Jung-Keun Kim

Effect of Rehmannia glutinosa Libosch extracts on bone metabolism

BACKGROUND Rehmannia glutinosa Libosch extracts (RGX) were investigated to determine if they play roles in bone metabolism. METHODS The effects on osteoblasts were determined by measuring (1) cell proliferation, (2) alkaline phosphatase (ALP) activity, (3) mRNA expression of bone-related proteins, (4) transcriptional activity of Runx2, and (5) osteoprotegerin (OPG) secretion. The effects on the osteoclasts were investigated by measuring (1) tartrate-resistant acid phosphatase-positive [TRAP(+)] multinucleated cell (MNC) formation and (2) resorption areas after culturing osteoclast precursors. Bone mineral density (BMD) measurements and histological observations on rats were also carried out. RESULTS RGX treatment showed a significant increase in both the proliferation and ALP activity of osteoblasts. RGX increased the expression of the bone-related genes. OPG secretion was markedly increased after RGX treatment. In addition, RGX treatment decreased the number of TRAP(+) MNCs and the resorption areas. In vivo studies using ovariectomy-induced osteoporotic rats revealed that RGX alleviated the decrease in the trabecular BMD, and increased the cortical bone thickness and trabeculation of the bone marrow spaces. CONCLUSIONS RGX stimulates the proliferation and activities of osteoblasts, while inhibiting the generation and resorptive activities of osteoclasts. It also shows preventive effects on osteoporotic bone loss induced by an ovariectomy. Although the active substances have not yet been identified, it is believed that the RGX seems to contain active components that have a potential to enhance the bone metabolism in osteoporosis.

Establishment and characterization of a stable cell line to evaluate cellular Runx2 activity

Runx2 is an essential transcription factor for osteoblast differentiation from early commitment step to final differentiation. Based on its crucial role in osteoblast differentiation, the transcriptional activity of Runx2 protein implies more valuable information for osteoblast differentiation than any other parameters, such as Runx2 mRNA or protein level. Thus, a sensitive, specific, and consistent method to determine the Runx2 transcriptional activity has long been expected. Here we suggest a stable cell line that carries 6xOSE2‐Luciferase reporter vector would be a good evaluation system to determine biological Runx2 transcriptional activity. The proliferation rate, cell shape, and the myogenic differentiation potential of the cloned cell line were similar to those of parental premyoblastic C2C12 cells. The cells specifically responded to Runx2 modulating agent such as FGF2. The stable cell line responded 5–6 folds more sensitively than the transiently transfected cells with Runx2. Though overexpression of any Runx gene stimulated the luciferase activity, Runx2 enhanced the reporter activity the highest. Collectively, the 6xOSE2‐luc stable cells would be a good biological evaluation system to assess the activity of extracellular Runx2 modulating stimulations as well as the signal transduction pathways involved in the stimulations.

Dissolution of poorly crystalline apatite crystals by osteoclasts determined on artificial thin-film apatite.

Poorly crystalline apatite (PCA) crystals introduced into bone tissue should be stable for a definite period before they are dissolved as a result of a host response. In this report, the dissolution of PCA crystals by the action of osteoclasts was studied on artificial thin films. These consisted of PCA crystals having similar crystallographic properties to bone crystals which were developed for assaying the osteoclast activity in vitro. The dissolution of minerals by osteoclasts decreased along with the decreased amount of labile phosphate and hydrogen phosphate domains of apatite crystals, which were caused by the crystal maturation temperature. A profound effect on mineral dissolution by pH in the culture medium was also shown. Low acidity considerably increased mineral dissolution, whereas a slight alkalinity totally blocked mineral dissolution. There was little difference in the mineral dissolution behavior of osteoclasts near the physiologic pH. In addition, it was determined whether mineral dissolution by osteoclasts was dependent on the destruction of the organic matrix. Nocodazole was introduced to inhibit the secretion of hydrolytic enzymes, and acetazolamide was added to inhibit acid production by the osteoclasts. There was no significant change as a result of nocodazole addition on mineral dissolution or by the addition of acetazolamide on degradation of collagen. These results indicate that small changes in the physicochemical properties of apatite crystals can decrease resorption by osteoclasts, which can be highly activated at low pH. These results also suggest that mineral dissolution and organic degradation by osteoclasts are self-regulating.