Cellular considerations for optimizing bone cell culture and remodeling in a lab-on-a-chip platform

Our lab has developed a lab-on-a-chip platform for bone remodeling that enables long-term culturing of bone cells out to 7 weeks and serves as a foundation toward a multicellular organ-on-a-chip system. Here, we optimized culturing protocols for osteoblasts, osteoclasts and osteocytes within the lab-on-a-chip and performed functional activity assays for quantifying bone formation and resorption. We analyzed cell seeding densities, feeding schedules and time in culture as a basis for optimizing culturing protocols. Further, we addressed concerns of sterility, cytotoxicity and leakage during the extended culture period within the polydimethylsiloxane chip. This system provides a method for quantifying the soluble effects of mechanically stimulated osteocytes on bone remodeling (formation/resorption).

Thermosensitive chitosan gels containing calcium glycerophosphate for bone cell culture

In this article, properties of thermosensitive chitosan hydrogels prepared with the use of chitosan chloride with β-glycerophosphate disodium salt pentahydrate enriched with calcium glycerophosphate are presented and compared with chitosan hydrogels with β-glycerophosphate disodium salt pentahydrate. The study is focused on the determination of hydrogel structure and biological testing of hydrogels with human osteoblasts line Saos-2. The structure of gels was visualized by scanning electron microscopy and was investigated by infrared spectroscopy. The crystallinity of gel structure was determined by X-ray diffraction analysis and thermal effects were determined using differential scanning calorimetry thermograms.

Ki-Energy (Life-Energy) Stimulates Osteoblastic Cells and Inhibits the Formation of Osteoclast-Like Cells in Bone Cell Culture Models

Some practitioners of the Nishino Breathing Method (NBM) were found to have a higher bone density than the average values of age- and gender-matched non-practitioners. Using bone cell culture models, we investigated a possible mechanism behind this observation. For the study of bone mineralization, we performed the following two experiments using cultured osteoblastic MC3T3-E1 cells: (i) Kozo Nishino, a Japanese Ki expert, sent Ki-energy to the cells once for 5 or 10 min after they were seeded in culture dishes in the presence of 10% fetal bovine serum (FBS). They were incubated for 72 h and the cells were counted. The number in the dish with 10-min Ki-exposure was significantly greater than that in the control (P< 0.01 withn= 8). We performed a reverse transcription-polymerase chain reaction (RT–PCR) study using these cells, but the mRNA expressions did not change significantly. (ii) After cells were incubated for 72 h without Ki-exposure (in the presence of FBS), they were further cultured for 48 h (in the absence of FBS) to promote differentiation. At the beginning of the second culture stage, Ki was applied once for 10 min. After 48 h, RT–PCR was performed. The mRNA expressions which are related to bone mineralization, such as Runx2, α1(I) collagen, alkaline phosphatase and osteocalcin, increased significantly (P< 0.05 andn= 4 for all). For the bone resorption study, we used mouse marrow cultures, which can form osteoclast-like cells in the presence of (1–34) parathyroid hormone (PTH), and stimulate resorption. We exposed these cells to Ki-energy twice for the duration of 5 or 10 min on day 0 and day 4. On day 7, the cells were counted. The number of osteoclast-like cells in dishes with Ki exposure was significantly smaller than those in control dishes (P< 0.05 withn= 5). The difference between 5-min exposure and 10-min exposure was not statistically significant. All of our data suggest that the Ki-effect on osteoporosis should be further explored.

Influence of Catalyst Type on Biocompatibility of Polyurethanes

Segmented polyurethanes (PUR) based on poly(ε-caprolactone)diol (PCL) as soft segments and cycloaliphatic diisocyanate were obtained. Two different types of catalysts were used for synthesis: dibutyltin dilaurate (DBTDL) and iron (III) acetylacetonate Fe(acac)3. Structure, thermal analysis, water absorption of obtained polyurethanes were studied. Polyurethanes catalyzed by Fe(acac)3 have lower glass transition temperature (Tg2) and melting temperature (Tm2) of hard domains in comparison with polyurethanes synthesized with DBTDL. Amount of absorbed water is in the range of 1,0-2,0% and is higher for PUR catalyzed by DBTDL. SEM observations show that type of catalyst used in synthesis affect structure of polyurethanes. Biological test XTT was done in order to determine influence of various metallic compounds used as catalyst on viability of human bone cell culture. The XTT results indicate no influence on viability of human osteoblasts cultured on the obtained PUR.