Effect of HBO therapy on adipose-derived stem cells, fibroblasts and co-cultures: In vitro study of oxidative stress, angiogenic potential and production of pro-inflammatory growth factors in co-cultures1.

BACKGROUND: A key moderator of wound healing is oxygen. Wound healing is a dynamic and carefully orchestrated process involving blood cells, cytokines, parenchymal cells (i.e. fibroblasts and mesenchymal stem cells) and extracellular matrix reorganization. Human adipose derived stem cells as well as human fibroblasts produce soluble factors, exhibit diverse effects on inflammation and anti inflammation response and are involved in wound healing processes.Hyperbaric oxygen therapy is an effective adjunct treatment for ischemic disorders such as chronic infection or chronic wounds. In vitro effects of hyperbaric oxygen therapy on human cells were presented in many studies except for those on mono- and co-cultures of human adipose derived stem cells and fibroblasts. OBJECTIVE: The aim of this study was to investigate the effects of hyperbaric oxygen therapy on mono- and co-cultures of human adipose derived stem cells and fibroblasts. METHODS: Mono- and co-cultures from human adipose derived stem cells and fibroblasts were established. These cultures were exposed to hyperbaric oxygen therapy every 24 h for five consecutive days. Measuring experiments were performed on the first, third and fifth day. Therapy effects on the expression of VEGF, IL 6 and reactive oxygen species were investigated. RESULTS: After exposure to hyperbaric oxygen, cell culturess showed a significant increase in the expression of VEGF after 3 and 5 days. All cultures showed significantly reduced formation of reactive oxygen species throughout the experiments. The expression of IL-6 decreased during the experiment in mono-cultures of human adipose derived stem cells and co-cultures. In contrast, mono-cultures of human skin fibroblasts showed an overall significantly increased expression of IL-6. CONCLUSIONS: Hyperbaric oxygen therapy leads to immunmodulatory and proangiogenetic effects in a wound-like enviroment of adipose derived stem cells and fibroblasts.

Correction to: Induced surface proteins of Staphylococcus epidermidis adhering to titanium implant substrata.

There is a mistake in the original published version of this article. The word 'Streptococcus' in the article title should have been 'Staphylococcus'.

Induced surface proteins of Staphylococcus [corrected] epidermidis adhering to titanium implant substrata.

OBJECTIVE: Staphylococcus epidermidis, as a primary colonizer, is strongly associated with infections of (dental) implants (i.e., peri-implantitis), but little is known about the surface proteome of this bacterium. For the identification of bacterial adhesins, this study investigated the surface proteome of S. epidermidis adhering directly to titanium implant substrata. MATERIALS AND METHODS: S. epidermidis strain ATTC 35984 was cultured either planktonically or on titanium implant specimens. The surface proteomes were isolated by mutanolysin digestion, and proteins were separated by 2D gel electrophoreses to reveal highly expressed proteins only. Protein spots were visualized by silver staining and proteins were identified by mass spectrometry. RESULTS: Surface proteome analyses of S. epidermidis on titanium identified six expressed proteins. Three proteins were highly expressed on the titanium implants including accumulation-associated protein Q8CQD9. These specific proteins could be potential pathogenicity factors of bacteria in peri-implant biofilms. CONCLUSION: For the first time, our study identified S. epidermidis surface proteins, which are expressed after adhesion to titanium implant materials. CLINICAL RELEVANCE: Our study reveals possible candidates for a newly protein-based vaccine against peri-implantitis.

Circulating serum CK level vs. muscle impairment for in situ monitoring burden of disease in Mdx-mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) consists of a lack in the expression of the subsarcolemmal protein dystrophin causing progressive muscle dysfunction. Among the widely applied animal models in DMD research is the C57BL/1010ScSn-Dmdmdx mouse, commonly referred to as the "mdx mouse". The potential benefit of novel interventions in this model is often assessed by variables such as functional improvement, histological changes, and creatine kinase (CK) serum levels as an indicator for the extent of in situ muscle damage. OBJECTIVE: Our objective was to determine to what extent the serum CK-level serves a surrogate for muscle dysfunction. METHODS: In this trial mdx mice were subjected to a four-limb wire-hanging test (WHT) to assess the physical performance as a reference for muscle function. As CK is a component of the muscle fiber cytosol, its serum activity is supposed to positively correlate with progressing muscle damage. Hence serum CK levels were measured to detect the degree of muscle impairment. The functional tests and the serum CK levels were analyzed for their specific correlation. RESULTS: Although physical performance decreased during the course of the experiment, latency to fall times in the WHT did not correlate with the CK level in mdx mice. CONCLUSION: Our data suggests that the serum CK activity might be a critical parameter to monitor the progression of muscle impairment in mdx mice. Further this study emphasizes the complexity of the DMD phenotype in the mdx mouse, and the care with which isolated parameters in this model should be interpreted.

EGR1 supports the osteogenic differentiation of dental stem cells.

