Amniotic membrane as part of a skin substitute for full-thickness wounds: an experimental evaluation in a porcine model.

BACKGROUND: We evaluated the use of human amniotic membrane (HAM) as a graft material for the treatment of iatrogenic full-thickness (FT) skin wounds in a porcine model with a view to reducing donor site morbidity in free flap transfer. METHODS: Forty experimental FT-wounds were covered with an autologous split-thickness skin graft (STSG) alone or in combination with a mono- or multilayer HAM or Integra(®). Untreated wounds served as controls. Clinical evaluation and biopsy-sampling for histological and immunohistochemical staining with von-Willebrand-factor (vWF) antibody, laminin antibody, Ki-67 antibody, and smooth muscle actin (αSMA) antibody were performed on days 5, 7, 10, 20, 40, and 60 after surgical intervention. RESULTS: Considerable disparities in the estimated criteria were observed between the various treatment groups of the FT-wounds. The use of HAM was found to have an accelerating impact on re-epithelialization. The multilayered amnion membrane showed better results than the Integra(®) and monolayer technique in terms of contraction rate, inflammation, and scarring and seemed useful as a dermal substitute in FT-wounds giving comparable results to STSG coverage alone. CONCLUSIONS: This study demonstrates the successful application of HAM as part of a skin substitute in FT-wounds in minipigs. The results offer promise as a simple and effective technique for the application of multilayer HAM in iatrogenic human skin defects and the acceleration of wound healing.

Human ß-defensins and psoriasin/S100A7 expression in salivary glands: anti-oncogenic molecules for potential therapeutic approaches.

BACKGROUND: Host defence peptides (HDPs), including human ß-defensins (hBDs) and psoriasin/S100A7, exert antimicrobial and immunoregulatory functions of the innate defense system. In addition to these functions, the search for cancer biomarkers has identified HDPs as playing a potential role in both tumor suppression and oncogenesis. Although HDPs are highly expressed in salivary glands, their role as molecules for potential diagnostic and therapeutic approaches has not yet been analyzed. OBJECTIVE: The aim of the present study was to investigate whether expression levels of putative pro- or anti-oncogenic hBDs, including hBD-1, -2, -3, and psoriasin/S100A7, are altered in salivary gland tumor tissue as potential targets for molecular-based therapeutic approaches. METHODS: We analyzed the expression levels of hBD-1, -2, -3, and psoriasin/S100A7 by quantitative real-time polymerase chain reaction (qrt-PCR) and immunohistochemistry in a case control study by comparing salivary gland tumor samples relative to healthy control specimens from 58 patients. Expression level analysis of hBD-1, -2, -3, and psoriasin/S100A7 by qrt-PCR was normalized to the endogenous 18S rRNA expression levels. RESULTS: The results demonstrate the significant downregulation of hBD-1 (p < 0.001), hBD-2 (p = 0.003), hBD-3 (p = 0.002), and psoriasin/S100A7 (p = 0.003) mRNA in human salivary gland tumors compared with healthy control specimens. Protein expression levels of hBD-1, -2, -3, and psoriasin/S100A7 in salivary gland tumor tissue were strongly reduced compared with healthy control specimens. CONCLUSION: The data indicates a putative role of the innate defense system in salivary gland tumor formation. The identification of immunoregulatory molecules as diagnostic biomarkers or therapeutic targets could provide new approaches for molecular-based diagnostic and therapeutic support to treat salivary gland tumors as well as other malignancies. We suggest that HDPs should be taken into consideration for use in molecular-based therapeutic approaches.

The human submandibular gland: immunohistochemical analysis of SNAREs and cytoskeletal proteins.

