Transmission and scanning electron microscopic analysis of mineralized loci formed by human periodontal ligament cellsin vitro

Abstract

Fibroblasts isolated from human periodontal ligament (PDL) were cultured under a medium supplemented with ascorbic acid, beta-glycerophosphate and dexamethasone. The cultures were assessed for their ability to elaborate a mineralized matrix. Cell cultures stained positive when analyzed for alkaline phosphatase activity throughout the culture period. After about 3 weeks in culture, the cells produced a calcified matrix. Light microscopy showed formation of clusters of different shapes and sizes. Von Kossa staining revealed mineral deposits as amorphous brown-black precipitates. Transmission electron microscopy showed cells in multilayers and mineralized formations in close association with a dense network of collagen fibers. Scanning electron microscopy revealed smooth formations rising over the cultures with an abundant fiber matrix. We conclude that human PDL fibroblasts can be induced to form a mineralized matrix which shares features with bone mineralized matrix but most likely represents a more immature type of in vitro mineralization. Moreover, the present study further supports the osteoblastic potential of these cells.

99mTc-Hydroxydiphosphonate quantification of extracellular matrix mineralization in 3D human mesenchymal stem cell cultures

“…Recent studies have evaluated some novel, non-destructive approaches to quantify the osteogenic potential of human mesenchymal stem cells (hMSCs) using ultrasound and µ-CT. 21-23 Other published methods are scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive x-ray microanalysis (EDX), and x-ray diffraction analysis (XRD). All of these methods are highly elaborate techniques to quantify cell-mediated osteogenesis, 24-28 but they have at least one or more disadvantages: 1) they are harmful and destructive for the cell culture and scaffolds, therefore eliminating the availability to undertake further experiments or analytical methods; 2) the equipment and the method are very costly; 3) most of these methods only allow a qualitative proof of osteogenesis; 4) if a quantitative proof of HA measured by these methods is possible, it is measured indirectly, which is a potential source of error for the exact assessment of the mineral content of the specimen.…”

Preliminary approach of real‐time monitoring in vitro matrix mineralization based on surface plasmon resonance detection

“…Although these conventional approaches have demonstrated meaningful results, their critical drawbacks can limit the potential use in practical applications. For example, the SEM, EDX, and XRD methods are known to have limitations in quantitative analysis (Basdra and Komposch 1999;Chou et al, 2005;Wang et al, 2005). ICP is a quantitative and sensitive analysis technique, but it is somewhat time-consuming due to the pretreatment procedure required to get rid of organic residues (Wang et al, 2005).…”