumor, p53, lymph-vascular invasion, estrogen/progesterone receptors (ER/PR), tumor grade, axillary lymph node status, and patient age. A total of 199 cases of invasive breast cancer studied at the UCLA Pathology Laboratory during 2003 were included in this study. Tumors with IHC 0, 1+, 2+, and 3+ scores were found to be FISH positive in 3.5%, 6.4%, 25.7%, and 81.5% of the respective groups. Our study showed a strong association between the FISH-negative and IHC scored 0 and 1+ tumors, suggesting that the FISH test may not be necessary in these cases (p 〈 0.0001). Although the concordance between IHC 3+ and FISH positive is high, 18% of the patients with overexpression of HER-2/neu fail to show gene amplification by FISH. HER-2/neu positivity was found to be proportionally associated with increasing grade in infiltrating ductal carcinoma (p 〈 0.0001). p53-positive tumors are more likely to be HER-2/neu amplified (p = 0.0003). Tumors that are negative for ER/PR are also associated with HER-2/neu positivity by FISH (31.15%, p = 0.0016). FISH-determined HER-2/neu status is not associated with histologic type, tumor size, nodal status, lymph-vascular invasion, or patient age.

Notes: Abstract:  Stromal invasion is identified with difficulty in routine hematoxylin-eosin-stained sections of core needle biopsy specimens from mammary intracystic papillary carcinomas. The goal of this study was to determine if nuclear grade, mitotic activity, and immunohistochemical stains for p53 and cyclin D1 would assist in differentiating intracystic papillary carcinomas without stromal invasion (ICPC) from tumors with stromal invasion (ICPC-INVA). Eight cases of ICPC and 12 cases of ICPC-INVA were reviewed. Hematoxylin-eosin slides were examined to determine the histologic features. Immunohistochemistry was performed using monoclonal antibodies to human p53 and cyclin D1. Fisher's exact test was used to compare the nuclear grade, mitotic activity, and immunoreactivity between ICPC and ICPC-INVA. High nuclear grade was more often associated with ICPC-INVA than with ICPC, although the difference was not statistically significant (p = 0.069). Frequent mitotic activity was associated with ICPC-INVA more than with ICPC (p = 0.0198). All cases of ICPC were negative for either p53 or cyclin D1, whereas 7 of 12 cases (58.3%) of ICPC-INVA were positive for either cyclin D1 alone (3 cases), p53 alone (3 cases), or both cyclin D1 and p53 (1 case) (p = 0.0147). Identical nuclear grade, mitotic activity, and immunostaining patterns were seen in the intracystic and the invasive components, and in the core biopsy and the excision of the same tumor. When any one of the positive indicators (high nuclear grade, frequent mitotic activity, or positive immunostains for cyclin D1 and/or p53) was present, the positive predictive value for stromal invasion was 91.7%. When none of the positive indicators was present, the negative predictive value was 87.5%. 

Notes: Abstract:  The purpose of this study was to assess the quality of life in women who had previously undergone a bilateral prophylactic mastectomy and to determine what factors predict quality of life in this population. Women in Ontario who had undergone prophylactic mastectomy between 1991 and 2000 were asked to complete several questionnaires that assessed current psychosocial functioning, including the Quality of Life Index (QLI). The mean score for the QLI was 23.34 (range 9.53–30.00). QLI scores were negatively correlated with cancer-related distress, body image difficulties, and psychological distress. Conversely, QLI scores were positively correlated with social support. Significant predictors of quality of life included psychological distress and one subscale of body image (vulnerability). Vulnerability and psychological distress are important predictors of quality of life in women who have previously undergone bilateral prophylactic mastectomy.

Notes: Abstract:  Background: Sentinel lymph node (SLN) biopsy is often used in the assessment of lymph node status in melanoma and early stage breast cancer. With the rapidly increasing use of the technique, we can now better characterize and assess the rate of adverse reactions to the dye. Methods: A retrospective review of all patients undergoing SLN mapping at the Columbia-Presbyterian Breast Center were identified from June 2000 to July 2002. All patients who experienced allergic reactions were documented and records examined. Results: In total, three out of 351 patients had allergic complications from the procedure. All three patients developed “blue hives” after injection with isosulfan blue. The incidence at our Breast Center was 0.9%. All were treated with intravenous corticosteroids and diphenhydramine and recovered within twenty-four hours. Conclusions: The increasing utilization of the sentinel lymph node technique will make these complications more common. A high index of suspicion and appropriate clinical management are recommended to minimize the potential morbidity of these reactions.

Notes: Early to mid-term fetuses heal cutaneous incisional wounds without scars; however, fetal response to burn injury has not been ascertained. We present a fetal model of thermal injury and subsequent analysis of fetal and lamb response to burn injury. A reproducible deep dermal burn injury was created in the fetus by application of water at 66 °C for 7 seconds, and at 82 °C for 10 seconds to the lamb. Macroscopically, the area of fetal scald was undetectable from day 7 post injury, while all lamb scalds were readily identified and eventually healed with scarring. Using a five-point histopathology scoring system for alteration in tissue morphology, differences were detected between control and scalded skin at all stages in lamb postburn, but no difference was detected in the fetal model after day 7. There were also large differences in content of α-smooth muscle actin and transforming growth factor-β1 between control and scalded lamb and these differences were statistically significant at day 14 (P 〈 0.01). This novel model of fetal and lamb response to deep dermal injury indicates that the fetus heals a deep burn injury in a scarless fashion. Further elucidation of this specific fetal process of burn injury repair may lead to improved outcome for patients with burn injury.

Notes: Our group has developed an ovine model of deep dermal, partial-thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross-linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a “response” to burn injury.

Notes: Introduction:  Chronic, nonhealing wounds are often observed in tissues with poor oxygen supply. Impaired reepithelialization is a hallmark of these wounds; however, the pathogenesis of the retarded reepithelialization in chronic, ischemic wounds remains poorly understood. Transforming Growth Factor beta (TGF-beta) is involved in both normal and hypoxic wound healing response and exogenous overexpression of Smad3, a TGF-beta signaling intermediate, has been known to accelerate reepithelialization. In a recent study, Ad-Smad3 injection in the rabbit ear dermal ulcer model showed enhanced reepithelialization and granulation tissue area suggesting a positive effect of Smad3 on wound healing. However, little is known about the role of Smad3 in the ischemic wound healing process. In this study we examined the effect of Smad3 in an ischemic wound model.Methods:  Ad-Smad3 or LacZ (108 pfu/wound) empty vector was injected in either ear of White New Zealand Rabbits. Twenty-four hours later, these ears were rendered ischemic using an established model and four 7-mm full-thickness punch wounds were made on each ear.Results:  Histological evaluation showed a highly significant increase in reepithelialization, epithelial ingrowth and percentage of epithelialization in Ad-Smad3 transfected wounds versus ischemic wounds transfected with LacZ-empty vector (p 〈 0.01).Conclusion:  Our data confirm the enhancing effect of Smad3 on reepithelialization in an ischemic wound model. The deficiency in reepithelialization, as evident in chronic ischemic wounds, could thus be ameliorated by excess Smad3. Therapeutic interventions using overexpressed Smad3 may improve wound healing through accelerated epithelialization in chronic wounds.

Notes: Proinflammatory cytokines (such as IL-1 [alpha], IL-1 [beta]) have been shown to play a significant role in early stages of wound healing. Previous studies also indicate that mucosal wounds heal faster in males. The purpose of this study was to assess gene expression of inflammatory cytokines before and during wound healing. Younger and older volunteers received two small oral wounds on the hard palate. One of the wounds (3.5 mm in diameter) was videographed daily until healed. The other wound (1 × 5 mm) was biopsied either 6 or 24 h postwounding. Real-time PCR (ABI Prism 7000 SDS) was performed on the unwounded and wounded tissues to study gene expression. Preliminary analyses revealed that compared to unwounded tissue, wounding induced rapid gene expression for these cytokines. In males, IL-1 [beta] gene expression peaked at 6 h and was higher than in females. Since males have been shown to heal this type of wound faster than females, this earlier peak of IL-1 [beta] may be beneficial to wound healing. Regardless of gender, IL-1ra and IL-1 [alpha] levels peaked at 6 and 24 h, respectively. Interestingly, cytokine levels in unwounded tissue correlated with wound healing parameters (e.g., wound sizes, time to heal), but in females only. Specifically, women with higher basal levels of IL-1 [beta] and IL-1ra, and lower levels of IL-1 [alpha] tended to heal faster. Thus, the expression of inflammatory cytokine genes in unwounded mucosal tissue may play a determinant role in wound healing outcomes.(Supported by NIH P50 DE-13749)