Submandibular acinar glands secrete numerous proteins such as digestive enzymes and defense proteins on the basis of the exocrine secretion mode. Exocytosis is a complex process, including a soluble NSF attachment protein receptor (SNARE)-mediated membrane fusion of vesicles and target membrane and the additional activation of cytoskeletal proteins. Relevant data are available predominantly for animal salivary glands, especially of the rat parotid acinar cells. The authors investigated the secretory molecular machinery of acinar (serous) cells in the human submandibular gland by immunohistochemistry and immunofluorescence and found diverse proteins associated with exocytosis for the first time. SNAP-23, syntaxin-2, syntaxin-4, and VAMP-2 were localized at the luminal plasma membrane; syntaxin-2 and septin-2 were expressed in vesicles in the cytoplasm. Double staining of syntaxin-2 and septin-2 revealed a colocalization on the same vesicles. Lactoferrin and α-amylase served as a marker for secretory vesicles and were labeled positively together with syntaxin-2 and septin-2 in double-staining procedures. Cytoskeletal components such as actin, myosin II, cofilin, and profilin are concentrated at the apical plasma membrane of acinar submandibular glands. These observations complement the understanding of the complex exocytosis mechanisms.

Decreased cognitive functioning in patients with advanced oral squamous cell carcinoma.

Cognitive impairment causes a delay in diagnosis and treatment of the various cancer entities, resulting in reduced surgical outcomes and patient survival. However, no investigations have been carried out as to whether an association exists between cognitive functioning and tumour size in patients with oral squamous cell carcinoma (OSCC). In this study, 46 patients with OSCC were evaluated by using a screening test for dementia, consisting of a combination of the mini-mental state examination and the clock test (81% sensitivity and 90% specificity). Test scores were correlated with tumour size according to the TNM staging system, which was categorized as being either limited (T1, T2; n=24) or advanced (T3, T4; n=22). No difference in age (P=0.172), sex (P=0.330), the percentage of drinkers (P=0.090) or the percentage of smokers (P=0.484) was evident between the groups. Patients with advanced tumour size scored significantly lower (median 5.5 of 9 possible points) when compared with those having tumours of a limited size (median 9 of 9 possible points; P=0.005). The median score of patients with T3/T4 tumours suggested the need for comprehensive neuropsychological evaluations for dementia. In conclusion, this study has demonstrated the correlation of reduced cognitive functioning in patients with advanced OSCC. As a consequence, instructions for the identification of early signs and of symptoms of oral cancer are strongly recommended for relatives and nursing staff of patients with cognitive impairment. Such patients might need immediate treatment for oral cancer but might not be able to understand the significance of their symptoms and therefore present late, often too late.

Use of microvascular flap technique in older adults with head and neck cancer: a persisting dilemma in reconstructive surgery?

OBJECTIVES: To compare perioperative problems and outcomes of reconstructive surgery with microvascular flaps of a group of older (≥ 70) and younger adults (20-69). DESIGN: Prospective clinical cohort study. SETTING: Maxillofacial surgical unit of a university teaching hospital in Munich, Germany. PARTICIPANTS: Two hundred fifteen people with head and neck carcinoma (older: n = 54, mean age 75.8, range 70-96; younger: n = 161, mean age 55.5, range 20-69) who underwent surgery between 2007 and 2009. MEASUREMENTS: Participant characteristics: age, sex, American Society of Anesthesiologists (ASA) status, tumor type, preoperative radiation or chemotherapy, medical comorbidities. Surgical variables: flap type, type of reconstruction (primary/secondary), length of operation (minutes). Postoperative variables: length of stay (minutes) on intensive care unit (ICU), reasons for ICU stay longer than 1,500 minutes (surgical or medical), length of hospitalization (days), and reasons for hospitalization longer than 20 days (surgical or /medical). Short-term outcome within 30 days: revisions, flap success, overall complication rate, mortality. RESULTS: Older adults had a higher ASA class (P < .001) and shorter duration of surgery (P = .02). Age as an independent factor prolonged stay on ICU (P = .008) and was associated with a higher complication rate (P = .003) but had no influence on length of hospitalization, flap success, need for revisions, or mortality. CONCLUSION: Although higher rates of peri- and postoperative difficulties must be expected when microvascular reconstructive surgery is considered for older adults, careful surgical technique, adequate postoperative surveillance, and immediate management of complications can facilitate outcomes comparable with those for younger adults.

