Application of cyclic strain for accelerated skeletal myogenic differentiation of mouse bone marrow-derived mesenchymal stromal cells with cell alignment.

The fabrication of biomimetic skeletal myocyte constructs continues to present a challenge to functional tissue engineering. The skeletal myogenesis of bone marrow-derived mesenchymal stromal cells (BMSCs) to mimic the native tissue architecture offers great therapeutic promise, but remains particularly difficult. The aim of this study was to examine the possibility of accelerating the skeletal myogenic differentiation of BMSCs with an aligned structure by applying cyclic strain. Mouse BMSCs (mBMSCs) were plated on silicone sheets that were coated with fibronectin and subjected to cyclic 10% uniaxial strain when they reached 80%-90% cell confluency. Cells cultured in a growth medium that were subjected to cyclic strain at a frequency of 0.17 Hz (10 times/min) demonstrated a shift of alignment within 48 h from a completely random orientation to a well-aligned morphology with well-organized actin stress fibers that were parallel to the strain vector. The cyclic strain restricted the motility and proliferation of the aligned mBMSCs in the growth medium, which resulted in tight cellular contact in the cell population. When mBMSCs were subjected to cyclic strain in a myogenic medium, reverse transcription-polymerase chain reaction analysis demonstrated the upregulation of skeletal myogenic marker genes (myogenic factor 5 [Myf5], myogenin, and myogenic regulatory factor 4 [MRF4]), but not smooth muscle marker genes (myocardin and α-smooth muscle actin). In addition, immunocytochemistry showed that the mBMSCs fused to form multinucleated myosin- and myogenin-positive myotubes in the direction of the applied tension within 5 days. These results demonstrate that our simple method of applying of cyclic strain to cells cultured in a myogenic medium greatly accelerates the skeletal myogenic differentiation of mBMSCs with an aligned structure, and they highlight the importance of cellular alignment for creating physiologically relevant environments to study the myogenesis of BMSCs and engineer skeletal muscle.

An analysis of the persistent presence of opportunistic pathogens on patient-derived dental impressions and gypsum casts.

PURPOSE: This study aimed to assess the persistent presence of microorganisms on patient-derived dental impressions and gypsum casts, while highlighting important human pathogens such as Candida, methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa. MATERIALS AND METHODS: The practices and opinions regarding cross-infection control from 59 general dentists in Japan were obtained via a questionnaire. Alginate impressions were made from 56 patients. Using a brain heart infusion agar medium, impression and imprint cultures were carried out to visualize the microbial contamination on the surfaces of the impressions and gypsum casts, respectively. The colonies on the surfaces of the 30 impression cultures and 26 imprint cultures were collected by swabbing and then inoculated onto selective agar plates to detect streptococci, staphylococci, Candida, MRSA, and P aeruginosa. RESULTS: The questionnaire showed that only 54% of general dentists had a cross-infection policy in their dental clinics, and only 30% to 40% were aware of the possible persistence of MRSA or P aeruginosa on impressions and gypsum casts. The impression/imprint cultures grew a large number of visible bacterial colonies on all of impression/gypsum cast samples investigated. Selective agar cultures demonstrated the presence of streptococci (100, 100%), staphylococci (56.7, 65.4%), Candida (30, 46.2%), MRSA (26.7, 15.4%), and P aeruginosa (6.7, 7.7%) on the impressions and the gypsum casts, respectively. CONCLUSIONS: This investigation showed that patient-derived dental impressions and gypsum casts are contaminated with numerous microbes, including Candida, MRSA, and P aeruginosa, which are known pathogens responsible for nosocomial and/or life-threatening infection in the immunocompromised host.

Clinical evaluation of the efficacy of removing microorganisms to disinfect patient-derived dental impressions.

PURPOSE: Disinfection of dental impressions is an indispensable procedure for the control of cross-contamination; however, there is limited information on the efficacy of disinfection under clinical conditions. The objective of this study was to clinically evaluate the disinfection efficacy of commercially available agents in removing oral pathogens from patient-derived impressions. MATERIALS AND METHODS: Impressions from 54 patients were divided into groups and either left undisinfected or underwent 1 of 5 disinfection treatments: (1) 2% glutaraldehyde (GA), (2) 1% sodium hypochlorite (SH), (3) 0.25% benzalkonium chloride (BC), (4) 1 ppm ozonated water (OW), or (5) the Hygojet/MD520 system (HJ). An impression culture technique using a brain heart infusion agar medium was used to visualize the microbial contamination on the surface of the impression cultures. The persistent presence of oral pathogens on the impression cultures was examined using selective isolation agar plates. RESULTS: The isolation frequencies of streptococci, staphylococci, Candida, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa species from undisinfected impressions were 100%, 55.6%, 25.9%, 25.9% and 5.6%, respectively. Disinfection with HJ and BC removed the microorganisms with the greatest efficacy, followed by GA, SH, and OW. Potential bacterial contamination could be detected even after disinfection had been performed. Combined use of BC plus GA or SH removed oral pathogens almost completely from dental impressions. CONCLUSIONS: This investigation showed that potential contaminants are still present, even after general disinfection procedures. Therefore, either HJ or the combined use of BC with GA or SH is recommended for clinical and laboratory use.

Downregulation of extracellular matrix-related gene clusters during osteogenic differentiation of human bone marrow- and adipose tissue-derived stromal cells.

Bone marrow- and adipose tissue-derived stromal cells (BMSCs and ASCs, respectively) exhibit a similar capacity for osteogenic differentiation in vitro, but it is unclear whether they share a common differentiation process, because they originate from different tissues. The aim of this study was to explore BMSC and ASC osteogenic differentiation by focusing on the expression of extracellular matrix-related genes (ECMGs), which play a crucial role in osteogenesis and bone tissue regeneration in vivo. We characterized the gene expression profiles of BMSCs and ASCs using a custom complementary deoxyribonucleic acid microarray containing 55 ECMGs. Undifferentiated BMSCs and ASCs actively expressed a wide range of ECMGs. Once BMSCs and ASCs were placed in an osteogenic differentiation medium, 24 and 17 ECMGs, respectively, underwent considerable downregulation over the course of the culture period. The remaining genes were maintained at a similar expression level to corresponding uninduced cell cultures. Although the suppression phenomenon was consistent irrespective of stromal cell origin, collagen (COL)2A1, COL6A1, COL9A1, parathyroid hormone receptor, integrin (INT)-beta3, and TenascinX genes were only downregulated in osteogenic BMSCs, whereas COL1A2, COL3A1, COL4A1, COL5A2, COL15A1, osteopontin, osteonectin, and INT-beta1 genes were only downregulated in osteogenic ASCs. During this time period, cell viability was sustained, suggesting that the observed downregulation did not occur by selection and elimination of unfit cells from the whole cell population. These data suggest that osteogenically differentiating BMSCs and ASCs transition away from a diverse gene expression pattern, reflecting their multipotency toward a configuration specifically meeting the requirements of the target lineage. This change may serve to normalize gene expression in mixed populations of stem cells derived from different tissues.