Notes: Background and Objectives:  Hyaluronan (HA), a ubiquitous glycosaminoglycan is synthesized by one of three distinct hyaluronan synthases (HAS). The purposes of this study were to determine whether a cause-and-effect relationship exists between the proinflammatory cytokine, interleukin-1β(IL-1β), and hyaluronan expression in normal jejunum-derived mesenchymal cells (JDMCs), to identify possible mechanisms involved and to determine the effects of glucocorticoids, a common therapy for intestinal inflammatory pathologies including Crohn’s disease and ulcerative colitis (UC), on cytokine-mediated hyaluronan expression.Experimental Procedures:  JDMCs were stimulated with IL-1β for 20 hr with or without pretreatment for 1 hr with mitogen activated protein kinase (MAPK) inhibitors or dexamethasone. HA levels from culture media were quantified using a slot blotting approach. HAS transcript levels were quantified using RPA. Immunoblotting was used to detect and quantify activation of MAP kinases.Results:  Preliminary data of sample tissue from patients with Crohn’s or UC indicated increased HAS transcript levels. Similarly, IL-1β induced an approximate 18-fold increase in both HAS2 steady state transcripts and HA levels. These increases were blocked by dexamethasone (95%). Knockdown with siRNA demonstrated that IL-1β induction of HA expression occurred via HAS2. Immunoblot analysis indicated that IL-1β activated the ERK1/2, p38 and JNK pathways. Inhibitors of the p38 and ERK1/2, but not JNK, blocked the IL-1β induction of HA expression (80 and 90%, repectively). Similarly, dexamethasone was also found to block activation of the ERK1/2 and p38 pathways indicating a mechanism for glucocorticoid steroid regulation of HAS expression.Conclusion:  This study provides evidence for proinflammatory cytokine regulation of HA expression in JDMCs. Furthermore, this study provides support for the hypothesis that HA may be one of the targets involved in the treatment of steroid-dependent inflammatory bowel disease.Support:  This work was supported by NIH GM 58530.

Notes: Introduction:  Early excision and closure is standard for severe burn management. Cadaver skin is generally used as temporary coverage if autograft donor sites are inadequate. However, it is associated with an inherent risk of antigenicity and infection and has a limited shelf life and availability. Use of Integra™, a dermal substitute, is well established for postburn reconstruction, but its efficacy as primary coverage for severe burns it is not well documented. Therefore the aim of the present study was to determine the short- and long-term efficacy of Integra™ as an acute cover.Method:  Twenty children with 〉40 total body surface area (TBSA) burn, were randomized to be grafted with Integra™ or with the autograft-allograft technique. Short-term outcome measures such as length of hospital stay, mortality, incidence of infection/sepsis, cardiac, respiratory, and metabolic indexes were compared between and within groups before and after Integra™/graft application. Long-term outcome measures such as number of reconstructive procedures and blinded scar scoring were performed up to 2 years postinjury. Statistical analysis was performed using paired and unpaired t-test. Values are expressed as Mean ± SEM and significance accepted at p 〈 0.05.Results:  There were no significant differences between groups, in burn size (Controls 74 ± 4% and Integra™ 70 ± 5%), mortality (Control 30%, Integra™ 40%), length of hospital stay (Control 39 ± 4 days, Integra™ 41 ± 4 days), and metabolic rates. In addition, there was no significant incidence of infection or sepsis in the Integra™ group compared to controls. Long-term follow-up (Integra n = 5, control n = 5) revealed a significantly improved scar, in terms of height, thickness, vascularity, and maturity, in the Integra™ group compared to the control group, at 12 months and 18–24 months postburn, (p 〈 0.01).Conclusion:  Integra™ can be safely used for immediate wound coverage in children with severe burns, with better functional and aesthetic outcomes up to 2 years postinjury.

Notes: TGFβ1 is a multipotent secreted cytokine that plays a pivotal role in wound healing. Previously, both positive and negative effects of TGFβ1 on cutaneous wound healing have been reported. The mechanisms underlying these effects of TGFβ1 remain to be elucidated. In the present study, we used a mouse model to examine the effects of cutaneous wound healing, wherein the latent form of TGFβ1 is constitutively expressed in basal keratinocytes of the mouse epidermis, targeted by the keratin 5 promoter (K5.TGFβ1). In wild-type mice with 6-mm full thickness excisional cutaneous wounds, TGFβ1 protein levels were elevated immediately after wounding, and reached a peak level on day 3 postwounding, which was 3–5-fold higher than that in the nonwounded skin, and sharply declined afterward. This elevated expression pattern suggests that only transient TGFβ1 overexpression is required for normal wound healing. In contrast, K5.TGFβ1 mice with prolonged TGFβ1 transgene expression at the level equivalent to the above peak level in wild-type mice exhibited a significant delay in healing of 6-mm full thickness excisional wounds when compared to wild-type mice. Histological analysis of wounds revealed delayed reepithelialization, increased inflammation and prolonged granular tissue accumulation in K5.TGFβ1wt mice. Immunohistochemistry analysis revealed excessive leukocyte infiltration in transgenic wounds when compared to wild-type wounds. Understandably, the accumulation of leukocytes was also accompanied by increased expression of inflammatory cytokines, chemokines, angiogenic factors and fibrotic factors as determined by qRT-PCR analysis. Although the above factors override the growth inhibitory effect of TGFβ1 on epidermal proliferation away from the wound edge, proliferation at the leading edge of the migrating keratinocytes was reduced in transgenic skin compared to the nontransgenic skin, evident by PCNA staining. Keratinocyte proliferation was also reduced in vitro when assessed by H3-thymidine incorporation. To assess the direct effect of TGFβ1 on keratinocyte migration, we performed an in vitro migration assay and found that K5.TGFβ1 keratinocytes had delayed migration when compared with wild-type cells. Our study suggests that prolonged TGFβ1 expression in keratinocytes delays cutaneous wound healing by excessive inflammation, coupled to a direct inhibitory effect on keratinocyte proliferation and migration.

Notes: The role of the EGF superfamily of growth factors and their receptors during wound healing continues to unfold. The aim of the study was to determine the effect of overexpression of erbB3 in wound healing and whether topical application of its various ligands enhances repair. Partial thickness porcine wounds were transfected with recombinant adenoviral particles containing erbB3 receptor gene or a vehicle β-galactosidase gene (Lac Z). Highly similar ligands: Epidermal growth factor (EGF), Epiregulin (EPR), Heparin-binding epidermal growth factor (HB-EGF), Heregulin (HER) were topically applied to wounds boosted with erbB3 receptors. Enhanced wound healing parameters were assessed 5 days after initial treatment for reepithelization, dermal depth, macrophage infiltration, proliferating cells, angiogenesis, and apoptotic cells. Treatment with EPR and HB-EGF promoted greater resurfacing of the epidermal layer than EGF, HER, erbB3 only or vehicle treated wounds. Neodermal formation as measured by dermal or granulation depth was significantly different in the EPR and HB-EGF treated wounds compared to EGF, HER, erbB3 only or vehicle. Numerous hallmarks of enhanced wound maturity (fewer infiltrating macrophages, significant reductions in neovascularization, fibroblastic proliferation) were noted in EPR- and HB-EGF-treated wounds transfected with erbB3 as compared to vehicle controls. Only erbB3 wounds treated with these particular ligands showed significant increases in apoptotic cells. Our results indicate that wounds treated with this erbB3 receptor form supplied with topical application of EPR and HB-EGF exhibit synergistic and accelerating reparative effects on porcine partial thickness wounds as compared to vehicle. These in vivo findings suggest the complexity and evidence of synergism between the erbB receptor and its various ligands on wound healing.(This research work was funded by the NIH GM40437.)

