Comparison Study of Bone Defect Healing Effect of Raw and Processed Pyritum in Rats.

To evaluate and compare the effect of raw and processed pyritum on tibial defect healing, 32 male Sprague Dawley rats were randomly divided into four groups. After tibial defect, animals were produced and grouped: sham and control group were orally administrated with distilled water (1 mL/100 g), while treatment groups were given aqueous extracts of raw and processed pyritum (1.5 g/kg) for successive 42 days. Radiographic examination showed that bone defect healing effect of the treatment groups was obviously superior compared to that of the control group. Bone mineral density of whole tibia was increased significantly after treating with pyritum. Inductively coupled plasma-optical emission spectrometry showed that the contents of Ca, P, and Mg in callus significantly increased in the treatment groups comparing with the control. Moreover, serological analysis showed that the concentration of serum phosphorus of the treatment groups significantly increased compared with that of the control group. By in vitro study, we have evaluated the effects of drug-containing serum of raw and processed pyritum on osteoblasts. It was manifested that both the drug-containing sera of raw and processed pyritum significantly increased the mRNA levels of alkaline phosphatase and collagen type I. Protein levels of phosphorylated Smad2/3 also increased. The mRNA levels of osteocalcin and transforming growth factor ß (TGF-ß) type I and II receptors, as well as the protein levels of TGF-ß1 in the processed groups, were higher than those in the control. In summary, both raw and processed pyritum-containing sera exhibited positive effects on osteoblasts, which maybe via the TGF-ß1/Smad signaling pathway. Notably, the tibia defect healing effect of pyritum was significantly enhanced after processing.

[Research on Sailong boneextracts on proliferation and apoptosis of osteoblast cells].

OBJECTIVE: To investigate the metabolic regulation and apoptosis of Sailong bone extracts on rat osteoblast cells in vitro. METHOD: Sailong bone fat-soluble extract, Sailong bone ethanol extract and Sailong bone aqueous extract were extracted with super critical fluid extraction (SCFE) , and Sailong bone boiling water component was extracted with distilled water directly. MTT assay was applied to determine the proliferation of the cell promoted by four Sailong bone extracts and PAS assay for the aqueous proportion of the cell at different doses. RESULT: Sailong bone fat-soluble and aqueous extract (each 10 mg x mL (-1)) could significantly improve the proliferation of rat osteoblast cells ROS 17/2. 8 (P < 0. 01). Compared with the blank, the proportion of xub-G, of the different extracts from Sailong bone is reduce evidently. The result have shown the extracts from Sailong bone could reduce the rate of aqueous of cell and could suspend the aqueous. CONCLUSION: Sailong bone can promoting the proliferation, degrading the rate of the apoptosis and delay the development of osteoblast to be the substitute of the bone of tiger as a Chinese materia medica.

Effect of antler extract on corticosteroid-induced avascular necrosis of the femoral head in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Deer antler, traditionally used as a tonic and valuable drug in oriental medicine, has been considered to possess bone-strengthening activity and effectively used in bone diseases therapy. AIM OF THE STUDY: The present study was designed to investigate therapeutic effect of antler extract on avascular necrosis of the femoral head (ANFH) induced by corticosteroids in rats. MATERIALS AND METHODS: Rats were intragluteally injected with dexamethasone at 50mg/kg twice per week for 6 weeks to induce ANFH. Then the rats were treated with antler extract by oral gavage at 200mg/kg, 400mg/kg and 800 mg/kg once per day for 60 days. The concentration of hydroxyproline and hexosamine in serum was measured and the ultrastructure of femoral head was examined. In vitro, effect of the drug-containing serum of antler extract on proliferation and differentiation of primary osteoblasts were investigated by MIT assay, ALP activity assay and cell cycle analysis. RESULTS: After treatment with antler extract, the degree of necrosis induced by dexamethasone was significantly reduced, hydroxyproline was significantly decreased, and hexosamine and the ratio of hexosamine/hydroxyproline were significantly increased. The drug-containing serum of antler extract promotes osteoblastic proliferation through regulation of cell cycle progression. CONCLUSIONS: The results suggest that antler extract has a positive curative effect on ANFH by promoting osteoblastic proliferation.

FMS*Calciumfluor specifically increases mRNA levels and induces signaling via MAPK 42,44 and not FAK in differentiating rat osteoblasts.