Microsurgical reconstruction of the oral cavity with free flaps from the anterolateral thigh and the radial forearm: a comparison of perioperative data from 161 cases.

BACKGROUND: Free flaps are commonly used for reconstruction of extensive tumor resection defects in the oral cavity. The radial forearm free flap (RFFF) is the most frequent choice. However, a major problem of RFFF is a limitation in its size. The anterolateral thigh free flap (ALTFF) has become popular as an alternative donor site in maxillofacial surgery. We have compared patient data after reconstructions of the oral cavity using the RFFF or ALTFF. MATERIALS AND METHODS: Perioperative data of 161 oral cancer patients with ALTFF (45) or RFFF (116) reconstructions were reviewed and statistically analyzed for the following characteristics: sex, histology, primary tumor localization, defect type, American Society of Anesthesiology score, success rates, revisions, wound healing disorders, fistula rates, type of reconstruction, prolonged stay in an intensive care unit (ICU) and in hospital, donor site, flap size, length of operation, and number of follow-up visits. RESULTS: Flap success was 97.8% (44 of 45) in the ALTFF group and 97.4% (113 of 116) for RFFF. The mean size was higher in ALTFF than in RFFF (110 vs. 29 cm(2); P < 0.001). ALTFF needed less postoperative care and developed fewer wound healing disorders (P = 0.005 and P = 0.035). Operative time was significantly shorter in RFFF reconstructions (P = 0.020). Intraoperative arterial spasm was the most significant complication in ALTF and postoperative venous thrombosis in RFFF. CONCLUSIONS: ALTFF has distinct advantages over RFFF with respect to intraoral reconstruction. However, the RFFF remains as a very reliable flap because of the minimal variability in its anatomy.

Expression of host defence peptides in the lip vermilion mucosa during early infancy.

Emerging resistance to antibiotics has become a major problem. Host defence peptides (HDPs), which are effector molecules of the innate immune system, show broad antimicrobial activity. Synthetic derivates are currently being investigated as new anti-infectious agents. In infants, the use of conventional antibiotics is limited to a few substances because of adverse reactions. The new HDP substances might become alternatives to conventional antibiotics, but knowledge of the physiological quantities of the HDPs in infants is essential because of a narrow therapeutic index of currently available derivates. This study compares the mRNA levels of five major HDPs between infants and adults to test the hypothesis that HDP gene expression differs between these groups. Expression profiles of human beta-defensin (hBD)-1, hBD-2 and hBD-3, psoriasin and RNase 7 were assessed in the lip vermilion mucosa of infants (n = 15) and adult controls (n = 15) using real-time polymerase chain reaction. A significantly lower expression of hBD-2 (P = 0.043), hBD-3 (P = 0.014) and psoriasin (P = 0.018) was found in infants. No difference between the groups was noted with respect to transcript levels of hBD-1 and RNase 7. In conclusion, several HDPs are expressed at lower levels in infants, but not all. The results emphasize the need to adjust the dose of agents based on the specific HDP level for the treatment of infantile infections.

Secondary maxillofacial reconstruction with the radial forearm free flap: a standard operating procedure for the venous microanastomoses.

