Information on the interconversion potential of adipocytes, the key in the understanding of bone turnover in metabolic diseases such as osteoporosis has been limited by the lack of pure adipocytic cell population. The object of the present study was i) to isolate pure populations of adipocytes from human bone marrow, ii) to clone single adipocytes from these populations, and iii) to examine in vitro the interconversion potential of the progeny of these single cloned adipocytes between the osteogenic and adipogenic phenotypes. Adipogenic colonies were isolated from the low-density floating fraction of normal bone marrow cultured in adipogenic media for 4 days. Single adipocytes were isolated and cloned by limiting dilution. The cloned adipocytes were found to dedifferentiate into fibroblastlike cells, and subsequently to differentiate into two morphologically distinct cell types: osteoblasts and adipocytes in appropriate culture systems. The adipocytic phenotype was confirmed by morphology, oil red O staining and immunocytochemistry using antiserum to aP2. The osteogenic phenotype was confirmed by alkaline phosphatase, osteocalcin immunostaing using an osteocalcin antiserum OS35 and mineralized nodule formation. These findings confirm that there is plasticity between the differentiation of adipocytic and osteogenic cells in human bone marrow stromal cell cultures. In conclusion, we have shown the ability of isolated clonal adipogenic cells to redifferentiate into cells of the osteogenic and adipogenic lineage and defined the conditions required for the in vitro modulation of adipogenic differentiation. These results provide further evidence that the osteogenic and adipogenic cells share a common multipotential precursor.
Adipocytes* ; Adipogenesis* ; Alkaline Phosphatase ; Bone Marrow* ; Clone Cells* ; Humans* ; Immunohistochemistry ; Mesenchymal Stromal Cells ; Metabolic Diseases ; Osteoblasts ; Osteocalcin ; Osteogenesis* ; Osteoporosis ; Phenotype ; Plastics*

No Abstract Available.
Chondrocytes* ; Phenotype*

Stem cells and regenerative medicine are emerging and promising fields both in academic and industry points of views. They are currently under active investigation worldwide and their market size is expected to grow rapidly in the near future. The Korean government is also investing a huge amount of money on these fields to promote R&D and product commercialization. However, its technical maturity is still in its infant state and many hurdles should be resolved to accelerate technology to business. I can definitely say that we have to focus in the future on innovations in technology, regulations and reimbursement. In particular, the importance of translational research and clinical studies are of no doubt in the stem cells and regenerative medicine. I am going to deal with some of these issues in more detail in the main text.
Commerce ; Humans ; Infant ; Regenerative Medicine ; Social Control, Formal ; Stem Cells ; Translational Medical Research

Ryanodine has different effects on the contractility of rat and guinea pig ventricular muscle. Thus we investigated the effect of ryanodine on the intracellular Ca2+ and Na+ activities of the rat and guinea pig ventricular myocytes with two specific aims; whether there are any differences in intracellular Na+ activities between rat and guinea pig ventricular muscle cells, and if any, how the differences in intracellular Na+ activities are related to the effect of Na(+)-Ca2+ exchange on the action potential configuration and excitation-contraction coupling of the rat and guinea pig ventricular myocytes. Ryanodine (10(-7) M) diminished the slow repolarization phase of the rat ventricular action potential while the duration of the rapid repolarization phase increased. Ryanodine (10(-7) M) significantly increased the plateau of the action potential. At the steady state of 0.2 cps, intracellular Na+ activities (aiNa) of the rat and guinea pig ventricular myocytes were 8.7 +/- 5.2 mM (n = 16, 4 rats) and 10.0 +/- 4.1 mM (n = 25, 7 guinea pigs) respectively, but there were no statistically significant differences. The contractility of the rat ventricular muscle nearly disappeared due to ryanodine (10(-7) M) with little changes in aiNa. Monensin (10 mM) not only increased the resting tension but also remarkably increased aiNa from 2.0 mM to 20 mM. Ryanodine (10(-7) M) continuously decreased aiNa of the guinea pig ventricular muscle after the contraction ceased to decrease. Monensin increased the contractility as well as aiNa. These results suggest that the contractility of rat and guinea pig ventricular myocytes is determined by the change in the action of the Na(+)-Ca2+ exchange mechanism depending upon the plateau of action potential and the intracellular Na+ and Ca2+ activities. So ryanodine could decreases the contractility via its effect on Na(+)-Ca2+ exchange transport which could be one of possible mechanisms of negative inotropism by ryanodine.
Action Potentials/drug effects ; Animal ; Female ; Guinea Pigs ; Heart/*drug effects ; Heart Ventricle ; Intracellular Membranes/metabolism ; Male ; Myocardial Contraction/*drug effects ; Myocardium/cytology/*metabolism ; Rats ; Ryanodine/*pharmacology ; Sodium/*metabolism ; Support, Non-U.S. Gov't

