Osteogenic induction protects rat bone marrow–derived mesenchymal stem cells against hypoxia-induced apoptosis in vitro

Background

Bone marrow–derived mesenchymal stem cells (BMSCs) undergo hypoxia-induced apoptosis when cells are transplanted from a normoxic to a hypoxic microenvironment in vivo. The effect of the osteogenic microenvironment on BMSCs under hypoxic conditions has not yet been revealed.

Materials and methods

In the current study, we investigated the effects on BMSCs of hypoxia and osteogenic induction (OI) individually and in combination. We isolated BMSCs from rat bone marrow and confirmed them by recognition of surface antigens using cytometry. After passaging the BMSCs to the third generation, we treated them with the following conditions: 1% oxygen and OI, normoxia and OI, and 1% oxygen without OI; normoxia without OI was the control condition. On days 3, 7, 14, and 21, we detected the expression levels of hypoxia inducible factor-1α and alkaline phosphate via Western blotting. Cellular apoptosis was detected by Hoechst staining and terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine, 5′-triphosphate nick end labeling; caspase activity was also detected.

Results

The expression of hypoxia inducible factor-1α was induced and up-regulated when BMSCs were grown under 1% oxygen. The incidence of terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine, 5′-triphosphatenick end labeling–positive cells in the hypoxia plus OI group was much lower than that in the hypoxia group without OI. Caspase activity increased on days 3, 7, 14, and 21. The absolute value of caspase was statistically higher in the BMSC hypoxia group than in the other three groups, whose values were similar to each other.

Conclusions

Osteogenic induction could protect BMSCs against hypoxia-induced apoptosis. Bone marrow–derived mesenchymal stem cells may be appropriate candidate cells for cytotherapy for skeletal diseases.     

Effects of 5-aminolevulinic acid–mediated photodynamic therapy on antibiotic-resistant staphylococcal biofilm: an in vitro study

Background

Treatments of infections are not always successful because of multi-antibiotic-resistant organisms. It is therefore particularly urgent to provide more effective anti-infective strategy against these organisms. 5-Aminolevulinic acid (ALA), with the chemical structure C₅H₉NO₃, is the only photodynamic therapy agent that is a biochemical precursor of a photosensitizer (protoporphyrin IX [PpIX]), which is naturally produced by the body. 5-Aminolevulinic acid–mediated photodynamic therapy (ALA-PDT) has been shown to have a strong effect on the treatment of localized cancerous and precancerous lesions, and further study demonstrated its efficacy for gram-positive and gram-negative bacteria. However, its effect on biofilm formed by antibiotic-resistant strains has not been reported.

Methods

In this study, we evaluated the effectiveness of ALA-PDT on biofilms formed by methicillin-resistant Staphylococcus aureus (ATCC 43300) and methicillin-resistant S epidermidis (MRSE 287). The strains were cultured with 40 mM of ALA in 24-well microtiter plates containing coverslips at 37°C for 24 h in the dark. PpIX fluorescence in biofilms formed by the two strains was observed by confocal laser scanning microscopy (CLSM). ALA-treated biofilms were irradiated at different doses (0, 100, 200, and 300 J/cm²) using a semiconductor laser. Biofilm exposed only to Tryptone Soy Broth or irradiation (300 J/cm²) was investigated. Viability determination, CLSM, and scanning electron microscopy were used to investigate the photodynamic inactivation of ALA-PDT.

Results

ALA was absorbed and converted to PpIX by both methicillin-resistant S aureus and methicillin-resistant S epidermidis. No cell inactivation was detectable in biofilms of either strain incubated with ALA without exposure to light, incubated with Tryptone Soy Broth only, or irradiated with red light only. However, a significant number of cells within biofilms were inactivated during irradiation with different doses of red light in the presence of 40 mM of ALA in a dose-dependent manner. The drastic reduction in cell survival within biofilms and the disruption of biofilms were confirmed by CLSM and scanning electron microscopy.

Conclusions

ALA-PDT has the potential to eliminate the biofilm of Staphylococcus, especially antibiotic-resistant strains, effectively. It will be suitable for the treatment of superficial local infections such as surface wounds, burns, oral and dental infections, dermatologic infections such as acne and rosacea, and soft tissue and bone infections with bone exposure.     

A polyhedral oligomeric silsesquioxane–based bilayered dermal scaffold seeded with adipose tissue–derived stem cells: in vitro assessment of biomechanical properties

Background

Although commercial skin substitutes are widely available, its use remains challenging at surgery and postoperatively. The high cost is also prohibitive. We designed and characterized a scaffold for dermal replacement, using advanced nanocomposite materials, which are known to have unique nanoscale features that enhance cellular behavior.

Methods

A bilayered scaffold was developed using the nanocomposite, polyhedral oligomeric silsesquioxane, incorporated into poly(caprolactone-urea)urethane, resulting in a mechanically robust bioabsorbable polymer; forming the inner layer, which was designed with a range of porosities. The removable outer layer contained nanosilver. Tensile testing, surface tension, permeability, and scanning electron microscopy were performed. Optimal pore morphology for cellular proliferation was elucidated through adipose tissue–derived stem cell culture and a cell viability assay. All tests were repeated on Integra Dermal Regeneration Template.

