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.
Highlights
Osteogenic cell culture systems are routinely used to study the tightly regulated cellular and extracellular matrix events of bone formation in a controlled environment
MTT assay showed that cell viability was negatively affected by the 3D culture and bioactive glass (BG) particles at 7 days
The present study confirmed that three-dimensional collagen and bioactive glass particles supported cell viability and favored Alkaline phosphatase (ALP) activity, mineralized nodule formation and osteoblastic phenotype expression of osteogenic cells derived from newborn rat calvaria
Summary
Osteogenic cell culture systems are routinely used to study the tightly regulated cellular and extracellular matrix events of bone formation in a controlled environment. A commonly used system to study bone formation in vitro is the primary culture of rat calvaria osteogenic cells (well characterized as an abundant source of osteoblasts and committed osteoprogenitor cells). Culture conditions, the temporal sequence of osteoblastic differentiation and expression of matrix proteins have been well defined for this system. The expression pattern of matrix proteins in vitro has been correlated with acquisition and maturation of the osteoblast phenotype [1]. In vitro studies on osteogenesis and matrix formation are frequently based on two-dimensional (2D) cultures. There are well-established differences between cells grown in 2D culture and a three-dimensional (3D) environment [2]. Collagen is the most useful biomaterial that has been used for tissue engineering purposes, due its excellent biocompatibility and safety associated with its biological characteristics, such as biodegradability and weak antigenicity materials in the cells [3]
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