Number Of Calcified Nodules Research Articles

Construction of human bone morphogenetic protein-7 gene fluorescent eukaryotic cell expression vector and test of bioactivity in vitro

To construct fluorescent eukaryotic cell expression vector with human bone morphogenetic protein-7 (hBMP-7) gene and to transfect mouse stromal cell line W-20-17 to detect the bioactivity of pEGFP-hBMP-7 in vitro. pEGFP-hBMP-7 plasmid was constructed by subcloning technique and identified by enzyme cutting and electrophoresis. W-20-17 cells were transfected with pEGFP-hBMP-7 by means of lipofectamine-2000 media methods. Transfection efficiency and gene expression were evaluated by fluorescent microscopy. ALP, von Kossa and osteocalcin (OC) were tested to determined the phenotypes of osteoblast. After 48 hours, the gene transfection efficiency was 40%. Based on GFP and immunofluorescence of pEGFP-hBMP-7, there was the expression of aim gene. After gene transfection, there were not significant different of cell morphology feature and cell proliferation. ALP activity, the number of calcium nodules and the expression of OC increased. pEGFP-hBMP-7 with bioactivity was constructed, which could induce W-20-17 cells to differentiate to osteoblasts.

Effects of pH on Mineralization Ability of Human Dental Pulp Cells

The purpose of this study was to investigate the effect of alkaline pH on calcification in human dental pulp (HDP) cells. HDP cells were cultured in pH 7.8 conditioned medium, and alkaline phosphatase (ALP) activity was measured. The ALP activity was higher in the pH 7.8 conditioned medium group than in the pH 7.2 conditioned medium group. Expression of mRNAs for bone morphogenetic protein (BMP)-2 was measured by the RT-PCR technique. The expression of BMP-2 in the pH 7.8 groups was greater than that in the pH 7.2 group. Furthermore, we determined Calcified nodule formation by von Kossa staining. The number of calcified nodules was increased in the pH 7.8 conditioned medium. These results suggest that HDP cell mineralization was enhanced in alkaline pH (pH 7.8) conditioned medium.

Transforming growth factor‐beta 1 (TGF‐β1) prevents the age‐dependent decrease in bone formation in human osteoblast/implant cultures

Titanium implants have been extensively used in orthopedic surgery and dentistry. Most of the patients who receive such implants are elderly with a compromised ability to heal and form new bone. By using an in vitro osteoblast/implant culture system, the potency of TGF-beta1 in enhancing mineralization of human osteoblast cultures from elderly subjects was investigated in this study. Primary human osteoblast (HOB) cells obtained from different age group human subjects [Young (Y), Middle (M), and Old (O)] were cultured on Ti alloy (Ti-6Al-4V) disks with or without continuous administration of 0.2 ng/mL TGF-beta1 in the medium for 2 or 4 weeks. TGF-beta1 significantly (p < 0.05) increased calcium content and the size of calcified nodules on implant disks in the O group, but had no effect on the Y or M groups. The number of calcified nodules was not different with or without TGF-beta1 in all age groups. As measured by Northern blot analysis and RT-PCR, TGF-beta1 significantly increased the expression of bone-specific extracellular matrix proteins, including alkaline phosphatase, Type I collagen, bone sialoprotein and osteocalcin, after both 2 and 4 weeks in the O group but not in the Y group. In conclusion, TGF-beta1 enhances mineralization on implant materials of osteoblast cultures from elderly human subjects.