Results

The physical construct was easy to handle and clinically applicable. Macroporosity and permeability of scaffolds was demonstrated, confirmed by scanning electron microscopy. Both tensile strength and surface tension were comparable with skin; outer layer demonstrated hydrophobicity and inner layer showed hydrophilicity. Cell assay confirmed cellular proliferation onto the scaffold, comparable with Integra.

Conclusions

We demonstrate that a porous bilayered dermal scaffold could form the basis of a new generation of skin substitute that is both mechanically robust and harbors the ability for enhancing cell regeneration.     

Do we need to cool the lung graft after ex vivo lung perfusion? A preliminary study

Background

After normothermic ex vivo lung perfusion (EVLP), pulmonary grafts are usually flush-cooled and stored on ice until implantation although evidence for this practice lacks. We compared outcomes between 2 post-EVLP preservation strategies in a porcine left single-lung transplantation model.

Material and methods

After cold flush and 2-h EVLP, donor lungs were prepared and split. In [C], (n = 5) lungs cooled on device to 15°C were preserved in ice-water; in [W] (n = 5), lungs were disconnected from EVLP at 37°C and kept at room temperature. The left lung was transplanted in a recipient animal. Posttransplant, 6 h-monitoring included hourly assessment of pulmonary vascular resistance, pulmonary artery pressure, plateau airway pressure, compliance, and oxygenation before and after exclusion of the right lung. Lung biopsies and bronchoscopy with bronchoalveolar lavage (BAL) were performed at retrieval, at the end of EVLP (R lung), and 1 and 6 h after reperfusion (L lung).

Results

Lungs in [W] showed the highest compliance (P < 0.05) and the lowest plateau airway pressure (not statistically significant) throughout the whole reperfusion period. Oxygenation and pulmonary artery pressure were similar between groups. Pulmonary vascular resistance was stable in [C], but rose after reperfusion in [W]. Histologic signs of lung injury and BAL neutrophilia were more pronounced in [C] at 1 h (not statistically significant and P < 0.05, respectively). BAL cytokine levels and lung tissue expression of intercellular adhesion molecule 1 did not differ between groups.

Conclusions

Normothermic preparation after EVLP results in similar graft performances compared with lung cooling after EVLP.     

Transfusion de sang frais total en temps de guerre : expérience du groupement médicochirurgical Warehouse durant la période 2006–2009

Objective

The specificities of military medicine have led to the maintenance of fresh whole blood (FWB) transfusion.

Study design

The aim of our study was to evaluate this practice at the French military hospital in Kabul between 2006–2009.

Patients and methods

During our study period, 19 FWB transfusions were performed and the data from 15 FWB transfusions could be analyzed. We studied the number of units by recipient, the characteristics of recipients, the results of blood tests performed after transfusion, the incidents in donors and recipients, the period for obtaining a unit of FWB and mortality of recipients.

Results

A total of 66 units of FWB were transfused in 15 patients. The median number of FWB units transfused was three per patient. Thirteen out of 15 (87%) were combat-related casualties. All units were tested before transfusion for HIV with rapid diagnostic tests. Every blood samples of donors were negative for pathogens screened at the French Blood Service. No incident in donors and in recipients was reported. The average time between collection and transfusion was 140 ± 197 minutes (median 43 min). Mortality in recipients was 27% (n = 4).

Conclusion

In our study, the FWB transfusion was not associated with incidents. Nonetheless, this practice should be used only for exceptional situations like military conflicts where risks of FWB are lower than the absence of transfusion.     

Facteurs associés au burnout en anesthésie–réanimation. Enquête 2009 de la Société française d’anesthésie et de réanimation

Objective

To assess factors related to burnout in anesthesia and intensive care.

Design

National prospective observational study.

Materials and methods

Questionnaire posted on the French Society of anesthesia website from 3rd June 2009 to 27th August 2009: Maslach Burnout Inventory (MBI), Fast Alcohol Consumption Evaluation (FACE) and The Harvard National Depression Screening Day Scale (HANDS) scales and questions to assess health, work and personal life.

Results

One thousand six hundred and three questionnaires returned: 1091 anesthetists (67.6%), 241 intensivists (14.9%), 204 nurses (12.6%), emergency physicians (2.8%), supervisor nurses (0.9%). Seven hundred and sixty three in a university hospital (47.3%), 259 in a regional hospital (16.1%), 405 in a private structure (25.1%), 71 in a non-lucrative private structure (4.4%), 75 in a military hospital (4.6%). Rest of safety: 69.2% of institutions. Depression: 38.7%. Drug or chemicals addicted: 10.6%. Alcohol addicts: 10.6%. Among them, 62.3% of individuals were in burnout. Burnout was linked to fragmented sleep (P < 0.00001), interpersonal conflicts (P < 0.00001), perception of rest of safety (P < 0.02), mental history (P < 0.00001), suicidal ideations (P < 0.00001), depression (P = 0.00001), alcohol (P < 0.002), drug consumption (P < 0.00002), and accidents after a nightshift (P < 0.05). Subjects in burnout intended more frequently to leave the profession (P < 0.00001). Leaving in couple had a protective effect (P < 0.005). The logistic regression model retained seven covariates independently associated with burnout: quality of work, of personal life, of fatigue, depression, conflicts with colleagues and patients, regretting the choice of specialty.

Conclusion

This study of the largest cohort of anesthesia personnel performed in France detects a high proportion of burnout. It highlights links with tensors that may constitute possibilities of prevention of the burnout syndrome.