Notes: Previous studies have shown that cyclic mechanical stretching exerts anti-inflammatory effects on rabbit chondrocytes. But whether mechanical stretching has similar effects on human tendon fibroblasts are not known. This study therefore aimed to test the hypothesis that cyclic mechanical stretching regulates IL-1β induced COX-2 gene expression in a stretching magnitude-dependent manner. In custom-made silicone dishes, human patellar tendon fibroblasts (HPTFs) were grown on microgrooved culture surfaces, with which the shape and organization of HPTFs in vivo were closely mimicked. To induce inflammatory responses in HPTFs, 10 pM of IL-1β was added to the culture medium. A 4% or 8% cyclic uniaxial stretching was then applied to silicone dishes for 4 hrs using a custom-design stretching apparatus. After the end of stretching, total RNA of stretched and nonstretched tendon fibroblasts was collected, and RT-PCR was performed for measuring COX-2 mRNA expression levels. We found that tendon fibroblasts subjected to 4% stretching decreased COX-2 mRNA expression level by 20%(p = 0.0002; n = 6) compared to that of nonstretched, IL-1β-treated cells. However, cells subjected to 8% stretching showed a 39% increase (p = 0.007, n = 6) in COX-2 mRNA expression. Thus, the results of this study suggest that small stretching (4%) has anti-inflammatory effects on HPTFs in a mildly inflammatory environment such as that induced by 10 pM of IL-1β. In contrast, large stretching (8%) may amplify inflammatory responses of tendon fibroblasts. Therefore, physical therapy with low levels of tendon stretching may be beneficial in reducing mild tendon inflammation.Supported by the Arthritis Investigator Award and NIHAR049921 (JHW)

Notes: Introduction:  As an initial pilot effort a prospective, case-controlled trial examining the effects of treprostinil sodium (Remodulin®), by continuous, subcutaneous infusion, was performed in diabetic patients with recalcitrant lower extremity wounds. While the primary outcomes for the trial were wound healing and limb preservation, evaluation of local tissue perfusion with transcutaneous oximetry and laser Doppler analysis (TcPO2/LD) were included as secondary objectives, and with the expectation they would be useful monitors of any interventional modality.Methods:  All patients enrolled had nonhealing wounds (〉3 months) in critically ischemic lower extremities (Fontaine Stage III-IV or Rutherford Stage 4, 5, or 6). Hyperbaric oxygen exposures were included as diagnostic/monitoring indices. In the last four enrolled subjects TcPO2/LD measurements were made at ambient pressure and at 2.0 or 2.4 ATA before Remodulin® was begun and at various points during drug infusion.Results:  Early, observational evaluation of the TcPO2/LD data shows:1 – These examinations prognostically profile healing in ischemic limbs.2 – TcPO2/LD values demonstrate the efficacy of Remodulin® in augmenting perfusion in ischemic wounds. While there is a broad range, all measured parameters increased on the drug (resting TcPO2 ⇑ 5–30 mmHg, the rate of TcPO2 rise following ambient pressure O2-challenge ⇑ 25–150% and periwound TcPO2 ⇑ 90–300 mmHg at 2.0 and 2.4 ATA).3 – The in-chamber TcPO2 on Remodulin® further defines patients who will not benefit from HBO2 therapy.4 – The TcPO2/LD data will assist in determining dosages, duration, and maintenance requirements for this drug both as a single agent and when used as an adjuvant to HBO2 therapy.Conclusions:  Treprostinil sodium (Remodulin®) has significant laudatory effects on local wound perfusion in ischemic lower extremities.Acknowledgments:  United Therapeutics, Inc provided support for this study through an unrestricted educational grant.

Notes: α1-antichymotrypsin (ACT) and its murine homolog spi2 are serine protease inhibitors present in plasma, that are locally up-regulated during wound healing. Ad-spi2 and Ad-ACT expression vectors were constructed to address the biological activity of locally expressed serpins. The condition medium of infected COS-1 cell at 2 d postinfection contained 1.23 ng/ml spi2 and 2.61 ng/ml ACT. Ad-LacZ or Ad-spi2 108 pfu was injected into the sponges after 3 d subcutaneous implantation in rats. Histological analysis showed that overexpression of spi2 improves granulation tissue organization and collagen deposition at d7 implantation. Moreover, overexpression of spi2 increases the contents of tissue DNA (23.8%, p 〈 0.05) and protein (25.3%, p 〈 0.05) in sponge implantation model in rats. In incisional wound models in both normal and STZ-induced diabetic rats, Ad-spi2 increases tensile strength by 26%(p = 0.011) and 29%(p = 0.022) as well as Ad-ACT increases by 10.3%(p = 0.037) and 32%(p = 0.004), respectively. In adriamycin-induced wound model in rats, histological analysis displayed less inflammatory cells infiltrating in Ad-ACT injected wound than in Ad-LacZ after 21 d injection of adriamycin. Inflammation enzymes MPO and elastase levels were decreased by 32.3% and 29.2% in Ad-ACT injected wound tissue. Our data concluded that overexpression of spi2 and ACT improves wound healing in rats probably via inflammatory resolution.(Supported by Switch Biotech and the Department of Veterans Affairs)

Notes: Cellular interaction with biomaterials determines the fate of the implant. Naturally derived biomaterials such as small intestinal submucosa (SIS) and renal capsule matrix (RCM) may provide optimal structure and composition for cellular repopulation in certain wound healing applications. Porcine renal capsule matrix (RCM) is an acellular, naturally occurring extracellular matrix undergoing characterization and preclinical investigation for the treatment of soft tissue injury. Early studies have demonstrated that RCM constructs induce host tissue infiltration, tissue-specific remodeling, and repair. RCM consists primarily of collagen, although the other proteins have been detected, including the growth factors basic FGF, VEGF, and CTGF.Transmission and scanning electron microscopy were used to envision the cellular environment provided by this biomaterial. Comparison of the images utilizing cryo and traditional preparation techniques show that RCM is a dense, heterogeneous matrix consisting of textured fibers of mixed size. Although the matrix architecture shows signs of collapse following lyophilization, much of the native structure seen in the hydrated isolate is preserved. These differences are maintained following rehydration in normal saline. The effects of these structural changes on cellular reaction and infiltration are unknown, but the naturally complex architecture likely facilitates host interaction at the cellular level. Further compositional characterization is under way.This work was supported by Cook Biotech Incorporated, West Lafayette, IN.

Notes: The use of fresh platelets has gained value in medicine as an essential part of wound treatments. This is not surprising since platelets contain a number of bioactive factors that contribute to the process of wound healing, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF). Fresh platelets’ short shelf life limits platelet-based therapies. If platelets can be stabilized in freeze-dried form (FDP) then long-term storage as well as pathogen inactivation methods become possible. Adlyfe and Oregon Freeze-Dry have been developing technology to stabilize freeze-dried human platelets that can be subjected to gamma irradiation and stored for a long duration. Upon reconstitution, irradiated FDP retained growth factors PDGF-AB, PDGF-BB, and TGF-B1 in quantities similar to fresh platelets as judged by capture ELISA. The rehydrated FDP promoted new DNA synthesis and cellular proliferation of primary human dermal fibroblasts and endothelial cells (HUVECs) similar to fresh platelets. The FDP also promoted remodeling of extracellular matrix by accelerating fibroblast-mediated contraction of collagen gels and stimulated HUVECs to undergo angiogenesis and form capillary structures in vitro. Therefore, we conclude that FDP and fresh platelets have comparable in vitro wound healing potential. Preclinical wound healing stud"ies in diabetic mice are under way and further development will allow FDP to be a safe and well-suited alternative to fresh platelets for wound healing applications.

Notes: To investigate changes in the mechanical properties of skin with injury, tissue was evaluated 70 days following full thickness wounding in juvenile female Yorkshire pigs (N = 14). Samples were taken in the axial (cranial-caudal) and transverse (dorsal-ventral) directions, for both scar tissue and uninjured skin. The samples were evaluated mechanically in vitro using a protocol of stress relaxation followed by tensile failure, in an INSTRON universal test machine. Specimens were subjected to a 10% elongation and held for a 4-min isometric period, after which they were returned to the original length and failed in tension.Axial normal skin exhibited a smaller force at 10% elongation (−58%, p = 0.002), and higher failure load (p = 0.027) and displacement (p = 0.028) than transverse. Scarring reduced the failure strength on average by 60% in the axial and 65% in the transverse directions (p 〈 0.001), with large decreases (〉50%) in displacement at failure in both directions (p 〈 0.001). Elongation of 10% produced a larger force in axial scars (p 〈 0.001) than their normal counterparts, indicating a reduction in small-displacement compliance. Axial scars exhibited greater failure strength (p = 0.009) than transverse scars, yet no significant differences were observed in elongation to failure or load at 10% elongation.These results indicate that normal skin exhibits higher compliance and failure strength in the axial direction. Following injury, the strength and compliance of the tissue are greatly reduced, and the strong compliance directionality originally observed, is lost. Therefore it appears that the healing response leads to increases in tissue stiffness, such that small displacements result in larger tissue loads. This is further complicated by the reduced ability of the scar to resist failure. Taken together, these findings illustrate the compounding negative effects of scar on tissue integrity and function.Acknowledgments:  Alberta Ingenuity Fund, CIHR and NSERC

