Bone matrix production and mineralization involves sophisticated mechanisms, including the initial formation of an organic extracellular matrix into which inorganic hydroxyapatite crystals are later deposited. Human embryonic stem (hES) cells offer a potential to study early developmental processes and provide an unlimited source of cells. In this study, four different hES cell lines were used, and two different approaches to differentiate hES cells into the osteogenic lineage were taken. Undifferentiated cells were cultured either in suspension, facilitating the formation of embryoid bodies (EBs), or in monolayer, and both methods were in the presence of osteogenic supplements. Novel to our osteogenic differentiation study was the use of commercially available human foreskin fibroblasts to support the undifferentiated growth of the hES cell colonies, and their propagation in serum replacement-containing medium. Characterization of the osteogenic phenotype revealed that all hES cell lines differentiated toward the mesenchymal lineage, because T-Brachyury, Flt-1, and bone morphogenetic protein-4 could be detected. Main osteoblastic marker genes Runx2, osterix, bone sialoprotein, and osteocalcin were up-regulated. Alizarin Red S staining demonstrated the formation of bone-like nodules, and bone sialoprotein and osteocalcin were localized to these foci by immunohistochemistry. Cells differentiated in monolayer conditions exhibited greater osteogenic potential compared to those from EB-derived cells. We conclude that in vitro hES cells can produce a mineralized matrix possessing all the major bone markers, the differentiation of pluripotent hES cells to an osteogenic lineage does not require initiation via EB formation, and that lineage potential is not dependent on the mode of differentiation induction but on a cell line itself.

Polyhexamethylene guanidine phosphate (PHMG-P) belongs to the polymeric guanidine family of biocides and contains a phosphate group, which may confer better solubility, a detoxifying effect and may change the kinetics and dynamics of PHMG-P interactions with microorganisms. Limited data regarding PHMG-P activity against periodontopathogenic and cariogenic microorganisms necessitates studies in this area. Aim is to evaluate polyhexamethylene guanidine phosphate antimicrobial activity in comparison to chlorhexidine. Quantitative suspension method was used enrolling Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus mutans and Lactobacillus acidophilus. Both tested antiseptics at their clinically-used concentrations, of 0.2% (w/v) and 1% (w/v), correspondingly provided swift bactericidal effects against S. aureus, P. aeruginosa, E. coli and C. albicans, A. actinomycetemcomitans and P. gingivalis with reduction factors higher than 6.0. Diluted polyhexamethylene guanidine phosphate and chlorhexidine to 0.05% continued to display anti-bacterial activity and decreased titers of standard quality control, periopathogens to below 1.0 × 103 colony forming units/ml, albeit requiring prolonged exposure time. To achieve a bactericidal effect against S. mutans, both antiseptics at all concentrations required a longer exposure time. We found that a clinically-used 1% of polyhexamethylene guanidine phosphate concentration did not have activity against L. acidophilus. High RF of polyhexamethylene guanidine phosphate and retention of bactericidal effects, even at 0.05%, support the use of polyhexamethylene guanidine phosphate as a biocide with sufficient anti-microbial activity against periopathogens. Polyhexamethylene guanidine phosphate displayed bactericidal activity against periopathogens and S. mutans and could potentially be applied in the management of oral diseases.