The effects of creatine (Cr) supplementation on primary rat osteoblast-like cells cultured as monolayer and micromass were investigated. Cr was added to the medium at concentrations of either 10 or 20 mM. At various time points, the cell cultures were analyzed morphologically, metabolically and biochemically.The degree of differentiation of primary osteoblast-like cell cultures was higher in micromass cultures compared to monolayer cultures, as judged by alkaline phosphatase (ALP) activity and extent of mineralization. In both culture systems, Cr supplementation showed positive effects, which were dependent on the organizational level of the osteoblast-like cells in such a way that the cells in monolayer culture showed significantly increased metabolic activity, ALP activity and mineralization in the presence of Cr than without the supplement. In micromass cultures, Cr also significantly enhanced ALP activity and mineralization, without affecting metabolic activity. The effect of Cr on ALP activity was more pronounced at higher concentrations of Cr, but 20 mM Cr already showed some adverse effects on cell viability. In conclusion, chemically pure Cr added to low serum cell culture medium has a stimulatory effect on metabolic activity, differentiation and mineralization of osteoblast-like cells indicating that Cr supplementation could also be used as a potential clinical intervention to stimulate cell growth, differentiation and mineralization during bone repair in vivo.

The effects of various cell isolation procedures, growth media and the cell culture density on the in vitro differentiation of neonatal rat calvarial osteoblast-like cells were investigated.Cells were isolated by enzymatic treatment, or after explant culture and inoculated as a monolayer or micromass in serum containing BGJ b , or Dulbecco's Modified Eagle Medium (DMEM). The cells were kept for up to 3 weeks in culture and were then characterized, both morphologically and biochemically.The isolation technique appeared to have no effect on the differentiation process. The calvaria could be used several times as explant cultures for a reliable source of differentiating osteoblast-like cells. The cultures kept in DMEM had a significantly higher DNA content, but significantly less alkaline phosphatase activity (ALP) per DNA and protein per DNA content than the BGJ b cultures. Monolayer cultures had a significantly higher DNA content than the micromass cultures, in both growth media. Furthermore, the micromass culture had a significantly higher ALP per DNA than monolayer cultures at 1 week. The morphology of all cell cultures at 3 weeks reflected the biochemical results. Only the cells grown in BGJ b formed abundant ALP positive and mineralized nodules in monolayer cultures. In contrast, cells grown as micromasses formed a dense calcified area, independently of the growth medium used.DMEM promoted the proliferation, whereas BGJ b stimulated the differentiation of osteoblast-like cells in monolayer cultures. Micromass cultures were less sensitive to nutritional conditions than monolayer cultures and promoted the differentiation of osteoblast-like cells.

Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its use is limited by a risk of cardiotoxicity, which is not completely understood. Recently, we have shown that DXR impairs essential properties of purified mitochondrial creatine kinase (MtCK), with cardiac isoenzyme (sMtCK) being particularly sensitive. In this study we assessed the effects of DXR on respiration of isolated structurally and functionally intact heart mitochondria, containing sMtCK, in the presence and absence of externally added creatine (Cr), and compared these effects with the response of brain mitochondria expressing uMtCK, the ubiquitous, non-muscle MtCK isoenzyme. DXR impaired respiration of isolated heart mitochondria already after short-term exposure (minutes), affecting both ADP- and Cr-stimulated respiration. During a first short time span (minutes to 1 h), detachment of MtCK from membranes occurred, while a decrease of MtCK activity related to oxidative damage was only observed after longer exposure (several hours). The early inhibition of Cr-stimulated respiration, in addition to impairment of components of the respiratory chain involves a partial disturbance of functional coupling between MtCK and ANT, likely due to interaction of DXR with cardiolipin leading to competitive inhibition of MtCK/membrane binding. The relevance of these findings for the regulation of mitochondrial energy production in the heart, as well as the obvious differences of DXR action in the heart as compared to brain tissue, is discussed.

During intramembranous bone formation, preosteoblasts condense, differentiate into osteoblasts and deposit bone matrix. We compared the differentiation process of rat calvarial osteoblast-like cells inoculated as micromasses, which mimic the in vivo condensation process, with cells inoculated as monolayers. The cells were analysed morphologically at 1,2 and 3 weeks by light microscopy (alkaline phosphatase activity, mineralization), by transmission electron microscopy, and biochemically (collagen typing, alkaline phosphatase activity, protein and DNA content). The cells inoculated as monolayers formed alkaline phosphatase positive and mineralized nodules during the culture period. The cells inoculated as a micromass formed a large mineralized area consisting of smaller nodules. The ultrastructure of the cells in both culture systems showed the typical features of osteoblasts and osteocytes. The main difference between monolayer and micromass cultures was found after 1 week in culture. The cells inoculated as a micromass formed a multilayer of cells. The cytoplasm contained rER, mitochondria, vesicles and ribosomes. There were abundant collagen fibrils in membrane folds and in the extracellular matrix. This was in contrast to the cells in monolayer culture which showed hardly any collagen fibrils in the extracellular matrix. The promotion of the differentiation was also confirmed by biochemical data showing that the DNA content was lower in the micromass than in the monolayer cultures during the culture period. These results show that micromass, as compared to monolayer, culture promotes the differentiation of rat osteoblast-like cells in vitro.

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