Notes: Fibroblasts from a healing ligament likely have differential phenotypical expression from those in an intact ligament, because fibroblasts at the healing site need to repair/regenerate the wounded matrix around them caused by injury, whereas normal fibroblasts just maintain an intact matrix. This study was designed to determine the differences in proliferation, collagen production, α-smooth muscle actin (α-SMA) expression, and contraction between healing and normal fibroblasts. Transected and sham-operated rat medial collateral ligaments (MCL) were used to obtain healing and normal fibroblasts, respectively. It was noted that healing and normal MCL fibroblasts in culture were seen to have a similar morphology. However, healing fibroblasts in monolayer culture proliferated 1.4-fold faster at 48 hours and had 1.7-fold greater protein expression of α-SMA than normal fibroblasts. In addition, it was noted that the proliferation of healing fibroblasts in collagen gels was not significantly different from that of normal fibroblasts at 24 hours, but it was at 48 hours. Furthermore, in collagen gels, healing fibroblasts produced 10% more type I collagen than normal fibroblasts and generated 1.6- and 1.7-fold larger contractile forces at 15 and 20 hours, respectively, than their normal counterparts. Taken together, the results of this study show that healing fibroblasts possess a differential proliferation, α-SMA protein expression, and contraction than normal fibroblasts. This study also highlights the importance of using healing fibroblasts to study the cellular and molecular mechanisms of ligament healing.Supported by the NIH grant AR049921 and Whitaker Foundation Grant (JHW)

Notes: Cultured skin substitutes (CSS) are used as an adjunctive treatment for closure of massive burn wounds. CSS are prepared with keratinocytes and fibroblasts combined with a collagen-based matrix. A cDNA microarray analysis was performed to characterize CSS at a molecular level. We hypothesized that combination of keratinocytes and fibroblasts in a three-dimensional organo typic culture system would result in changes in gene expression compared with cells grown in monolayer culture. Human skin samples were obtained from breast and abdominal tissue of healthy adult females. Fibroblasts and kera tinocytes were isolated and grown in monolayer cultures; CSS, prepared with cells from four independent donors, were cultured for 2 weeks in vitro. Microarray analysis was performed on RNA from fibroblasts, keratinocytes, and CSS using the Affymetrix Human Genome U133A gene chip representing over 22,000 human genes. Cluster analysis was performed to identify gene expression “signatures” for each group, and further analysis was performed to identify genes that were significantly increased or decreased in CSS compared with either cell type. Microarray analysis revealed significant changes in gene expression upon combination of fibroblasts and keratinocytes in organotypic culture. Genes belonging to several classes were significantly increased in CSS compared with fibroblasts and keratinocytes. For example, genes associated with epidermal differentiation, including fillagrin, corneodesmosin, involucrin, and multiple keratins (1, 10, 13, 16, 23), were increased in CSS. Several genes that are overexpressed in psoriasis (S100A7, SERPINB3, PSORS1C1), as well as cytokines IL- 6 and IL-8, were also increased in CSS. Further, multiple MMP genes were up-regulated in CSS. The results suggest that epithelial-mesenchymal interactions contribute to morphogenesis of CSS, including the remodeling of the dermal layer and stratification and differentiation of the epidermal layer. This analysis reveals that CSS exhibit many characteristics of normal or inflamed human skin.

Notes: There are only a few models for studying epidermal migration in vitro, and their interpretation is made difficult by reliance on cell monolayers and the choice of a specific substrate. In this study, the process of keratinocyte migration and epiboly were investigated by using a bilayered bioengineered skin construct, consisting of human neonatal foreskin keratinocytes and dermal fibroblasts (Apligraf, Organogenesis, Canton, MA). At baseline, 6-mm punch biopsies of the construct were placed in serum-free media (AIM-V) or DMEM with or without 10% FBS. At varying time points the bioengineered skin samples were processed and analyzed by histology and immunostaining. By 72 hours, in a time-dependent manner, the epidermis had migrated over and enveloped the entire dermis (full epiboly). Full epiboly was partially inhibited by serum and was maximal in serum-free medium. Epiboly was preserved 5 days after stated expiration date and was equivalent to that seen in unexpired construct when stored at room temperature. The process of epiboly was downregulated in a dose-dependent manner by neutralizing antibodies to EGF and TGF-beta 1. The migrating epithelium was characterized by decreased keratinocyte proliferation (as per Ki67 immunostaining) and increased expression of vitronectin (epibolin). Increasing concentrations of antibodies of vitronectin blocked the process of epiboly, as did antibodies to the alpha5-betaV integrin receptor, which mediates vitronectin-driven keratinocyte locomotion. Interesting, epiboly was also blocked by preseeding human dermal fibroblasts on the dermal side of the construct. We propose that the process of epiboly in this model can be used to better understand and assess the mechanisms involved in keratinocyte migration and may be used as an assay for establishing construct functional viability.

Notes: We find that the ELR-negative CXC chemokines, CXCL10 (IP-10) and CXCL11(IP-9), expressed during wound repair, with IP-9 being produced by redifferentiated keratinocyte. Immunohistochemical analysis for IP-9 on human full thickness wound tissues collected from day 5 confirmed that IP-9 is a wound response protein. We have shown in in vitro coculture systems that IP-9 can serve as an autocrine factor promoting motility in keratinocyte while limiting motility in dermal fibroblasts. These effects occur via modulation of the intracellular proteases calpain isoforms that regulate cell adhesion during motility. Thus, the function of these chemokines in vivo is not obvious. To probe this, we have explored wound repair in mice lacking the receptor for these ligands, CXCR3. Quantitative analysis of IP-9 expression in wounds of these mice collected from day 2 to day 20 showed a significant increase in IP-9 levels in CXCR3-/- mice compared to wild type, which is expected if receptor-mediated feedback attenuation is lost. Full and partial thickness wound healing experiments on these mice showed a significant delay in CXCR3-/- mice when compared to wild type. Histochemical analysis of tissues collected from both full and partial thickness wounds demonstrated differences in epithelialization, granulation tissue formation, and angiogenesis in CXCR3-/- mice when compared to wild type. Collagen, the major extracellular matrix protein organization were analyzed by trichrome and picrosirus red staining methods. We are also examining the effect of this lost receptor on promotility signaling from EGF receptors. These in vivo and in vitro studies confirm the pathophysiologic role of the chemokines IP-9 and IP-10 and their cognate receptor CXCR3 during wound repair.

Notes: In this study, we evaluated and compared the effectivity of two modalities, gene therapy with adenovirus-mediated transforming growth factor-β and extracorporal shock waves (ESW) therapy, to treat ischemically challenged epigastric skin flaps in a rat model. Thirty male Sprague-Dawley rats were divided into three groups of ten rats each. An epigastric skin flap model, based solely on the right inferior epigastric vessels, was used as the model in this study. Rats received either subdermal injections of adenovirus encoding TGF-β(Ad-TGF-β) or ESW treatment with 750 impulses at 0.15 mJ/mm2. The third group received no treatment and served as a control group. A flap measuring 8 × 8 cm was outlined on the abdominal skin. The TGF-β injections were given subdermally in the left upper corner of the flap just prior to flap elevation, whereas the ESW treatment was immediately after the flap was sutured back to its bed. Flap viability was evaluated seven days after the initial operation. Digital images of the epigastric flaps were taken and areas of necrotic zones relative to total flap surface area were measured and expressed as percentages by using a software program. Overall, there was a significant increase in mean percent surviving area in the Ad-TGF-β group and the ESW-group compared to the control group (ESW-group: 97.7 ± 1.8% vs. Ad-TGF-β: 90.3 ± 4.0% and control group: 82.6 ± 4.3%; p 〈 0.05). Furthermore, in the ESW group mean percent surviving areas were significantly larger than in the Ad-TGF-β group (ESW-group: 97.7 ± 1.8% vs. Ad-TGF-β: 90.3 ± 4.0%; p 〈 0.05).We conclude that treatment with ESW enhances epigastric skin flap survival significantly more than Ad-TGF-β treatment and, thus, represents a modality that is more feasible, cost effective and less invasive compared to gene therapy with growth factors to improve blood supply to ischemic tissue.

Notes: Homeobox genes are transcription factor that form nested patterns in specific regions throughout the body. Homeobox genes are important for wound healing study because some have been shown to be related to scarless fetal wound healing. Muscle segment homeobox-2 (Msx-2) is a homeobox gene commonly expressed at sites of epithelial-mesenchymal interactions during tissue morphogenesis. Overexpression of Msx-2 leads to craniosynostosis, epidermal dysplasia, and abnormal hair growth. A knockout of this gene causes pleiotropic defects in bone growth, cyclic alopecia, and a thinner dermis. While Msx-2 has been found to play many critical roles in development, no functional study has been conducted on Msx-2 and wound healing. We hypothesize that Msx-2 regulates skin morphogenesis during wound repair which was tested by studying excisional skin wound closure. Isolated keratinocytes and dermal fibroblasts of Msx-2 WT and KO mice were also studied to determine the effects of Msx-2. Results showed that Msx-2 mRNA was induced in epidermis and dermis during wound repair. Additionally, Msx-2 KO mice exhibited enhanced re-epithelialization and faster wound closure than the WT control. These repair phenotypes may be attributed to keratinocyte motility and fibroblast interaction with collagen matrix as, in vitro, Msx-2 KO keratinocytes exhibited increased motility, and fibroblasts show stronger collagen matrix contraction and expressed higher levels of α2β1 than WT control. Msx-2 KO fibroblasts were refractory to BMP4 in collagen matrix contraction, demonstrating a disruption in the Msx-2 pathway. In conclusion, Msx-2 may regulate skin morphogenesis during repair at both epithelial and mesenchymal levels.This work was supported by NIH grant R01GM055081(TLT).

