Traditional transcutaneous approaches in head and neck surgery.

The treatment of laryngeal and hypopharyngeal malignancies remains a challenging task for the head and neck surgeon as the chosen treatment modality often has to bridge the gap between oncologically sound radicality and preservation of function. Due to the increase in transoral laser surgery in early tumor stages and chemoradiation in advanced stages, the usage of traditional transcutaneous approaches has decreased over the recent past. In addition, the need for a function-sparing surgical approach as well as highest possible quality of life has become evident. In view of these facts, rationale and importance of traditional transcutaneous approaches to the treatment of laryngeal and hypopharyngeal malignancies are discussed in a contemporary background. The transcutaneous open partial laryngectomies remain a valuable tool in the surgeon's armamentarium for the treatment of early and advanced laryngeal carcinomas, especially in cases of impossible laryngeal overview using the rigid laryngoscope. Open partial laryngetomies offer superior overview and oncologic safety at the anterior commissure, especially in recurrencies. In select advanced cases and salvage settings, the supracricoid laryngectomy offers a valuable tool for function-preserving but oncologically safe surgical therapy at the cost of high postoperative morbidity and a very demanding rehabilitation of swallowing.In hypopharyngeal malignancies, the increasing use of transoral laser surgery has led to a decline in transcutaneous resections via partial pharyngectomy with partial laryngectomy in early tumor stages. In advanced stages of tumors of the piriform sinus and the postcricoid area with involvement of the larynx, total laryngectomy with partial pharyngectomy is an oncologically safe approach. The radical surgical approach using circumferent laryngopharyngectomy with/without esophagectomy is indicated in salvage cases with advanced recurrences or as a primary surgical approach in patients where chemoradiation does not offer sufficient oncologic control or preservation of function. In cases with impending reconstruction, fasciocutaneous free flaps (anterolateral thigh flap, radial forearm flap) seem to offer superior results to enteric flaps in cases where the cervical esophagus is not involved leading to better voice rehabilitation with fewer complications and postoperative morbidity. In salvage situations, the Gastroomental Free Flap has proven to be a valuable tool.In conclusion, the choice of a surgical treatment modality is influenced by the patient's anatomy, tumor size and location as well as the surgeon's personal expertise.

TGF-ß1 antisense impacts the SMAD signalling system in fibroblasts from keloid scars.

AIM: To identify the effect of a TGF-ß1 antisense treatment of keloid fibroblasts on the SMAD signalling system. MATERIAL AND METHODS: In this cross-sectional study, keloid and adjacent healthy tissue was harvested from 9 patients with keloid scars after otoplasty. Keloid fibroblasts were placed in monolayer cultures. Expression of SMAD2, -3, -4, -6, and SMURF2 were analysed by immunohistochemistry. Analysis of treatment with antisense oligonucleotides was conducted by immunohistochemistry, and RT-PCR. RESULTS: Immunohistochemical investigation demonstrated increased expression of SMAD2, -3 and -4, and decreased expression of SMURF2. TGF-ß1 antisense therapy significantly down-regulated SMAD2 and SMAD4, up-regulated SMURF2 and showed no effect on SMAD3 and SMAD6. CONCLUSION: TGF-ß1 led to elevated levels of the SMAD signalling cascade, indicating an abnormal sensitivity of keloid-derived fibroblasts to this cytokine. Abrogation correlated with potential suppression of the fibro-proliferative progress. There is growing evidence for an abnormal response to this cytokine in the intracellular signal transduction in keloid-derived fibroblasts.

Effect of transforming growth factor-beta1 antisense oligonucleotides on matrix metalloproteinases and their inhibitors in keloid fibroblasts.