AIM: To evaluate whether and how the transcription factor early growth response gene 1 (EGR1) affects the osteogenic differentiation of dental stem cells. METHODOLOGY: Dental stem cells from apical papilla (SCAPs) and from the dental follicle (DFCs) were transfected with EGR1-specific siRNA or EGR-1 expression plasmid. Gene regulation was verified at protein level by Western blotting. The expression of the transcription factors distal-less homeobox 3 (DLX3), alkaline phosphatase (ALP) and bone morphogenetic protein 2 (BMP2), which are all regulators and markers of the osteogenic differentiation in dental stem cells, was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). To investigate mineralization, SCAP long-term cultures were stained with alizarin red after EGR1 over-expression. RESULTS: EGR1 was induced in SCAPs during osteogenic differentiation. DLX3 and bone morphogenetic protein 2 (BMP2) were up-regulated after EGR1 over-expression and down-regulated after EGR1 depletion. The expression of ALP was also down-regulated after EGR1 depletion. The over-expression of EGR1 in SCAPs promoted mineralization after osteogenic differentiation. CONCLUSIONS: EGR1 supported the osteogenic differentiation of dental stem cells by potentially regulating the expression of DLX3 and BMP2.

Pressure monitoring during lipofilling procedures.

BACKGROUND: Grafting of autologous lipoaspirate for various clinical applications has become a common procedure in clinical practice. With an estimated mortality rate of 10-15 percent, fat embolism is among the most severe complications to be expected after lipofilling therapies. OBJECTIVE: The aim of this study was to determine the level of interstitial pressure after the injection of defined volumes of lipoaspirate into the subcutaneous tissue of female breasts. It was hypothesized, that interstitial pressure levels exceed the physiologic capillary pressure during lipofilling procedures and hence increase the potential risk for fat embolism. Further it was investigated if external tissue expansion has the potential to significantly reduce interstitial tissue pressure. METHODS: Interstitial pressure was monitored in 36 female patients, that underwent autologous fat injections into the breast. Measurements were conducted with a sensor needle connected to a pressure transducer (LogiCal Pressure Monitoring Kit, Smiths medical int. Ltd., UK). Patients were divided into 4 subcohorts differing in their pre-treatment regimen or local tissue conditions. Pre-treatment consisted of tissue expansion, achieved with the Brava™ (Brava LLC Miami, Fla., USA) vacuum-chamber. RESULTS: The increase in interstitial pressure after injection volumes of 100 ml (p = 0.006), 200 ml (p = 0.000) and between 100 ml and 200 ml (p = 0.004) respectively, were significant in non-mastectomized patients without pre-treatment. Patients pre-treated with Brava™ did not show such statistically significant differences in interstitial pressures before and after the injection of 100 ml and 200 ml of lipoaspirate (p = 0.178). The difference in interstitial pressure in mastectomized patients between 0 ml and 100 ml (p = 0.003), as well as 0 ml and 200 ml (p = 0.028) was significant. The difference in pressures between pre-treated patients and patients without pre-treatment did not differ significantly in the mastectomized patient cohort. CONCLUSION: During lipofilling procedures interstitial pressures are reached that exceed pressure limits defined as hazardous for fat embolism. To date it is unknown what pressure levels need to be considered critical for complications in soft tissue interventions. Further the results indicate higher interstitial pressures for patients that had undergone mastectomy, whereas pre-treatment with external tissue expansion seemed to diminish pressure values.

Migration of human dental follicle cells in vitro.

BACKGROUND AND OBJECTIVES: The objective of this study was to elucidate the effects of different growth factors on the migration of dental follicle cells (DFCs). DFCs are ectomesenchymally derived easily accessible multipotent stem cells. Cell migration is a crucial step in many biological processes but also for tissue engineering. Growth factors such as epidermal growth factor (EGF), bone morphogenetic protein-2 (BMP2) or transforming growth factor ß1 (TGF-ß1) can be used to modify the behavior of cells. MATERIAL AND METHODS: We used different migration assays (gel spot assay, scratch assay, transwell assay) to evaluate the influence of EGF, BMP2 and TGF-ß1 on the migration of DFCs. We investigated the expression of migration-related genes after growth factor stimulation using the PCR array human cell motility. RESULTS: DFCs treated with BMP2 or TGF-ß1 migrated faster than DFCs treated with EGF. Additionally, more migration-related genes are regulated after treatment with BMP2 or TGF-ß1 than with EGF. TGF-ß1 additionally functions as a chemoattractant for DFCs. Osteogenic differentiation markers were regulated after BMP2 treatment only. CONCLUSION: Whereas the strong migration induced by BMP2 was accompanied by beginning osteogenic differentiation the strong migration induced by TGF-ß1 was directional. EGF exhibited not only the weakest migration stimulation but also the weakest induction of differentiation into mineralizing cells.

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines.

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133(+) cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.