Notes: Percutaneous devices play a central role in modern medicine, but are associated with significant risk of infection. One approach to circumvent this risk is to heal the wound around the device by creating a seal at the skin/device interface. We previously showed, using cell adhesion assay and organ culture model, that surface modification of poly(2-hydroxyethyl methacrylate)[poly(HEMA)] with 1,1′-carbonyldiimidazole (CDI) dramatically enhanced keratinocyte attachment to poly(HEMA). Although the organ culture model addresses epithelial response to an implanted biomaterial, to fully evaluate the cutaneous interaction with biomaterials, we tested implants in a mouse model. Porous poly(HEMA) rods with 40 μm pore size were implanted into two different sites on dorsal skin of forty eight, 6–8 week old male C57BL/10 J mice. Porous poly(HEMA) rods were treated with 1) PBS, 2) CDI, and 3) CDI plus laminin 5. Samples were harvested at 7, 14, and 28 days after surgery and analyzed morphologically using H&E and immunohistochemistry. Implanted rods remained intact and infection free at all time points. Morphological analysis showed diffuse dermal cellular infiltration in all samples and evidence of epidermal integration into the pores of CDI treated poly(HEMA) and CDI plus laminin 5 treated poly(HEMA). Immunohistochemistry showed laminin 5 expression at the skin/poly(HEMA) interface resembling the junctional epithelium of the tooth. Engineering this type of attachment to percutaneous devices is attractive since the tooth is a natural example of percutaneous material, where bacterial invasion of bone is prevented by epithelial attachment.Acknowledgments:  NSF EEC 9529161 [University of Washington Engineered Biomaterials (UWEB)], NIH DK 59221, the George F. Odland Endowed Research Fund, and the Marvin and Judy Young Research Fund

Notes: Introduction:  Clinically, it has been observed that treatment with an occlusive dressing that results in hydration of the epidermis reduces scarring. A keratinocyte/fibroblast coculture system in which the epidermis is hydrated results in increased TNF production by keratinocytes, and reduced collagen synthesis by fibroblasts. We wished to test the hypothesis that in a nonhydrated coculture system, increasing TNF expression would result in decreased collagen synthesis by fibroblasts. Both keratinocytes and fibroblasts play essential role in scar formation. However, little is known about how TNF-alpha specific communication between these cell types modulates scar formation. To better elucidate this interaction, we used a keratinocyte/fibroblast coculture system to assess fibroblast function in a milieu of TNF-alpha overexpression by keratinocytes.Methods:  Primary human keratinocytes were transfected with adenovirus (MOI = 10) containing cDNA for TNF-alpha (AdTNF-alpha) or with LacZ and then cocultured with human dermal fibroblasts for 6 days.Results:  TNF-alpha transfected keratinocytes and their corresponding conditioned media showed a 3000-fold increase of TNF-alpha expression and synthesis in QRT-PCR and ELISA. Subsequent antibody blocking experiments with a specific TNF-alpha antibody showed a dose-dependent increase in collagen type 1 protein in the conditioned media. Col-1A1 mRNA expression in dermal fibroblasts was significantly reduced (p 〈 0.05).Conclusion:  Our data demonstrate that in a coculture setting, TNF-alpha expression by keratinocytes reduces Col-1A1 expression in fibroblasts and suggests a mechanism for clinical finding on the scar-reducing effects of hydration. This may bolster support for a TNF-alpha-mediated therapeutic intervention to ameliorate wound scarification.

Notes: A delicate balance between synthesis of extracellular matrix (ECM) and degradation by matrix metalloproteinases (MMPs) is a key factor in maintaining the structural integrity of normal skin. Epidermal-mesenchymal interactions play a critical role in controlling the expression of MMPs during development and healing of skin. Disruption of this interaction increases the frequency of developing fibrotic conditions such as hypertrophic scarring and keloids. In the absence of epithelialization, ECM continues to accumulate until dermal fibroblasts receive signal(s) from epidermal cells to slow down the dynamic process of maturation and remodeling of the healing wound. We have recently demonstrated that keratinocyte releasable stratifin stimulates MMP-1 expression in dermal fibroblasts. However, the molecular mechanism by which stratifin protein induces MMP-1 expression in fibroblasts is unknown. Therefore, the purpose of the present study is to identify elements of the signaling pathway mediating stratifin stimulation of fibroblast MMP-1 expression. We did so by examining the three distinct MAPK pathways: ERK1/2, JNK, and p38 as well as the expression of the main components of the AP-1 dimers, c-Jun and c-Fos, which are mediated by distinct MAPK pathways. Our data shows that treatment of fibroblasts with stratifin resulted in rapid and transient up-regulation of c-jun and c-fos mRNA levels. Furthermore, we show that stratifin activates fibroblast MMP-1 expression at the mRNA and protein levels and this is mediated by p38 MAP kinase. Subsequent cDNA microarray analysis of fibroblasts treated with stratifin show an increase in a ternary complex factor, Sap-1. In conclusion, our results describes the mechanism by which stratifin activates MMP-1 in fibroblast, and demonstrates that p38 MAP kinase is an important regulator of MMP-1 gene expression involved in epidermal-mesenchymal interactions in wound remodeling.Acknowledgment:  This work was supported by the Canadian Institute of Health Research (CIHR).

Notes: The Hypoxia Inducible Factor (HIF) system has been characterized as the principal tissue level response to hypoxia. Posttranslational regulation of HIF-1alpha has been reported to act though a Hypoxia Responsive Element (HRE) promoter region on a range of hypoxia-induced genes. A plasmid was constructed consisting of a fivefold HRE repeat conjugated to a luciferase gene, used as a marker for HRE activation. Plasmid HRE-luciferase was then transfected into a well-established ischemic rabbit ear wound healing model. Ischemia was induced using a variety of published models for interruption of arterial inflow and compared with a nonischemic control. Central artery, caudal artery and rostrocaudal artery models were tested for induction of ischemia. Tissue specimens were harvested from the transfected areas and solubilized. Luminescence of each specimen was measured using a standard luminometer to quantify luciferase induction. To provide correlation on a regional level, tissue level oxygen tension was measured directly for each wound model. Profound and statistically significant differences were found in the induced luciferase production. Marked differences in the tissue hypoxia between the various ischemic wound healing models correlated in graded fashion with the ischemia induced on an anatomic basis. The least ischemic model showed no significant difference in hypoxia readings versus control. The intermediate model showed a significant fourfold greater ischemia signaling. The most ischemic model showed a highly significant 72-fold greater ischemic response compared to controls. The use of gene transfection is described as a sensitive and effective method for quantification of tissue hypoxia at a cellular level in ischemic wounds.Acknowledgments:  This study was funded by the Wound Healing Research Laboratory.

Notes: Impaired wound healing is a problem for immobilized patients, diabetics, and the elderly. The 43 amino acid angiogenic peptide thymosin β4 has previously been found to promote accelerated dermal wound repair in rats, aged mice and db/db diabetic mice, and corneal repair in normal rats. It has been found in great abundance in wound fluid. Here, we hypothesized that thymosin β4 may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Western blot analysis of keratinocytes, endothelial cells, and fibroblasts that were treated with increasing concentrations of thymosin β4 showed changes in the expression of the MMP-1, −2, and −9. Zymographic analysis of whole excised mouse wounds taken after homogenization also showed changes in MMP-2 and-9 expression over a 3-day period. These results were confirmed in 2-day-old wounds by RT-PCR. We conclude that part of the wound healing activity of thymosin β4 resides in its ability to increase protease activity. Since thymosin β4-induced protease activity can be further controlled by inflammatory cytokines, a regulatory role for thymosin β4 is proposed in wound healing. These studies suggest that thymosin β4 may play a pivotal role in extracellular matrix remodeling during wound repair and may be effective in the treatment of chronic wounds in humans.

