MC3T3

Abstract: Objective: The aim of this study was to investigate the effects of 670-nm, 780-nm, and 830-nm laser irradiation on cell proliferation of normal primary osteoblast (MC3T3) and malignant osteosarcoma...

Abstract: The behavior of MC3T3 osteoblasts and human bone marrow stem cells on non-coated and hydroxyapatite (HAP)-coated carboxymethyl chitosan (CMCS) scaffolds was investigated in this study. Four HAP-coated scaffolds with different coating morphology and coverage were prepared by mineralization for 1 week in four different mineralizing solutions. Viability, attachment, proliferation, and differentiation of the osteoblasts on these scaffolds were evaluated, and an osteogenic gene expression analysis was carried out to investigate the osteoblastic differentiation of the stem cells. No cytotoxic effects were observed with both the non-coated and coated scaffolds. The non-coated CMCS scaffold supports attachment, proliferation, and differentiation of the osteoblasts and directs stem cell differentiation to osteoblast. Coating the scaffold with HAP substantially enhances these effects on the osteoblasts and stem cells. The main improvement was in the late stage of osteoblast differentiation since osteoblastic differentiation of the osteoblasts and stem cells in this stage was significantly enhanced by the coatings regardless of the variation in morphology and coverage. On the other hand, high HAP coverage was beneficial in stimulating osteoblast attachment and proliferation. This study demonstrates the good potential of HAP-coated CMCS scaffolds as osteogenic scaffolds to stimulate bone healing.

Abstract: Osteoblasts are cells responsible for matrix deposition during bone development and although temporal expression of many genes has been related to osteoblast differentiation, a complete description of osteoblast-specific gene regulation will lead to a better understanding of osteoblast function. In this study, microarray technology was used to analyze gene expression on a broad scale during osteoblast differentiation. Expression analysis of 9596 sequences revealed 342 genes and expressed sequence tags (ESTs) to be modulated differentially during a time course experiment in which murine C2C12 mesenchymal progenitor cells were induced to differentiate into mature osteoblasts by treatment with bone morphogenetic protein 2 (BMP-2). By means of hierarchical clustering, these genes were grouped by similarities in their expression profiles, resulting in subsets of early, intermediate, and late response genes, which are representative of the distinct stages of osteoblast differentiation. To identify new bone markers, the bone specificity of the late response genes was determined by comparing BMP-induced expression in C2C12 and MC3T3 osteoblasts with that in NIH3T3 fibroblasts. This resulted in the identification of nine novel genes and ESTs that were induced specifically in osteoblasts, in addition to the well-known markers ALP and osteocalcin. For at least one of these novel genes, Wnt inhibitory factor 1, and two of the ESTs, expression in developing bone was verified in vivo by in situ hybridization of E16.5 mouse embryos. In conclusion, by a combination of in vitro and in vivo screening approaches, a set of new genes related to osteoblast differentiation and skeletal development has been identified.

Abstract: Vitamin K2 likely exerts its protective effects during osteoporosis by promoting osteoblast differentiation and mineralization. However, the precise mechanism remains to be fully elucidated. Autophagy maintains cell homeostasis by breaking down and eliminating damaged proteins and organelles. Increasing evidence in recent years has implicated autophagy in the development of osteoporosis. The aim of the present study was to verify whether vitamin K2 (VK2) can induce autophagy during the differentiation and mineralization of osteoblasts. In the present study, MC3T3-E1 osteoblasts were treated with various doses of VK2 (10−8−10−3 M) for 1–5 days. The results revealed no cytotoxicity at concentrations below 10−5 M, but cell viability was reduced in a dose-dependent manner at concentrations above 10−5 M. Furthermore, MC3T3-E1 osteoblasts were seeded in 6-well plates in complete medium supplemented with dexamethasone, β-glycerophosphate and vitamin C (VC) for osteogenic differentiation. MC3T3-E1 osteoblasts treated with different concentrations (10−5, 10−6 and 10−7 M) of VK2 for 24 h on days 1, 3, 5 and 7 of the differentiation protocol. It was confirmed that VK2 promoted osteoblast differentiation and mineralization by using alkaline phosphatase (ALP) and alizarin red staining. Using western blotting, immunofluorescence, monodansylcadaverine staining and reverse transcription-quantitative polymerase chain reaction, it was observed that VK2 induced autophagy in osteoblasts. The results revealed that VK2 (1 µM) significantly increased ALP activity and the conversion of microtubule associated protein 1 light chain 3-α (LC3)II to LC3I in MC3T3-E1 osteoblasts (P<0.05) at every time point. The number of fluorescent bodies and the intensity increased with VK2, and decreased following treatment with 3-MA+VK2. There was an increase in the mRNA expression levels of ALP, osteocalcin (OCN) and Runt-related transcription factor 2 in VK2-treated cells (P<0.01). The present study further confirmed the association between autophagy and osteoblast differentiation and mineralization through treatment with an autophagy inhibitor [3-methyladenine (3-MA)]. Osteoblasts treated with 3-MA exhibited significant inhibition of ALP activity and osteogenic differentiation (both P<0.05). In addition, ALP activity and osteogenesis in the VK2+3-MA group was lower compared with VK2-treated cells (P<0.05 for both). The present study confirmed that VK2 stimulated autophagy in MC3T3 cells to promote differentiation and mineralization, which may be a potential therapeutic target for osteoporosis.