The homeopathic compound of resonance FMS*Calciumfluor (FMS*) reportedly promotes osteogenic differentiation of rat pre-osteoblasts in vitro. Here, we show that the continuous exposure of differentiating rat osteogenic cells (ROB) to FMS* modulates the level of expression of mRNAs for 7 of the 8 osteogenic markers tested. Alkaline phosphatase (AP), osteocalcin (OC), metalloproteinases (MMP-2 and -14), procollagenase C (BMP-1), biglycan (BG) and integrin 1 are expressed at higher levels in FMS*-treated osteoblasts than in control cultures. MMP-2 and -14 mRNA are not down-modulated at mineralization. Also, the pattern of expression induced by FMS* for some of these genes (BMP-1, BG and integrin 1) is changed, but collagen type I (Coll I) mRNA levels are not affected by treatment with FMS*. This suggests that FMS* modulates mRNA levels and that this is not generalized, but gene(s) specific. We also report that exposure to FMS* rapidly and transiently induces activation of mitogen-activated protein kinases (MAPKs) 42,44 in populations of early osteoblasts, but not in pre-osteoblasts, with a cell differentiation stage-dependent and pertussis toxin (PTX)-sensitive response. Subsequent to FMS* MAPK signaling activation, an increase in AP and MMP-14 mRNA is detected, which is also inhibited by PTX, suggesting that FMS* activation of MAPK signaling could be an early event required for the induction of these genes. Exposure to FMS* does not cause changes in the activity of p125 (FAK)-mediated signaling.

Osteogenesis in vitro in rat tibia-derived osteoblasts is promoted by the homeopathic preparation, FMS*Calciumfluor.

We studied the effects of in vitro treatment of differentiating osteogenic cells with FMS*Calciumfluor, to determine whether it caused changes in proliferative or differentiation potential of osteoblasts. FMS*Calciumfluor was developed for the therapy of post-menopausal and age-related osteoporosis on the basis of the principles of resonance homeopathy and VTR Vega test. Its daily prescribed therapeutical usage is about 30,000-fold less in fluoride concentration than that recommended for NaF associated with calcium monophosphate. Rat tibial osteoblast (ROB) primary cultures represent populations of early osteoblasts and their derivative cultures of more than 60 cumulative population doubling (CPD) represent more mature osteogenic cells. Both these populations were shown to undergo in vitro differentiation, as monitored by the sequential expression of markers that define the stages of the osteogenic progression. Here we report that continual treatment of ROB during osteogenesis with FMS*Calciumfluor modulated the expression of critical osteogenic markers: alkaline phosphatase (AP), an indicator of osteoblast maturation, and(45)Ca incorporation into the matrix and nodule formation, events of the last phase of osteogenesis and a measure of osteoid mineralization. Treatment did not affect proliferation, or expression and activation of metalloproteinases (MMP). AP activity and levels of AP mRNA were increased by treatment with FMS*Calciumfluor; the incorporation of radiolabelled Ca into the matrix was also increased and the formation of nodules occurred in a shorter time and with higher frequency than in untreated control cultures. The effects of FMS*Calciumfluor were concentration dependent and specific for its modalities of preparation, and were observed at a concentration about three orders of magnitude lower than similar effects reported in the literature by treatment of osteoblast cultures in vitro with NaF.

Velvet antler polypeptides promoted proliferation of chondrocytes and osteoblast precursors and fracture healing.

AIM: To study the effects of velvet antler (VA) total polypeptides (VATP) and VA polypeptides, VAP-A, VAP-B, and VAP-C on proliferation of chondrocytes and osteoblast precusors. METHODS: Chondrocytes (rabbit and human fetus) and osteoblast precusors (chick embryo) were incubated in the culture medium containing VATP or VAP-A, VAP-B, and VAP-C. [3H]TdR incorporation into DNA was measured. Fracture healing-promoting action of VATP was determined in rats. RESULTS: VATP 50-200 mg.L-1 and VAP-B 12.5, 25, and 50 mg.L-1 showed most marked proliferation-promoting activity for rabbit costed chondrocytes and increased incorporation of [3H]TdR from (73 +/- 9) Bq (control group) to (272 +/- 55), (327 +/- 38), and (415 +/- 32) Bq, respectively (P < 0.01). The activity of VAP-A was weaker than that of VAP-B, and VAP-C had no activity. VATP 10 and 20 mg.kg-1 by local injection into the cross-section fracture area accelerated healing of radial fracture. The healing rate of VATP-treated group was higher (75%) than that of control group (25%) (P < 0.05). CONCLUSION: VATP accelerated fracture healing by stimulating proliferation of chondrocytes and osteoblast precursors.