OBJECTIVE: To identify changes in the expression of matrixmetalloproteinases (MMPs) and their specific inhibitors tissue inhibitors of metalloproteinases (TIMPs) after targeting of transforming growth factor-beta1 (TGF-beta1) with antisense oligonucleotides. STUDY DESIGN: Cross-sectional study. SETTING: The study was performed on tissue samples from nine patients with keloid scars after otoplasty presenting to the Otolaryngology-Head and Neck Surgery Department of the University Hospital in Mannheim, Germany. SUBJECTS AND METHODS: Keloid fibroblasts and normal fibroblasts were harvested from auricular keloid scars and healthy skin regions of the same patients during resection procedure of the keloid. Cells were placed in monolayer cultures. Expression of MMPs and TIMPs were analyzed by immunohistochemistry. The effect of TGF-beta1 targeting using antisense oligonucleotides on the expression of both protein groups in keloid-derived fibroblasts was analyzed by enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction. RESULTS: Immunohistochemical investigation demonstrated increased expression of MMP-2, -3, -9, and -13 and TIMP-1 and -2. TGF-beta1 antisense therapy significantly down-regulated MMP secretion in vitro. CONCLUSION: Usage of TGF-beta1 antisense oligodeoxynucleotides (ODNs) may show a potential chemopreventive or therapeutic option for keloids by blocking the effect of TGF-beta1. Furthermore, antisense ODNs can be used as an investigative approach toward a better understanding of molecular mechanisms in keloid pathophysiology.

Effect of the abrogation of TGF-beta1 by antisense oligonucleotides on the expression of TGF-beta-isoforms and their receptors I and II in isolated fibroblasts from keloid scars.

Disequilibrium of dermal wound repair can result in continued accumulation of ECM and excessive scar formation. In susceptible genetically predisposed individuals, keloid formation can be observed. Keloid disease represents a benign dermal fibroproliferative tumor that is unique to humans. TGF-beta is known to play a key role in the pathogenesis of this disease which is still not fully understood. The isoforms TGF-beta1 and TGF-beta2 have profibrotic properties, whereas TGF-beta3 may have antifibrotic functions. TGF-beta exerts its influence by binding to type I and type II TGF-beta receptors, thereby forming a complex and activating specific downstream effector molecules. The aim of this study was to investigate the effect of TGF-beta1 targeting by antisense oligonucleotides on the RNA synthesis and protein expression of TGF-beta isoforms and their receptors in keloid-derived fibroblasts. In tissue samples with normal fibroblasts (NFs) serving as control samples, expression of TGF-beta1 and -beta2 was decreased when compared to keloid fibroblasts (KFs), while expression of TGF-beta3 and of TGF-betaRII was significantly higher in NFs. In the ELISA assay, abrogation of TGF-beta1 led to a significant decrease in TGF-beta1 and -beta2 (p<0.05). Expression of TGF-beta2 mRNA was reduced. Expression of TGF-beta3 mRNA revealed contrary patterns in KFs from different patients while expression of TGF-betaRI was found to be equal during the measurement period. TGF-betaRII mRNA expression was increased after 48 and 72 h respectively. There is growing evidence for a regulatory mechanism between TGF-beta1 and its receptors. Our findings support this theory by suggesting interrelations between the different TGF-beta isoforms and their receptors. Abnormal response of KFs to TGF-betamight reflect a modification in the regulatory pathway that occurs at the receptor level or during intracellular trans-duction. Improving the understanding of TGF-beta in keloid disease could lead to the development of clinically useful therapeutic modalities for treatment of keloid disease or even allow identification of preventive strategies.

Evaluation of valid reference genes during stimulation with static magnetic fields in human myoblast cultures.