No abstract available.
Female ; Mammaplasty*

Recent electrophysiological data have provided the evidences that background currents such as Na(+)-Ca2+ exchange can significantly modulate cardiac pacemaker activity. In this study, the effects of extracellular Na+ and Ca2+ concentrations on the pacemaker activity were investigated by measuring the intracellular Na+ activity (aiNa) with Na(+)-selective microelectrodes and the results are summarized as follows. 1) In the rabbit SA node, aiNa was 3.2 +/- 0.3 mM and mean MDP (maximal diastolic potential) was -63.3 +/- 1.4 mV. 2) Graded decreases of external Na+ concentration resulted in the loss of spontaneous beating, hyperpolarization and the decrease of aiNa. 3) An increase in extracellular Ca2+ concentration in low Na+ solution augmented the transient decrease of aiNa, about 3 minutes in low Na+ solution, until aiNa started to increase. 4) In low Na+ solution, which had extracellular Ca2+ concentration according to the calculation based on the equilibrium state of Na+-Ca2+ exchange, aiNa was continuously decreased. It was concluded that intracellular Na+ activity modulated by Na+-Ca2+ exchange could play an important role in the initiation of pacemaker potential.
Action Potentials ; Animal ; Biological Transport ; Calcium/*metabolism/physiology ; Electrophysiology ; Female ; Male ; Pacemaker, Artificial ; Rabbits ; Sinoatrial Node/*metabolism/physiology ; Sodium/*metabolism/physiology ; Support, Non-U.S. Gov't

Bone formation by osteoblast may be closely related to the increase in intracellular Ca2+ activity of osteoblast. In order to study the effects of changes in Ca2+ activity of osteoblast-like cell on fracture healing, we changed intracellular Ca2+ activity of osteoblast-like cells by vanadate and verapamil. And the process of fracture healing was observed after injection of the treatment osteoblast-like cells into the fracture site by hematoxylin-eosin (H-E) stain and bromodeoxyuridine (BrdU) stain. The results were as follow: 1) The most effective range of concentration which could facilitate bone formation was 10-6 to 10-5 M. 2) H-E stain showed more abundant osteoblast and osteoid tissues, more active mitotic division of osteoblast, and earlier appearance of chondroblast and chondroid tissue, making the maturation of woven bone faster in the vanadate-treated group than in the control group. The opposite was true in the verapamil-treated group compared with the control group. 3) BrdU labeling index showed more active osteoblastic proliferation in the vanadate-treated than in the control group. The opposite was observed in the verapamil-treated group compared with the control group. From these results, the fracture healing appears to be facilitated and decelerated by vanadate which apparently increase intracellular Ca2+ activity of osteoblast and verapamil which decreases it, repectively. Therefore the change of intracellular Ca2+ activity of osteoblast can be considered to be one of fracture healing mechanisms and expected to be applied for clinical purpose.
Bromodeoxyuridine ; Chondrocytes ; Fracture Healing ; Osteoblasts ; Osteogenesis ; Vanadates ; Verapamil

