Bioactive Glass Particles in Two-Dimensional and Three-Dimensional Osteogenic Cell Cultures

Abstract This study aimed to investigate the influence of a three-dimensional cell culture model and bioactive glass (BG) particles on the expression of osteoblastic phenotypes in rat calvaria osteogenic cells culture. Cells were seeded on two-dimensional (2D) and three-dimensional (3D) collagen with BG particles for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity was performed. Cell morphology and immunolabeling of noncollagenous bone matrix proteins were assessed by epifluorescence and confocal microscopy. The expressions of osteogenic markers were analyzed using RT-PCR. Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay. Experimental cultures produced a growing cell viability rate up to 14 days. Although ALP activity at 7 days was higher on BG cultures, cells on 3D and 3D+BG had an activity decrease of ALP at 14 days. Three-dimensional conditions favored the immunolabeling for OPN and BSP and the expression of ALP and COL I mRNAs. BG particles influenced positively the OC and OPN mRNAs expression and calcified nodule formation in vitro. The results indicated that the 3D cultures and BG particles contribute to the expression of osteoblastic phenotype and to differentiated and mineralized matrix formation.

Resumo O objetivo deste estudo foi investigar a influência do modelo de cultura celular tridimensional e das partículas de vidro bioativo (BG) sobre a expressão fenotípica de culturas de células osteogênicas da calvária de ratos. As células foram mantidas em culturas sobre superfícies colágenas bi-dimensionais (2D) e em géis de colágeno tridimensional (3D) com e sem partículas de BG até 14 dias. Foram avaliadas: viabilidade celular, atividade de fosfatase alcalina (ALP), morfologia celular e imunomarcação de proteínas da matriz não-colágena do osso através de epifluorescência e microscopia confocal. As expressões de marcadores osteogênicos foram analisadas utilizando RT-PCR. A formação de nódulos mineralizados foi visualizada através de microscopia e o conteúdo de cálcio foi avaliado quantitativamente pelo Alizarina Red. As culturas experimentais produziram uma taxa crescente de viabilidade até 14 dias. Embora a atividade ALP em 7 dias tenha sido maior em culturas com BG, as células em 3D e 3D+BG apresentaram uma diminuição da atividade ALP aos 14 dias. As condições tridimensionais favoreceram a imunomarcação para OPN e BSP e a expressão de mRNAs para ALP e COL I. As partículas de BG influenciaram positivamente a expressão do mRNAs para OPN e OC e a formação de nódulos calcificados in vitro. Os resultados indicaram que as culturas em 3D e partículas BG contribuíram para a expressão do fenótipo osteoblástico e para a diferenciação e formação de matriz mineralizada.

Mycobacterium leprae downregulates the expression of PHEX in Schwann cells and osteoblasts

Neuropathy and bone deformities, lifelong sequelae of leprosy that persist after treatment, result in significant impairment to patients and compromise their social rehabilitation. Phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX) is a Zn-metalloendopeptidase, which is abundantly expressed in osteoblasts and many other cell types, such as Schwann cells, and has been implicated in phosphate metabolism and X-linked rickets. Here, we demonstrate that Mycobacterium leprae stimulation downregulates PHEX transcription and protein expression in a human schwannoma cell line (ST88-14) and human osteoblast lineage. Modulation of PHEX expression was observed to a lesser extent in cells stimulated with other species of mycobacteria, but was not observed in cultures treated with latex beads or with the facultative intracellular bacterium Salmonella typhimurium. Direct downregulation of PHEX by M. leprae could be involved in the bone resorption observed in leprosy patients. This is the first report to describe PHEX modulation by an infectious agent.

Petroleum ether extract of Cissus quadrangularis (Linn. ) enhances bone marrow mesenchymal stem cell proliferation and facilitates osteoblastogenesis

OBJECTIVE: To evaluate the effects of the petroleum ether extract of Cissus quadrangularis on the proliferation rate of bone marrow mesenchymal stem cells, the differentiation of marrow mesenchymal stem cells into osteoblasts (osteoblastogenesis) and extracellular matrix calcification. This study also aimed to determine the additive effect of osteogenic media and Cissus quadrangularis on proliferation, differentiation and calcification. METHODS: MSCs were cultured in media with or without Cissus quadrangularis for 4 weeks and were then stained for alkaline phosphatase. Extracellular matrix calcification was confirmed by Von Kossa staining. marrow mesenchymal stem cells cultures in control media and osteogenic media supplemented with Cissus quadrangularis extract (100, 200, 300 µg/mL) were also subjected to a cell proliferation assay (MTT). RESULTS: Treatment with 100, 200 or 300 µg/mL petroleum ether extract of Cissus quadrangularis enhanced the differentiation of marrow mesenchymal stem cells into ALP-positive osteoblasts and increased extracellular matrix calcification. Treatment with 300 µg/mL petroleum ether extract of Cissus quadrangularis also enhanced the proliferation rate of the marrow mesenchymal stem cells. Cells grown in osteogenic media containing Cissus quadrangularis exhibited higher proliferation, differentiation and calcification rates than did control cells. CONCLUSION: The results suggest that Cissus quadrangularis stimulates osteoblastogenesis and can be used as preventive/alternative natural medicine for bone diseases such as osteoporosis.