Antibacterial effects of electrospun chitosan/poly(ethylene oxide) nanofibrous membranes loaded with chlorhexidine and silver.

To prevent percutaneous device associated infections (PDAIs), we prepared electrospun chitosan/poly(ethylene oxide) (PEO) nanofibrous membrane containing silver nanoparticles as an implantable delivery vehicle for the dual release of chlorhexidine and silver ions. We observed that the silver nanoparticles were distributed homogeneously throughout the fibers, and a fast release of chlorhexidine in 2days and a sustained release of silver ions for up to 28days. The antibacterial efficacy of the membranes against Staphylococcus aureus showed that the membranes exhibited an obvious inhibition zone upon loading with either chlorhexidine (20µg or more per membrane) or AgNO3 (1 and 5wt% to polymer). Furthermore, long-term antibacterial effect up to 4days was verified using membranes containing 5wt% AgNO3. The results suggest that the membranes have strong potential to act as an active antibacterial dressing for local delivery of antibacterial agents to prevent PDAIs.

The effects of seeding density and osteoclastic supplement concentration on osteoclastic differentiation and resorption.

The bone resorbing osteoclasts are a complex type of cell essential for in vivo bone remodeling. There is no consensus on medium composition and seeding density for in vitro osteoclastogenesis, despite the importance thereof on osteoclastic differentiation and activity. The aim of this study was to investigate the relative effect of monocyte or peripheral blood mononuclear cell (PBMC) seeding density, osteoclastic supplement concentration and priming on the in vitro generation of functional osteoclasts, and to explore and evaluate the usefulness of commonly used markers for osteoclast cultures. Morphology and osteoclast formation were analyzed with fluorescence imaging for tartrate resistant acid phosphatase (TRAP) and integrin ß3 (Iß3). TRAP release was analyzed from supernatant samples, and resorption was analyzed from culture on Corning® Osteo Assay plates. In this study, we have shown that common non-standardized culturing conditions of monocyte or PBMCs had a significant effect on the in vitro generation of functional osteoclasts. We showed how increased osteoclastic supplement concentrations supported osteoclastic differentiation and resorption but not TRAP release, while priming resulted in increased TRAP release as well. Increased monocyte seeding densities resulted in more and large TRAP positive bi-nuclear cells, but not directly in more multinucleated osteoclasts, resorption or TRAP release. Increasing PBMC seeding densities resulted in more and larger osteoclasts and more resorption, although resorption was disproportionally low compared to the monocyte seeding density experiment. Exploration of commonly used markers for osteoclast cultures demonstrated that Iß3 staining was an excellent and specific osteoclast marker in addition to TRAP staining, while supernatant TRAP measurements could not accurately predict osteoclastic resorptive activity. With improved understanding of the effect of seeding density and osteoclastic supplement concentration on osteoclasts, experiments yielding higher numbers of functional osteoclasts can ultimately improve our knowledge of osteoclasts, osteoclastogenesis, bone remodeling and bone diseases.

Tuning the resorption-formation balance in an in vitro 3D osteoblast-osteoclast co-culture model of bone.

The aim of the present study was to further improve an in vitro 3D osteoblast (OB) - osteoclast (OC) co-culture model of bone by tuning it towards states of formation, resorption, and equilibrium for their future applications in fundamental research, drug development and personalized medicine. This was achieved by varying culture medium composition and monocyte seeding density, the two external parameters that affect cell behavior the most. Monocytes were seeded at two seeding densities onto 3D silk-fibroin constructs pre-mineralized by MSC-derived OBs and were co-cultured in one of three different media (OC stimulating, Neutral and OB stimulating medium) for three weeks. Histology showed mineralized matrix after co-culture and OC markers in the OC medium group. Scanning Electron Microscopy showed large OC-like cells in the OC medium group. Micro-computed tomography showed increased formation in the OB medium group, equilibrium in the Neutral medium group and resorption in the OC medium group. Culture supernatant samples showed high early tartrate resistant acid phosphatase (TRAP) release in the OC medium group, a later and lower release in the Neutral medium group, and almost no release in the OB medium group. Increased monocyte seeding density showed a less-than-proportional increase in TRAP release and resorption in OC medium, while it proportionally increased TRAP release in Neutral medium without affecting net resorption. The 3D OB-OC co-culture model was effectively used to show an excess of mineral deposition using OB medium, resorption using OC medium, or an equilibrium using Neutral medium. All three media applied to the model may have their own distinct applications in fundamental research, drug development, and personalized medicine.

Osteoblast-osteoclast co-cultures: A systematic review and map of available literature.

Drug research with animal models is expensive, time-consuming and translation to clinical trials is often poor, resulting in a desire to replace, reduce, and refine the use of animal models. One approach to replace and reduce the use of animal models is to use in vitro cell-culture models. To study bone physiology, bone diseases and drugs, many studies have been published using osteoblast-osteoclast co-cultures. The use of osteoblast-osteoclast co-cultures is usually not clearly mentioned in the title and abstract, making it difficult to identify these studies without a systematic search and thorough review. As a result, researchers are all developing their own methods, leading to conceptually similar studies with many methodological differences and, as a consequence, incomparable results. The aim of this study was to systematically review existing osteoblast-osteoclast co-culture studies published up to 6 January 2020, and to give an overview of their methods, predetermined outcome measures (formation and resorption, and ALP and TRAP quantification as surrogate markers for formation and resorption, respectively), and other useful parameters for analysis. Information regarding these outcome measures was extracted and collected in a database, and each study was further evaluated on whether both the osteoblasts and osteoclasts were analyzed using relevant outcome measures. From these studies, additional details on methods, cells and culture conditions were extracted into a second database to allow searching on more characteristics. The two databases presented in this publication provide an unprecedented amount of information on cells, culture conditions and analytical techniques for using and studying osteoblast-osteoclast co-cultures. They allow researchers to identify publications relevant to their specific needs and allow easy validation and comparison with existing literature. Finally, we provide the information and tools necessary for others to use, manipulate and expand the databases for their needs.