PURPOSE: Bone and cartilage were manufactured by using tissue engineering of mesenchymal stem cell (MSC) which can differentiate into variety of cell types. MATERIAL AND METHOD: MSC was isolated and cultured from the rabbit weighing 500g, and it was seeded into PGA mesh and pre-cultured for 1 week with different TGF- beta 3 treated conditions. It was implanted into nude mice and tissues generated were recovered from 1, 2, 3, 4, 8 ,and 12 weeks respectively. Degree of bone and cartilage formation was analyzed with histology and immunohistochemistry assay. RESULT: Pre-culture condition with TGF- beta 3 treatment showed early start of chondrogenic differentiation, and degree of bone and cartilage formation was promoted as time passed. But both of the cases differentiated into complete bone after 12 weeks. CONCLUSION: The results show that pretreatment of TGF- beta 3 promotes the differentiation process in vivo condition under the in vivo system where MSC differentiate into bone via cartilage formation.
Animals ; Cartilage ; Immunohistochemistry ; Mesenchymal Stromal Cells* ; Mice ; Mice, Nude ; Tissue Engineering

Articular chondrocytes have been known to have heterogeneity in articular cartilage. Four layers are generally recognized from the articular surface to the subchondral bone. We have used Percoll density gradients to separate chondrocytes from articular cartilage into distinct subpopulations. Non-fibrillated articular cartilage was obtained from rabbit knee. The cells were carefully layered on the top of the preformed gradient and spun. After centrifugation, we obtained four fractions: Fraction A referred boundary between 0% and 10%, fraction B from between 10% and 20%, fraction C from between 20% and 30%, and fraction D from between 40% and 50%. In the A fraction, cells are relatively larger and round in shape, while their nuclei are relatively smaller. In the cytoplasm many lipid droplets were found and rough endoplasmic reticulum were disrupted. In the D fraction, chondrocyte is small, with large nucleus which surrounded by well-developed rough endoplasmic reticulum. The type II collagen proteins were expressed strongly and more proteoglycans synthesized in fractions A and B. And chondrocytes from the fraction D divided more slowly than those from the fractions A, B, and C. We have succeeded in separating chondrocytes from articular cartilage into distinct subpopulations by Percoll density gradients, as well as characterized growth rate, histological appearances and phenotypic expression. This study is the first report about the Percoll density gradients to separate articular chondrocytes.
Cartilage, Articular ; Centrifugation ; Chondrocytes* ; Collagen Type II ; Cytoplasm ; Endoplasmic Reticulum, Rough ; Knee ; Population Characteristics ; Proteoglycans

The removal of Ca2+ from the cardioplegic solutions could cause the danger of inducing a "calcium paradox" during reperfusion. Since intracellular Ca2+ activities are coupled to Na+ activities via Na(+)-Ca2+ exchange, an increase in intracellular Na+ activities during the cardioplegia could cause an abrupt Ca2+ influx when reperfused. To study the effects of Na+ and Ca2+ concentrations in cardioplegic solutions on intracellular Ca2+ activities during the cardioplegia and subsequent recovery period, the membrane potential and intracellular Na+ and Ca2+ activities of guinea pig ventricular papillary were measured. 1) A cardioplegia with low Ca2+ cardioplegic solution significantly decreased the overshoot and duration of the first action potential after cardioplegia, but the changes in action potential configuration were minimized after a cardioplegia with Ca2+ concentration adjusted according to the Na(+)-Ca2+ exchange mechanism. 2) Intracellular Na+ activity was continuously decreased during the cardioplegia, and the intracellular Na+ activity 20 minutes after cardioplegia was the highest with low Ca2+ cardioplegic solution. 3) Intracellular Na+ and Ca2+ activities were continuously decreased during the cardioplegia with Ca2+ concentration adjusted according to the Na(+)-Ca2+ exchange mechanism. 4) During a reperfusion of Tyrode solution after cardioplegia intracellular Na+ and Ca2+ activities were increased. Intracellular Ca2+ activity was increased more rapidly than intracellular Na+ activity. 5) The rate of increase in intracellular Ca2+ activity with reperfusion of Tyrode solution was dependent upon intracellular Na+ activity during cardioplegia, in such a way that the higher the intracellular Na+ activity was, the faster the intracellular Ca2+ activity increased. These data suggest that Na(+)-Ca2+ exchange mechanism may play an important role in the regulation of intracellular Ca2+ activity during recovery after cardioplegia.
Animal ; Calcium/*pharmacology ; Cardioplegic Solutions/*pharmacology ; Ions ; *Myocardial Reperfusion ; Osmolar Concentration ; Papillary Muscles/cytology/*drug effects ; Sodium/*pharmacology ; Solutions/pharmacology