Notes: The large bulk of lost craniofacial soft tissue is mainly adipose tissue. Current surgical approaches following tumor resection and trauma have limitations. Adipose derived-stromal cells (ADSCs), also named as preadipocytes, have been demonstrated to be a good cell candidate for adipose tissue engineering. However, characteristics of scaffold played important role in a successful tissue engineered adipose tissue.Objectives:  The present study is to investigate whether in vitro adipogenesis could be induced both in a monolayer and 3-D by ADSCs.Methods:  Healthy adult ADSCs were isolated by liposuction from fat pads. After discarding the unattached cells, the ADSCs were cultured in DMEM supplemented with 10% FBS and 1% antibiotics until they reached confluence. After trypsinization, ADSCs were subcultured at a density of 105 cells/100 mm-dish as a first passage. The monolayer and 3-D adipogenesis were assessed after seeding 105 ADSCs into each well of 6-well plates and 5 × 106 ADSCs/ml into biodegradable collagen sponges, respectively, followed by replacement of basic medium with adipogenic one.Results:  In both monolayer and 3-D scaffolds, lipids containing adipocytes were observed 1 week after adipogenic stimulation, and further increased over the time. By contrast, no adipogenesis was observed in ADSCs cultured in basic medium.Conclusions:  Human adipose-derived stromal cells isolated from fat pads can readily be induced toward adipogenesis in vitro. The in vivo adipogenesis potential of ADSCs is being investigated.(Supported by Whitaker Biomedical Engineering Foundation, March of Dimes Foundation)

Notes: Background:  Apligraf, a bioengineered living skin construct, has been shown to accelerate the healing of venous leg and diabetic foot ulcerations. It appears to function as cell-based therapy (e.g., delivering growth factors), rather than as a true graft. Given this mechanism of action, its use could be expanded to areas in which traditional skin grafting is not routinely employed.Methods:  A 64-year-old wheel-chairbound Caucasian male presented to the wound clinic with a grade III left trochanteric pressure ulcer which had been present since 1976. He had been seen by numerous physicians and treated with debridement, topical antimicrobials, negative pressure therapy, growth factors, and a variety of dressings. We spent several months preparing the wound bed: maintaining adequate moisture balance, performing serial debridement, utilizing offloading surfaces and reducing the bacterial burden. His nutritional status was optimized. The wound bed developed a healthy granulating base but failed to close.Results:  A single unit of Apligraf was meshed at a 1:1.5 ratio and applied to the wound. It was fixed in place using a nonstick silicone dressing, foam, and a self-adhesive covering. The dressing was changed weekly in the wound clinic. The initial response was a decrease in wound depth followed by steady epithelialization. Complete closure occurred 17 weeks after grafting. The wound has not recurred.Conclusion:  This single case study suggests that bilayered cell therapy may have application in difficult wounds, such as pressure ulcerations. Further study into the efficacy of Apligraf in pressure ulcerations is ongoing.

Notes: Various matrices containing both synthetic or natural polymers are currently used for local regional drug delivery to a number of disease locations such as chronic cutaneous wounds, joints, bone, muscle, and nerves. Insert Therapeutics has developed a novel synthetic matrix which consists of a copolymer of β-cyclodextrin and polyethylene-glycol (PEG). Addition of di- or multifunctionalized adamantane-PEG molecules results in noncovalent cross-linking through inclusion complex formation. The resulting hydrogel showed rheological characteristics amenable to topical application or local-regional injection into a variety of tissues (Bellocq et al. (2004) Bioconjug Chem 15(6): 1201–1211).This matrix was shown to be biocompatible, allowing for cellular growth and migration in vitro. It also allowed for efficient delivery of an adenovirus gene therapy vector to dermal fibroblast cells. In vivo, the matrix demonstrated efficient delivery of biotherapeutics such as recombinant adenovirus and non-viral gene therapy vectors after intradermal injection. The matrix was well tolerated and was as efficient as collagen in promoting wound healing when an adenoviral gene therapy vector expressing PDGF-bb was delivered to cutaneous wounds of diabetic mice.The matrix can additionally be modified to incorporate therapeutic small molecules, peptides, or proteins, either through inclusion complex formation or chemical linkage. Using biodegradable linker chemistry, the release rate of covalently attached molecules can be controlled. The cyclodextrin-PEG matrix is therefore an attractive alternative to existing matrices that is biocompatible, biodegradable, tunable with regard to its physicochemical properties, and can be designed to deliver multiple therapeutic agents to a variety of tissues in a controlled fashion.

Notes: Neuropathic ulcers represent a significant cause of morbitity for diabetics in the United States. Diabetic wounds are notoriously difficult to resolve. Treatment options are limited due in part to the lack of knowledge about the molecular events that regulate successful resolution of wounds. To identify differences between normal and diabetic gene regulation in wounds, we examined the level of expression of 12,000 genes in granulation tissue and wound-edge epithelium in normal animals and in an experimental model of diabetes following induction of full thickness skin injuries. Full thickness injuries in diabetic mice were characterized in part by altered regulation of structural genes and genes that regulate energy metabolism and utilization. The energy cycle contributes backbone structures for the synthesis of a variety of amino acids, the molecular building blocks essential for wound repair. A direct consequence of a diminished ability to efficiently exploit energy supplies would be decreased availability of regulatory and structural proteins necessary for efficient healing in diabetic wounds. We have successfully enhanced healing in diabetic wounds by treatment with the angiotensin II peptide NorLeu3A(1–7). NorLeu3A(1–7) accelerates healing of full thickness injuries in diabetic mice. While NorLeu3A(1–7) enhances healing by accelerating collagen deposition and reepithelialization, gene array studies show that NorLeu3A(1–7) acts locally within the wound site by altering gene expression within the granulation tissue. Specifically at day 7 after injury, NorLeu3A(1–7) reduced the expression of MMP genes and increase the expression of genes involved in energy metabolism.Supported by PHS grant NIAMS 2 R44 AR47481

Notes: Antimicrobial peptides are major components of the innate defense system that present microbicidal activity against gram positive and negative bacteria, yeast, and some enveloped viruses. The human cathelicidin LL-37 is expressed in skin upon wounding and, in addition to its role in antimicrobial defense, it has also been previously demonstrated that this peptide might be involved in reepithelization. We have generated an adenoviral vector containing the complete sequence of LL-37 along with an Ires-GFP expression cassette. The efficacy of this vector was probed by testing the conditioned medium from adenoviral transduced keratinocytes in preventing bacterial growth. We have studied the in vitro effects of LL-37 on HK migration and proliferation. In addition, we have efficiently transduced human skin grafts (in immunodeficient mice) using an in vivo gene transfer approach and we are currently testing the involvement of this peptide in wound repair.

Notes: Fluorescence correlation spectroscopy was used to measure the binding and diffusion of growth factors in model extracellular matrices in order to investigate the importance of protein-matrix interactions in regulating signaling molecules within a three-dimensional matrix. Two important growth factors were studied, transforming growth factor β1 and basic fibroblast growth factor, which are known to have specific affinities for fibronectin and the heparan-sulfate-proteoglycan perlecan, respectively. Collagen-based matrices were prepared by polymerizing type I collagen in the presence of fibronectin or perlecan, and we measured diffusion constants and binding constants of the two growth factors. The binding constant measured for transforming growth factor β1 with fibronectin-containing matrices was in good agreement with that measured using affinity chromatography. However, the interactions measured between fibroblast growth factor and perlecan were significantly weaker than expected. Surprisingly, the strongest interactions by far were with monomeric collagen solutions and fibrillar collagen matrices. Our findings suggest a central role for the three-dimensional fibrillar collagen matrix in growth factor interactions, with modulatory roles for fibronectin or perlecan.

