Enhanced Osteogenic Differentiation in Zoledronate-Treated Osteoporotic Patients

Abstract

Bisphosphonates are well known inhibitors of osteoclast activity and thus may be employed to influence osteoblast activity. The present study was designed to evaluate the in vivo effects of zoledronic acid (ZA) on the proliferation and osteoblastic commitment of mesenchymal stem cells (MSC) in osteoporotic patients. We studied 22 postmenopausal osteoporotic patients. Densitometric, biochemical, cellular and molecular data were collected before as well as after 6 and 12 months of ZA treatment. Peripheral blood MSC-like cells were quantified by colony-forming unit fibroblastic assay; their osteogenic differentiation potential was evaluated after 3 and 7 days of induction, respectively. Circulating MSCs showed significantly increased expression levels of osteoblastic marker genes such as Runt-related transcription factor 2 (RUNX2), and Osteonectin (SPARC) during the 12 months of monitoring time. Lumbar bone mineral density (BMD) variation and SPARC gene expression correlated positively. Bone turnover marker levels were significantly lowered after ZA treatment; the effect was more pronounced for C terminal telopeptide (CTX) than for Procollagen Type 1 N-Terminal Propeptide (P1NP) and bone alkaline phosphatase (bALP). Our findings suggest a discrete anabolic activity supported by osteogenic commitment of MSCs, consequent to ZA treatment. We confirm its anabolic effects in vivo on osteogenic precursors.

Keywords: Runt-related transcription factor 2 (RUNX2); bone turnover; differentiation; gene expression; mesenchymal stem cells; zoledronic acid.

Figure 1

Schematic representation of the experimental design.

Figure 2

Mean values of bone metabolism/osteogenic differentiation markers across the time points. Biochemical values were obtained from blood samples; qRT-PCR values obtained from differentiating cells were utilized as gene expression markers. For each marker, mean values were calculated from the standardized z-score. The lumbar BMD increase during treatment correlated significantly with the expression of osteogenic marker SPARC (p < 0.05); COL1A1 gene expression showed an analogous trend without reaching any statistical significance (p > 0.05).

Figure 3

Peripheral blood derived-MSCs and osteogenic differentiation after ZA infusion. Cellular and molecular analyses display the reduction of Colony Forming Unit-Fibroblastic (CFU-F) (A) and the increase of osteogenic expression (B) during the treatment. The values are representative of 22 patients at each observation time. Three independent qRT-PCRs were performed at the same time for each gene. The overall information suggests an enhanced osteoblastogenesis in ZA-treated patients. (* p < 0.05 vs. pre-therapy; ^# p < 0.05 vs. 6 months).

Figure 4

Linear correlation between lumbar spine bone mineral density (BMD) and SPARC gene expression levels (represented as fold change). The correlation was statistically significant (p < 0.05).