Mechanism Of Osseointegration In Hydroxyapatite Induced Murine Pre-Osteoblasts Cell Line (Mc3t3-E1) Stimulated With Recombinant Interleukin 6 And/Or Interleukin 17A
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Date
2015-07
Authors
Sri Tharan, Shaminea
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
Cytokines are gaining momentum due to their possible role in enhancing osseointegration and their potential role in bone remodelling. The compatibility of the implant affects osseointegration in many ways. Therefore, extensive studies are being carried out to enhance osteoblast formation minimizing such complications. HA has been widely utilized due to its osteogenic property in current osseointegration applications. Receptor activator of NF-κB ligand (RANKL) has been shown to regulate osteoclast differentiation and its function. While osteoprotegerin (OPG) blocks the binding of RANKL thus inhibiting the differentiation of osteoclasts which then favours osteogenesis. Interleukin-6 (IL-6) and Interleukin-17 (IL-17), the key regulators of immune system have been known to play potential roles in bone remodelling. Hence, the aim of the current study was to determine the potential role of rIL-6 and rIL-17A in regulating the OPG/RANKL system of the murine osteoblast cell line (MC3T3-E1) in the presence of hydroxyapatite (HA). Cell proliferation and differentiation activity was measured by MTS and ALP assays respectively. Gene and protein expression was performed on RANKL and OPG markers using qPCR, Western blot and ELISA. In addition, SEM was carried out to observe the overall osteoblast cell attachment on the HA scaffold. This study involved the interaction of osteoblasts with cytokines alone and those seeded on HA and treated with cytokines. Analyses of MTS and ALP assays showed
that osteoblasts treated with 10 ng/ml of rIL-6 or rIL-17A significantly induced proliferation and ALP activities; thus 10 ng/ml was found to be optimal for downstream experiments. Gene expression analysis showed significant up-regulation of OPG and ALP in all the treated groups (rIL-6, rIL-17A, rIL-6 + rIL-17A, HA + rIL-6, HA + rIL-17A and HA + rIL-6 + rIL-17A). In contrast, treatment of cells with rIL-6 and/or rIL-17A showed down-regulation of RANKL expression. Osteoblast cells treated with combinations of rIL-6 + rIL17A showed marked increase in OPG/RANKL ratio. Similar pattern was observed in the expression of protein in osteoblasts treated with combinations of rIL-6 + rIL-17A and rIL-17A. However, a retroactive mechanism was observed in rIL-6 as detected by Western blotting and ELISA. This pattern was further supported by the SEM analysis where rIL-6 + rIL-17A and rIL-17A were found to be perfectly attached and intercalated with HA. On the other hand, blebs and ruffles were more prominent in osteoblast treated with rIL-6 compared to the other treated and control groups. Overall, the results of this study revealed that the combination of cytokines (rIL-6 + rIL-17A) show promising outcome compared to all the other treated samples. Hence, it can be concluded that the synergistic effect of rIL-17A towards IL-6 favours bone regulation. These findings suggest a new mechanism of regulation by these cytokines on the expression of OPG and RANKL, which could promote osteogenesis and diminish osteoclastogenesis with or without the presence of HA.
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Keywords
Cytokines are gaining momentum , in enhancing osseointegration.