Investigation of gene expression using real-time PCR (qRT-PCR) requires normalization with genes that are continuously expressed (reference genes; RGs). For accurate measurements, it is exceedingly important that RG expression is invariant under the investigated experimental conditions. It has recently become evident that RG expression may vary considerably under different culture conditions, which results in inaccurate qRT-PCR measurements. Static magnetic fields (SMFs) have been shown to enhance myogenic cell differentiation in the rat cell line L6, and may also induce differentiation in human myoblast cultures. In order to perform precise qRT-PCR measurements in human myoblast cell cultures stimulated with SMFs, one prerequisite is to find the most suitable RG. In this study, qRT-PCR was used to investigate the gene expression of six widely used RGs in human myoblast cell cultures stimulated with SMFs, with the aim of identifying the most stable among them. The mRNA concentration of ß-actin (ACTB), ß-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO) were quantified, and the most suitable RGs were identified using the geNorm and NormFinder software programs. Results were verified by BestKeeper software. mRNA expression of the following genes of interest was analyzed: myosin, heavy chain 1, skeletal muscle, adult (MYH1); myosin, heavy chain 3, skeletal muscle, embryonic (MYH3); myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), as well as the immunoreactivity of MYH1 (irMYH1). Using geNorm, PPIA and B2M were found to be the most stable genes, followed by GAPDH. NormFinder identified PPIA as the most stable gene, followed by B2M and GAPDH. Finally, BestKeeper revealed TBP and PPIA to be the most stable genes, while B2M was ranked third.

Keloids: current concepts of pathogenesis (review).

Excess scar formation occurs after dermal injury as a result of abnormal wound healing. Hypertrophic scars and keloids both represent fibrotic skin conditions which can be very difficult, even frustrating, to treat. Identification of differences between hypertrophic scars, keloids and normal scars are a prerequisite for finding the correct therapeutical concept. Despite the relatively high prevalence of keloids in the general population, the mechanisms underlying keloid formation are only partially understood. This fact is reflected in the multiple treatment modalities, of which no single treatment has proven to be widely effective. Advances in our understanding of the wound healing process reveal new pathophysiological concepts for keloid formation. Our article presents an overview on physiological wound healing and the pathogenesis of scar formation, differentiates keloids from hypertrophic scars and reviews current hypotheses for keloid formation. This information might assist in deciphering the complexity of keloid pathogenesis and help in the development of an efficacious therapeutical strategy.

Identification of valid reference genes during the differentiation of human myoblasts.

BACKGROUND: Analysis of RNA expression using real-time PCR (qRT-PCR) traditionally includes reference genes (RG) as an internal control. This practice is being questioned as it becomes increasingly clear that RG may vary considerably under certain experimental conditions. Thus, the validity of a particular RG must be determined for each experimental setting. We used qRT-PCR to measure the levels of six RG, which have been reported in the literature to be invariant. The RG were analyzed in human myoblast cultures under differentiation conditions. We examined the expression by qRT-PCR of mRNA encoding Beta-actin (ACTB), Beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO). The mRNA expression of the following genes of interest (GOI) were analyzed: skeletal muscle alpha 1 actin (ACTA1), myogenin/myogenic factor 4 (MYOG), embryonic skeletal muscle myosin heavy chain 3 (MYH3) and the activity of creatine phosphokinase (CK). The geNorm, NormFinder and BestKeeper software programs were used to ascertain the most suitable RG to normalize the RNA input. RESULTS: Using the geNorm program, RPLPO and TBP were found to be the most stable genes, additionally a suitable normalization factor (NF) was calculated. The NormFinder software showed that RPLPO was the most stable, whereas TBP ranked second. BestKeeper program also revealed that RPLPO and TBP as stable genes, but PPIA was the most stable reference gene, whereas GAPDH and ACTB were the worst ranked. CONCLUSION: RNA expression analyses including three independent softwares revealed that RPLPO, TBP as reference genes or NF calculated by geNorm software, are suitable to normalize the mRNA expression in myoblast after culture under differentiation conditions. Significant correlations can be identified between the differentiations markers ACTA1, MYOG, MYH3 and creatine phosphokinase (CK) activity, when the expression is normalized with the NF calculated with RPLPO and TBP.

Apoptosis in bone for tissue engineering.

