The Susceptive Alendronate-Treatment Timing and Dosage for Osteogenesis Enhancement in Human Bone Marrow-Derived Stem Cells

Abstract

Recent studies indicated that alendronate enhanced osteogenesis in osteoblasts and human bone marrow-derived stem cells. However, the time- and dose-dependent effects of Aln on ostegenic differentiation and cytotoxicity of hBMSCs remain undefined. In present study, we investigated the effective dose range and timing of hBMSCs. hBMSCs were treated with various Aln doses (1, 5 and 10 µM) according to the following groups: group A was treated with Aln during the first five days of bone medium, groups B, C and D were treated during the first, second, and final five days of osteo-induction medium and group E was treated throughout the entire experiment. The mineralization level and cytotoxicity were measured by quantified Alizarin Red S staining and MTT assay. In addition, the reversal effects of farnesyl pyrophosphate and geranylgeranyl pyrophosphate replenishment in group B were also investigated. The results showed that Aln treatment in groups A, B and E enhanced hBMSC mineralization in a dose-dependent manner, and the most pronounced effects were observed in groups B and E. The higher dose of Aln simultaneously enhanced mineralization and caused cytotoxicity in groups B, C and E. Replenishment of FPP or GGPP resulted in partial or complete reverse of the Aln-induced mineralization respectively. Furthermore, the addition of FPP or GGPP also eliminated the Aln-induced cytotoxicity. We demonstrated that hBMSCs are susceptible to 5 µM Aln during the initiation stage of osteogenic differentiation and that a 10 µM dose is cytotoxic.

In vitro effects of alendronate on fibroblasts of the human rotator cuff tendon

“…The decreased cell viability was mainly caused by apoptosis involving the caspase-3 pathway. Alendronate-induced cytotoxicity has been reported in several studies involving oral keratinocytes [18], gingival fibroblasts [21], human periodontal ligament fibroblasts [18], endothelial cells [22], and human bone-marrow-derived stem cells [23]. Those studies all found that the highest concentrations of alendronate, ranging from 10 −11 M to 100 M, had various levels of cytotoxicity to the studied cells.…”

Alendronate Can Improve Bone Alterations in Experimental Diabetes by Preventing Antiosteogenic, Antichondrogenic, and Proadipocytic Effects of AGEs on Bone Marrow Progenitor Cells

“…By this mechanism it reduces osteoclastic stress fiber and focal adhesion density, leading to a disruption of the actin cytoskeleton [38, 39]. However, low doses of alendronate have also been shown to stimulate osteoblast function in vitro [20, 40] and to modulate BMPC phenotypic induction by increasing osteogenesis and decreasing adipogenesis [41–44]. Our present results are in agreement with these previous studies: in vitro exposure of BMPC to alendronate alone (at doses that have been reported in the serum and resorption lacunae of patients treated with this N-BP) [45–47] increased their osteoblastic differentiation.…”

Enhancement of Bone Marrow-Derived Mesenchymal Stem Cell Osteogenesis and New Bone Formation in Rats by Obtusilactone A

“…The PCR reaction was carried out with specific primers for each gene and a thermostable DNA polymerase (Gibco BRL, Bethesda, MD, USA). According to our previous report, the osteogenic marker gene changes in mRNA expression levels of BMP2 , osteocalcin , type I collagen and Cbfa1/Runx2 were then analyzed [ 21 , 22 ]. The following mouse primer pairs were used: Osteocalcin (OC) (5′-CTTGGTGCACACCTAGCAGA-3′ forward and 5′-CTCCCTCATCGTGTT GTC CCT-3′ reverse) and Cbfa1/Runx2 (5′-CGCTCCGCCCACAAATCTC-3′ forward and 5′-CCGCACGACAACCGCACCAT-3′ reverse), BMP2 (5′-TGCGGTCTCCTAAAGGTCG-3′ forward and 5′-GAGGACCTGGGGAAGCA-3′ reverse), Collagen I (5′-GGGGCAAGACAGTCATCGAA-3′ forward and 5′-GGGTGGAGGGAGTTTACACG-3′ reverse) and GAPDH (5′-ATACGGCTACAGCAACAGGG-3′ forward and 5′-GCCTCTCTTGCTCAGTGTCC-3′ reverse) as a house-keeping gene.…”