Notes: Platelet-derived growth factor (PDGF-B) is a potent cell mitogen and chemotactic signal involved in normal wound healing. PDGF-B is present at decreased levels in diabetic wounds and has been shown to improve wound healing when applied in a topical form. However, clinical results with topical PDGF in the treatment of diabetic wounds have been equivocal. In this study, we investigated whether application of PDGF-B via gene therapy can effectively promote wound healing in the diabetic environment in an animal model. Retroviral vectors carrying the gene for human PDGF-B were constructed using the LNCX virus and the G418 resistance gene for selection (LN-PDGF-B), and transduced into mouse dermal fibroblasts. LN-PDGF-B transduced fibroblasts were seeded into alginate hydrogel scaffolds at a concentration of 25 × 106 cells/implant and implanted into 2 cm × 2 cm full thickness excisional dermal wounds created on the dorsae of genetically diabetic db/db mice. At 21 days, animals treated with LN-PDGF-B transduced cells suspended in alginate hydrogel demonstrated a significantly smaller epithelial gap (0.97 +/− 0.34 cm) than animals receiving untransduced mouse dermal fibroblasts alone (1.44 +/− 0.37 cm; P 〈 0.05) or animals receiving dermal fibroblasts transduced with LNCX viral vector alone (1.40 +/− 0.20 cm; P 〈 0.01). These results suggest tissue-engineered retroviral gene therapy with PDGF-B specifically promotes diabetic wound healing and may yield advances in the effective treatment of diabetic wounds.Supported by a grant from the American Diabetes Association

Notes: Nitric oxide (NO) is a critical mediator of normal tissue repair and is inhibited by homocysteine (Hcy). Following 8 weeks of lower extremity ulcer (LEU) treatment with human fibroblast-derived dermal substitute (Dermagraft®) for 12 (n = 12) patients, a correlation was observed between patients with a poor wound healing response to Dermagraft and elevated serum homocysteine (Hcy). The responders group (R) to Dermagraft treatment (n = 6) was observed with robust granulation tissue, epidermal migration and a 67% rate of healed ulcerations. All R-group patients (6/6) had normal serum Hcy. The nonresponders (NR) group (n = 6) exhibited poor granulation tissue formation and epidermal migration and a complete absence of healed wounds. Five of the NR patients (5/6) were observed with elevated serum Hcy. There was no significant (p 〈 0.05) difference between the wound areas (cm2 ± SE) of each group [35.43(±28.99)-R vs. 19.90(±7.55)-NR]. After 2 weeks of treatment, the R-group demonstrated significantly greater %Δwound area than the NR-group [62.17 (±7.96)-R group vs. 23.17(±8.82)-NR group; p 〈 0.05]. Wound fluid nitrate (WFNOx), a surrogate measurement (μM ± SE) for local NO bioactivity, was significantly lower for the NR-group than the R-group [3.17(±1.46)μM-NR group vs. 12.98(±1.73)μM-R group; p 〈 0.05]. In a large group of LEU patients (n = 138) we observed a 50% incidence of elevated Hcy; 69% for diabetic neuropathic LEU patients and 47% for nondiabetic LEU patients. Hcy inhibits NO production by multiple pathways and may also inhibit wound repair by occupying the fibronectin domain of fibrin during provisional matrix formation. Our study suggests that elevated serum Hcy is a common finding in chronic LEU patients. The high Hcy may contribute to impaired wound healing by decreasing wound NO production and, perhaps, by inhibition of fibronectin-fibrin-mediated wound matrix development.

Notes: Atherosclerotic plaque formation involves recruitment and adhesion of circulating monocytes to sites of blood vessel wall injury followed by their migration into the subendothelial space stimulated by MCP-1 (monocyte-chemoattractant-protein1). Monocytes differentiate into macrophages, cells that release a variety of factors and take up oxidized LDL, becoming foam cells. Smooth muscle cells of the vessel wall differentiate and migrate to the subendothelial space, interact with foam cells and components of the ECM to form a “fatty streak,” the precursor of plaque. Adiponectin, a protein secreted by adipocytes, circulates in the blood, maintains blood vessel wall stability and inhibits foam cell formation. Cigarette smoke is a major risk factor of atherosclerosis, although the mechanisms remain unknown. We showed previously that both firsthand and secondhand cigarette smoke stimulate MCP-1 in endothelial cells and others have shown that adiponectin levels in smokers with cardiovascular disease are reduced. We hypothesized that a localized increase in MCP-1 and a decrease in adiponectin are critical for cigarette smoke-induced plaque formation. To test this possibility, we used mice transgenic for human ApoB100, a model system that closely mimics human conditions that lead to atherosclerosis, and a smoking machine that closely simulates human smoking. We found that, in smoking mice, the micro vessels in the heart tissue are often filled with lipids, the areas surrounding the plaque-prone regions have numerous neutrophils, that these cells express high levels of MCP-1, and that the plaques are larger than in control mice. In addition, the level of the adiponectin monomer in the blood of smoking mice is lower and the presence of this protein in the heart tissue is also decreased. In conclusion, the decrease in adiponectin and the increase in MCP-1 levels may be responsible for cigarette smoke-induced atherogenesis.

Notes: The treatment of venous ulcers must start with compression and if the ankle brachial index is greater than 0.8 high compression bandages can be applied. Despite edema control, there are a number of venous ulcers that do not heal at the expected rate. Patients with venous ulcers of greater than 4 weeks duration were treated with a prolonged release absorptive nanocrystalline silver dressing (Acticoat 7) under the 4 layer bandage, Profore for 12 weeks, or until healing. Biopsies were obtained from the ulcer base at week 0 for histology and bacterial burden. Duplicate biopsies for quantitative bacteriology were performed with one submitted whole and the second bisected into superficial and deep components. The paired biopsies were then repeated after a median of 6.5 weeks (range 2 to 12 weeks). The histological specimens were examined by the histopathologist (SR). Inflammatory infiltrates were identified in the superficial, middle and deep segments of the biopsies. Acute infiltrates were identified through the concentration of neutrophils and chronic infiltrates by the presence of lymphocytes. Each biopsy and each segment was graded for infiltrates on a four point semi quantitative score. A total of 15 patients (9 male, and 6 female) were enrolled into the study. The median age was 63 years (range 30–83 years). The median duration of current ulceration was 17.3 weeks (range 4 weeks to 11 years) and the median ulcer area was 4.8 cm2(range 1.8–43.9 cm2). The median exposure to Acticoat 7 was 82 days (range 8–86 days).There were 12 sets of paired biopsies that were analyzed. There was a statistically significant reduction (p = 0.0114) in the log10(total bacterial count) between the baseline and final biopsies (median 4.48 and 3.00, respectively). Four patients healed, 8 patients continued to the end of the 12-week study period and three patients were discontinued early. For all patients, the median percentage reduction in ulcer area was 94.4% and the median final ulcer area was 0.4 cm2. Analysis of the histology and bacteriology data demonstrated that the presence of a high neutrophilic infiltrate in skin biopsies was associated with high bacterial counts (superficial compartment of the quantitative biopsies) at week 4 and delayed healing (p = 0.037). In the week 0 biopsy, increased lymphocytic infiltrates within the superficial and middle segments were associated with accelerated healing in the first 4 weeks (p = 0.26 and 0.09). The nanocrystalline silver dressing has demonstrated an anti-bacterial and permissive but selective anti-inflammatory action allowing lymphocytic infiltrates to increase associated with an accelerated reduction in ulcer size.

Notes: Advocate Christ hospital is a 650-bed, level-one tertiary-care center that created a comprehensive wound care program in November 1998. Over the first 3 years of the program there were over 140 admissions per year directly from the wound center along with numerous hospital to hospital transfers for complex wound management. Prevalence rates therefore increased linearly over this time frame. The wound team calculated a system-wide (eight hospitals) spend of $2.1 million per year for specialty rental beds. Incidence rates for the system were at, but not lower than, national levels despite the high cost incurred. A $1.3 million capital purchase for KCI atmos-air 9000/〉 mattresses resulted in the complete replacement of all medical surgical beds in all eight hospitals. An internal prevalence and incidence team was created and data were collected both at the hospital and system-wide level on a quarterly basis. Hospital and system incidence rates fell to or below 7%(targeted goal) and the entire project will achieve a return on investment in 18 months. Data to be presented include prevalence and incidence results, rental bed costs pre- and postbed purchase, and a detailed review of the process of creating an electronic prevalence and incidence tracking system.Acknowledgments:  This study was not supported by any outside grants.

Notes: We have previously shown that resident fibroblasts from human chronic wounds are phenotypically abnormal, displaying an impaired response to TGF-beta 1, decreased TGF-beta Type II receptor expression, and decreased phosphorylation of Smad2, Smad3, and p42/44 MAPK. We now report that these human cells delay healing by approximately 30% when implanted in mouse incisional wounds and in a dorsal flap model. These new findings point to the need for the transplantation of new healthy cells in human chronic wounds. Bioengineered living skin constructs can deliver new cells to chronic wounds, at least for a limited period of time. However, most available bioengineered skin constructs consist of already differentiated allogeneic cells, which do not allow for the possibility of engraftment and reconstitution of wound structures. Recently, in a preliminary report, we have shown that autologous bone marrow-derived cells can dramatically enhance the healing of recalcitrant chronic wounds when all other treatments have failed. Our present efforts are now focused on characterizing the bone marrow cells involved in this stimulation of wound healing and in developing better ways for their topical delivery to the wound. Here we present evidence that a fibrin construct may be an ideal way of applying bone marrow-derived cells to human chronic wounds. Autologous bone marrow cells are allowed to grow in culture under conditions favoring the survival and proliferation of mesenchymal stem cells (MSC), as shown by surface markers and functional studies. This usually requires two weeks after bone marrow harvesting. The cellular component is then placed in a fibrinogen solution which, when simultaneously combined with thrombin through a CO2 driven flow, delivers a fine cell-containing fibrin polymer film to the wound. In vitro and in vivo experiments, both in mouse models and in human wounds, show the feasibility of this approach.

