Ulrich Reinhart Goessler

Identification of valid reference genes during the differentiation of human myoblasts

BackgroundAnalysis 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.ResultsUsing 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.ConclusionRNA 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.

Rhinophyma: diagnosis and treatment options for a disfiguring tumor of the nose.

Rhinophyma is a benign dermatologic disease of the nose affecting primarily Caucasian men in their fifth to seventh decades of life. It is characterized by a slowly progressive enlargement with irregular thickening of the nasal skin and nodular deformation. It is assumed to be the end stage of chronic acne rosacea. Main reasons that urge the patients to seek help are plastic cosmetic and functional impairments such as nasal obstruction. Surgical removal of the hyperplastic tumor mass is the treatment of choice for rhinophyma. In a retrospective review, the authors describe the pros and cons of the main treatment modalities that have been described in literature and present their own clinical experience.

Perspectives of Gene Therapy in Stem Cell Tissue Engineering

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain or improve tissue function. It is hoped that forming tissue de novo will overcome many problems in plastic surgery associated with such areas as wound healing and the immunogenicity of transplanted tissue that lead to dysfunctional repair. Gene therapy is the science of the transfer of genetic material into individuals for therapeutic purposes by altering cellular function or structure at the molecular level. Recently, tissue engineering has been used in conjunction with gene therapy as a hybrid approach. This combination of stem-cell-based tissue engineering with gene therapy has the potential to provide regenerative tissue cells within an environment of optimal regulatory protein expression and would have many benefits in various areas such as the transplantation of skin, cartilage or bone. The aim of this review is to outline tissue engineering and possible applications of gene therapy in the field of biomedical engineering as well as basic principles of gene therapy, vectors and gene delivery.

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 morphological, metabolic and functional alterations can be present as a result of 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. Systemic effects of alcohol interact with local changes in the morphology and function of the salivary glands. In addition, alcohol leads to the accumulation of pathological 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, leucoplakia and carcinoma. Chronic alcohol consumption has been correlated with an increased risk of cancer and 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 chapter.

In Vitro Analysis of Differential Expression of Collagens, Integrins, and Growth Factors in Cultured Human Chondrocytes

OBJECTIVES: Tissue engineering represents a promising method for the construction of autologous chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation and proliferation of the cells are critical factors that influence the generation of transplants. The aim of our study was to find and characterize markers for cell proliferation and dedifferentiation in cultured chondrocytes. STUDY DESIGN AND SETTING: Human chondrocytes were isolated from septal cartilage and held in primary cell culture. Cells were harvested after 1, 6, and 21 days. The differentiation of the cells was investigated with bright-field microscopy, the expression patterns of various proteins using immunohistochemistry, and the expression of distinct genes with the microarray technique. RESULTS: The chondrocytes showed a strong proliferation. After 6 and 21 days, collagen 9 and 10 were downregulated; collagen 11 was activated. Collagen 1 and 2 were downregulated after 6 days but were reactivated after 21 days. Tumor growth factor β (TGF-β)1 was strongly expressed on days 1, 6, and 21, TGF-β2 was never expressed, and TGF-β3 and -β4 were upregulated from day 1 to day 21. The TGF-β receptor III was expressed on days 1, 6, and 21. Integrin β1, β5, and α5 were upregulated from day 1 to day 21; integrin β3 was downregulated. CONCLUSION AND SIGNIFICANCE: Collagens 3, 4, 8, 9, and 11 might be new markers for the dedifferentiation of chondrocytes. Collagen 2 might be a marker for the synthetic activity of the cells rather than the dedifferentiation. TGF-β3 and -β4 might influence the dedifferentiation, which is fortified by the expression of TGF-β receptor III. Integrin β1, β5, and α5 might be involved in signal transmission for the dedifferentiation.

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.

Soft palate implants as a minimally invasive treatment for mild to moderate obstructive sleep apnea.

Conclusion. The palatal implant method originally designed to reduce snoring can significantly reduce the apnea-hypopnea index (AHI) in some patients with mild to moderate obstructive sleep apnea (OSA) in a single office-based procedure. Objective. An initial study designed to evaluate the short-term efficacy and safety of palatal implants as primary treatment for patients with mild to moderate OSA. Materials and methods. This was a prospective, non-randomized study of 16 previously untreated and undiagnosed patients with sleep apnea. The inclusion criteria were an AHI of 10–30/h and a body mass index (BMI) ≤ 30. Results. The mean AHI was reduced following implantation, from 16.1 to 11.8 (p<0.01). A reduction in AHI was achieved in 13 patients (81%). Ten of 16 patients had their AHI reduced to <10.0. Snoring intensity decreased from 8.3±1.8 to 4.7±2.5 on a visual analog scale (p<0.001) and daytime sleepiness dropped from 7.2±2.5 to 4.6±3.2 on the Epworth Sleepiness Scale (p<0.05). No significant adverse events were reported.

Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells

The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-α1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM-MSCs. The strongest DES expression was observed using the 30% conditioned cell culture medium. The detection of myogenic markers using different cell culture media as stimuli was only achieved in the AT-MSCs, but not in the BM-MSCs. The strongest myogenic differentiation, in terms of the markers examined, was induced by the 30% conditioned cell culture medium.

CD44 as a stem cell marker in head and neck squamous cell carcinoma

In the recent past, evidence is increasing indicating the existence of a subpopulation of resistant tumor cells in head and neck squamous cell carcinoma (HNSCC) that cannot be eradicated by established antineoplastic treatments. These cancer stem cells (CSCs) have features of somatic stem cells such as selfrenewal, proliferation and differentiation. CD44+ cells in tumors of the head and neck are referred to as CSCs of HNSCC. Expression profiling of CD44 in 29 HNSCC tumors was performed by fluorescence microscopy. ELISA analysis was performed to detect concentration of soluble CD44 in the peripheral blood of 29 HNSCC patients and 11 healthy controls. Expression of CD44 was determined in all HNSCC tissue samples (n=29). In all samples a surface staining pattern was found. The concentration of CD44 in the peripheral blood of HNSCC patients was significantly higher compared to a healthy control group (mHNSCC =13.5 ± 0.5 ng/ ml; mCont = 9.3 ± 0.6 ng/ml; P=0.6 x 10(-12)). The role of CD44 as a marker for CSCs in HNSCC remains to be ascertained. Further experiments might reveal its role as a diagnostic and prognostic factor, and possibly as a therapeutic target.

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.