Bone loss due to congenital defects, trauma, improper fracture fixation, metabolic disturbances, infections, or after tumor resection represents a major clinical problem in head and neck surgery. To address these issues, different types of scaffolds, growth factors and cell sources -- alone or in various combinations -- have been applied for development of bioartificial bone tissues. Although these applications have received increasing interest, use of autologous bone grafts is still considered as the gold standard for tissue repair. Despite progress in some areas of tissue regeneration, significant translation into clinical practice has not been achieved. Reasons for this impass include rejection of engineered tissue implants by the immune system, limited blood supply, or morbidity of the donor site. During the process of bone regeneration, approximately 50-70% of osteoblasts undergo apoptosis. Apoptosis is a naturally occurring cell death pathway induced in a variety of cell types and is associated with caspase activation or caspase mediation. It is recognized as an important component of embryogenesis and tissue morphogenesis and, in adult skeletons, it contributes substantially to physiological bone turnover, repair, and regeneration. Intracellular mechanisms are orchestrated by a variety of proteins, the interplay of which seems to vary, depending on the differentiation state of the cell or the current status of the tissue. Closing gaps in current knowledge of the apoptosis of bone and understanding the mechanisms of cell death in tissue engineered bone will improve results in the translation from bench to bedsite. This review aims to provide a broad overview of the current general concepts in apoptosis with a special focus on its regulation in osteoblasts and its significance for bone tissue engineering.

Tgf-beta antisense therapy increases angiogenic potential in human keratinocytes in vitro.

BACKGROUND: Transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. The purpose of this study was to analyze the effect of TGF-beta targeting on the expression of angiogenic vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, and in vitro angiogenic activity. METHODS: Expression of angiogenic VEGF in tissue samples from chronic dermal wounds was investigated by immunohistochemistry. The effect of TGF-beta targeting using antisense oligonucleotides on the expression of VEGF was analyzed by ELISA and RT-PCR in cultured human keratinocytes. Human endothelial cells (EC) were grown in conditioned medium produced from the treated keratinocytes. EC migration was measured using a modified Boyden chamber, EC tube formation was analyzed under the light microscope. RESULTS: Immunohistochemical investigation demonstrated a decreased expression of VEGF protein in tissue samples from chronic dermal wounds compared to normal human skin. Antisense TGF-beta oligonucleotide treatment upregulated VEGF secretion in vitro. Addition of conditioned medium from TGF-beta antisense-treated keratinocytes resulted in an increase of endothelial cell migration and tube formation. CONCLUSIONS: Our results demonstrate that TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for stimulation of angiogenesis in chronic wounds.

Current status of genetic modulation of growth factors in wound repair.

Growth factors are members of a large functional group of polypeptide regulatory molecules secreted by different cells. They are important players in orchestrating all stages of wound healing exerting their influence through autocrine and paracrine fashions within sites of injury and repair. They are mitogen, chemotactic, they regulate cell-cell interactions and influence synthesis and composition of extracellular matrix components. The use of growth factors to stimulate wound healing is a promising therapeutic approach to repair chronic tissue defects. The delivery of genetic material offers an attractive treatment modality to produce an appropriate amount of growth factor proteins within the wound site. Gene therapy might become a significant treatment modality for those wound healing pathologies refractory to other wound management approaches. This review discusses several methods of growth factor gene transfer into wound tissue.

Alcohol-related diseases of the mouth and throat.

Chronic consumption of alcoholic beverages is an accepted social custom worldwide. In the upper aerodigestive tract, local morphologic, metabolic and functional alterations are present due to alcohol consumption. A clinical link between the chronic consumption of alcohol and head and neck cancer has been observed for decades. While alcohol was described initially as a risk enhancer only in smokers, a number of epidemiological studies have now provided sufficient evidence that chronic alcohol consumption increases the risk of head and neck cancer independent of exposure to tobacco smoke. The systemic effects of alcohol interact with local changes in the morphology and function of the salivary glands. In addition, alcohol leads to accumulation of pathologic microbes within the mucosa, leading to chronic infection. Susceptibility to carcinogens and cell proliferation in the mucosa are increased, resulting in genetic changes with the development of dysplasia, leukoplacia and carcinoma. Chronic alcohol consumption is correlated with an increased risk of cancer and an increased mortality in a dose-effect relationship. A number of biologically plausible mechanisms exist by which alcohol may cause cancer. These mechanisms are discussed in this article.