Notes: Bone marrow stromal cells (MSCs) are a promising cell resource of osteoprogenitor cells for bone tissue engineering. However, the diverse characteristics of osteoprogenitor cells within the bone marrow of individual subjects require varying treatments to stimulate osteogenic differentiation. Thus, an effective monitoring system is needed to identify the progression of osteogenesis. Magnetic resonance (MR) microscopy was used in the present study to monitor osteogenesis of tissue engineering (TE) constructs prepared by human bone MSCs seeded on scaffolds of gelatin sponges. The characteristics of MR images and parameters corresponded to osteogenic progression of TE constructs exposed to differentiation medium, significantly differing from control groups exposed to basic medium. Upon quantification, MR image and parameters correlated well to cell seeding densities and alkaline phosphatase activities of various TE constructs. In conclusion, MR can effectively detect the biochemical cascades of osteogenic differentiation of TE constructs and may be a promising, noninvasive monitoring system to provide three-dimensional information for bone tissue engineering.

Notes: Reactive oxygen species (ROS) play a major role in recruiting and activating inflammatory cells, inducing angiogenesis, fibroblast proliferation, and collagen synthesis, all processes essential to wound healing. However, the same mediators have been implicated in pulmonary and hepatic fibrosis, vascular occlusion, and necrotic tissue damage. We have previously shown that hyperbaric oxygen caused a prolonged elevation of VEGF and delayed wound healing in a rat model of tissue ischemia. In this study we examine the effect of the ROS scavenger, n-acetylcysteine (NAC), on ischemic wound healing. NAC is a virtually nontoxic free radical scavenger that has been used in many experimental studies to block ROS-mediated signaling.Methods:  Male Sprague-Dawley rats underwent creation of the ischemic flap previously validated by this laboratory. This model provides two ischemic and two nonischemic excisional wounds. Rats were treated daily with HBO for 90 minutes at 2.4 atm, HBO plus 150 mg/kg NAC intraperitoneal, or control (neither HBO nor NAC). Wounds were analyzed for surface area, lactate, and VEGF.Results:  The HBO/NAC-treated animals had markedly impaired healing of their ischemic wounds at day 7 (0.105 ± .005 cm vs. 0.068 ± .002 cm for ctl and 0.064 ± .006 for HBO) and a twofold elevation of VEGF (120 pg/mg protein vs. 60 pg/mg protein). Lactate levels were not altered by NAC treatment and non-ischemic wounds showed no difference in healing, lactate, or VEGF.Conclusion:  Reactive oxygen species are required for wound healing. Our initial hypothesis was that the delay in wound closure with HBO treatment was due to excess ROS and that NAC would improve healing. The finding of elevated VEGF and delayed wound healing in the presence of a potent free radical scavenger suggests that blocking ROS signaling prevents the normal mitogenic response to VEGF. Additional studies to examine the VEGF receptor and tyrosine kinase activation will further elucidate the mechanism of ROS signaling in response to HBO treatment.

Notes: The Thermage system is a radiofrequency (RF) device designed to treat human skin. The Thermage ThermaCool TC™ is a noninvasive, nonablative, nonlaser system that combines cryogen cooling and radiofrequency (RF) energy to volumetrically heat dermal and subdermal tissue. Thermage treatment causes collagen contraction resulting in immediate skin tightening. It has also been clinically shown to tighten skin over time. This long-term tightening effect is believed to result from the body’s wound healing response. We performed studies in a juvenile pig to characterize the wound healing response after ThermaCool treatment in order to elucidate the cellular and molecular events resulting from treatment. Histological markers of the wound healing process were examined over a 1-month period. Collagen gene expression was examined by quantitative PCR.Macrophages and mast cells are important inflammatory cell types that stimulate fibroblast recruitment, collagen synthesis and blood vessel growth. Three to 5 days following treatment, macrophages and mast cells were present in the dermis. By 28 days, both increased dermal blood vessel density and epidermal thickness was observed. Collagen I and collagen III genes were expressed at elevated levels by 21 days posttreatment.ThermaCool™ treatment stimulates dermal remodeling by initiating a wound-healing response. This study demonstrates that thermage treatment results in the recruitment of important cellular mediators of wound healing and increased collagen synthesis in an animal model. These events are presumed to be responsible for dermal remodeling and skin tightening over time in human subjects. Because the Thermage device noninvasively delivers heat energy, it may be a unique tool for the examination of aseptic thermally induced wound models.

Notes: Induced organ regeneration is de novo synthesis of a physiological, or nearly physiological, organ at the same anatomical site as the organ that is being replaced. Regeneration of skin, peripheral nerves, and the conjunctiva have been accomplished using nothing more than biologically active scaffolds (regeneration templates) seeded with epithelial cells; devices for regeneration of the first two organs are in clinical use. Templates appear to function by blocking contraction well as by acting as temporary configurational guides for synthesis of new stroma that resembles that of the organ under replacement. The combined evidence supports a theory which predicts that selective blocking of the adult healing response uncovers part, at least, of the latent fetal response to injury and, in the presence of the appropriate scaffold, eventually leads to organ regeneration. An independent theory suggests that loss of regenerative potential, which is observed during the mammalian fetal-adult transition, is associated with simultaneous acquisition of individual immunocompetence.

Notes: Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated collagen matrix contraction is independent of Rho kinase whereas platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase dependent. The current studies were carried out to learn more about the molecular motors responsible for LPA- and PDGF-stimulated fibroblast-collagen matrix contraction. Fibroblasts whose MLC kinase was knocked down using siRNA were used to measure matrix contraction in the presence of LPA or PDGF with or without the Rho kinase inhibitor added. The extent of contraction of MLC kinase-silenced cells was not detectably different from control cells. Other experiments were carried out to test the effects of LPA and PDGF on MLC phosphorylation with and without Rho kinase inhibitor. After 15 min of growth factor stimulation, levels of diphosphorylated MLC were highest in cells in LPA-containing medium and lowest in PDGF containing medium. Prior addition of Rho kinase inhibitor markedly reduced phosphorylation in every case. These observations suggested that stimulation of collagen matrix contraction required neither growth factor stimulation of MLC phosphorylation nor MLC kinase.

Notes: Although the pathogenesis of chronic ulcer still remains undefined, one of the characteristics is poor granulation of wound bed, in other words, disruption of wound angiogenesis. Experimental studies have shown that bone marrow (BM) derived endothelial progenitor cells take part in postnatal neovascularivation in cutaneous wound healing. BM cells also contain mesenchymal stem/progenitor cells with pluripotency differentiating into myofibroblasts and fibroblasts. We treated nonhealing leg and foot ulcers sustained over one year by topical transplantation of autologous fresh unfractionated BM-impregnated collagen sponge (BMiCS). In all patients, the treatments led to rapid generation of well-vascularized granulation tissue. All of the wounds were healed up completely with conservative treatment or skin grafting. We suggest that direct transplantation of autologous bone marrow cells on the wound may represent a novel procedure for regeneration of disrupted wound healing on recalcitrant cutaneous ulcers.

Notes: Aim:  Omental implantation, a surgical procedure in which a perforated gastric or duodenal ulcer is repaired by drawing and implanting a portion of the omentum into the digestive tract, accelerates ulcer healing and inhibits ulcer recurrence compared with omental patch from clinical results. To clarify these mechanism and differences, we investigated ulcer healing in two groups.Methods:  In two groups of rats in which acetic acid-induced gastric ulcers were perforated. Omental implantation was used for repaired in one group and omental patch was employed in the other group. Basic fibroblast growth factor (bFGF) mRNA-positive cells were identified and localized by in situ hybridization. Fibroblast growth factor receptor (FGFR)-positive cells were identified and localized by immunohistochemical analysis.Results:  Antiinflammatory and angiogenic activity and accelerated collagen synthesis were seen in the omental implantation group. BFGF mRNA-positive cells (macrophages, fibroblasts, and endothelial cells) and FGFR-positive cells were seen within the omentum, resulting in abundant collagen production and rapid epithelial regeneration. In the omental patch group, extensive neutrophilic infiltration and ulcer recurrence were seen. Few bFGF mRNA-positive cells and FGFR-positive cells were seen within the omentum, resulting to inhibit omentum becoming to be granulation tissue and ulcer healing.Conclusions:  These results indicated that omental implantation accelerated ulcer healing, and the presence of bFGF mRNA and FGFR played a significant role in this phenomenon.