BACKGROUND: The radial forearm free flap (RFFF) is commonly used in reconstructive surgery. With respect to the maxillofacial region, several venous anastomotic techniques (e.g., single or double anastomoses) have been described, but the debate as to which alternative is preferable is ongoing. A complicating factor is the unpredictable anatomical situation in patients undergoing secondary operation. No recommendations are available for the surgical strategy in such cases. We present a standard operating procedure (SOP) applicable for secondary reconstructions, postulating double anastomoses as the method of choice, and evaluate its efficacy. METHODS: The following parameters were retrospectively analyzed for 120 patients with secondary (41; 34.2%) or primary (79; 65.8%) reconstruction following the instructions of SOP and compared between the study groups: age; sex; history of radiotherapy; side of the donor arm; flap size; preparation and use of the cephalic vein and reasons for its non-inclusion; included venae comitantes; recipient veins; arterial anastomoses; revisions, flap survival, and mortality within thirty days after operation. RESULTS: The method of choice was applicable in 26 (63.4%) secondary and 52 (65.8%) primary reconstructions (no difference; P = 0.841), resulting in 100% flap viability in both groups. In the remaining cases, single venous anastomoses were performed, resulting in 73.3% flap viability in secondary and 100% in primary reconstructions. Flap survival in secondary reconstructions was significantly higher when double anastomoses were conducted (P = 0.012). CONCLUSIONS: The results suggest the necessity of double venous anastomoses in secondary maxillofacial reconstructions with RFFF.

Repair of oronasal fistulas with human amniotic membrane in minipigs.

We evaluated the use of multilayer human amniotic membrane (HAM) as a grafting material for the repair of mid-palate oronasal fistulas in seven Berlin minipigs. After two weeks, three animals had the fistulas repaired with multilayered HAM grafts, three had them repaired with a collagen-based dermal substitute (INTEGRA((R)), Integra Life Sciences, Plainsboro, NJ, USA), and one fistula was left untreated to serve as a control. Grafts were interposed between the oral and nasal mucosa, traversing the fistulas. After healing for 40 days, the pigs were killed for clinical, histological, and immunohistochemical examination. Two of the three fistulas closed with HAM were successful, the diameter of the third was reduced in size, and there was no change in the diameter of the fistula in the control. This study shows successful closure of oronasal fistulas in minipigs using interposed grafts of cryopreserved HAM, and offers promise as a simple and effective technique for tension-free closure of such fistulas.

Expression profile of human beta-defensin 3 in oral squamous cell carcinoma.

Although it is known that innate immunity is important for protecting the body against foreign agents such as bacteria, little is known about elements of the innate immune system that have antitumor activity. This prospective study was designed to investigate the function of human beta-defensin 3 (hBD-3), an important component of the innate immune response, in oral squamous cell carcinoma (OSCC). Paired cancerous and noncancerous specimens of 45 patients who underwent surgical treatment for OSCC were examined for hBD-3 expression on protein and mRNA. Clinical and pathological features such as age, gender, tumor and lymph node status, UICC stage, and histological grading were correlated. hBD-3 was significantly overexpressed in tumors in comparison to healthy tissue examined with real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis (p = .004). Immunohistochemical stain for hBD-3 was much more pronounced in tumors than in corresponding healthy mucosa. The results illustrate that hBD-3 is frequently overexpressed in oral squamous cell carcinomas and seems to be related to oncogenesis. Increased expression of hBD-3 in oral squamous cell carcinomas suggests its potential role in the pathogenesis of oral cancer. This might be a starting point for novel pharmacological/molecular treatment modalities.

Psoriasin (S100A7) up-regulation in oral squamous cell carcinoma and its relation to clinicopathologic features.

Proteomic analysis recently suggested aberrant psoriasin (S100A7) expression in oral squamous cell carcinoma (OSCC). In this study, OSCC specimens and matching normal oral tissues from 45 patients who had undergone ablative surgery were examined. Increased psoriasin expression at mRNA level was observed in OSCC samples by quantitative real-time RT-PCR (p=0.015). Immunofluorescence analysis with psoriasin antibody confirmed these observations. Moreover, significantly increased mRNA ratios between malignant and normal samples were correlated with early UICC stage (p=0.006), T1/T2 tumour classification (p=0.043), absence of cervical lymph node metastasis (p=0.027) and age under 65 (p=0.009). Additionally, well-differentiated tumour tissues demonstrated a significantly higher psoriasin expression than moderate and poor differentiated carcinomas (p=0.018). Based on this data, we conclude that psoriasin is a positive marker for oral cancerogenesis and early tumour progression.