Positive Effects of Bisphosphonates on Osteogenic Differentiation in Patient-Derived Mesenchymal Stem Cells for the Treatment of Osteoporosis

Abstract

Background: Recent evidence from in vitro and in vivo studies indicates that bisphosphonates may promote osteoblastic bone formation and potently inhibit osteoclast activity. However, little is known about the potential effect of bisphosphonates on the recruitment of osteoblastic precursors from patient-derived bone marrow stromal cells due to difficulties in accessing human bone marrow from healthy and disease subjects.

Methods: In this study, we evaluated the potential of using FDA-approved and clinically utilized bisphosphonates such as alendronate, ibandronate, and zoledronate to enhance the development of bone forming osteoblasts from osteoporosis patient- and healthy-person derived hBMSCs (op-MSCs and hp-MSCs, respectively). hBMSCs were obtained from postmenopausal women without endocrine diseases or receiving hormone replacement therapy. Cells were treated with or without a bisphosphonate (alendronate, ibandronate, and zoledronate) and analyzed over 21 days of culture.

Results: hBMSC from osteoporosis-patient with bisphosphonates treatment demonstrated a significant increase in Alizarin red staining after 7 days compared to that from healthy-person. Calcium contents and alkaline phosphatase (ALP) enzyme activity also demonstrated an increased propensity in hMSCs from osteoporosis-patient compared to those from healthy-person, although there were inter-individual variations. Gene expression levels varied among different donors. There were no significant differences in the effect on the osteoblastic differentiation of hBMSCs among alendronate, ibandronate, and zoledronate. Statistical significance in the osteoblastic differentiation of hBMSCs between the positive control group cultured in osteogenic medium alone and groups cultured in osteogenic medium supplemented with bisphosphonate was not shown either. These results might be due to various cell types of hBMSCs from individual clinical patients and concentrations of bisphosphonate used.

Conclusion: Our study using a clinically relevant in vitro model suggests that bisphosphonate treatment is more effective for patients with osteoporosis than its preventive effect for healthy person. In addition, patient-specific responses to bisphosphonates should be considered rather than bisphosphonate type prior to prescription. Further investigations are needed to determine how bisphosphonates influence hBMSCs function to mediate bone quality and turnover in osteoporotic patients. Such studies can generate novel approaches to treat age-related osteoporotic bone loss.

Keywords: Bisphosphonates; Mesenchymal stem cell; Osteoblast; Osteoporosis.

Fig. 1

Optical microscopy of osteogenic differentiation with culture time. A At 7 days after culture. B At 21 days after culture. After 7 days after culture, op-MSCs appeared to have induced deposition of mineral like particles. A dramatic course of mineralization was observed over 21 days of culture. Mineralization of hp-MSCs was detected over 21 days of culture. PC positive control, NC negative control, ALN alendronate, IBN ibandronate, ZON zoledronate

Fig. 2

Mineralization capacity. Mineralization was effectively induced in op-MSCs treated with bisphosphonates. A Alizarin Red staining after 7 days culture. B Alizarin Red staining after 21 days of culture. C Calcium concentration measured after 7 days culture and D 21 days culture. PC positive control, NC negative control, ALN alendronate, IBN ibandronate, ZON zoledronate

Fig. 3

A ALP staining at 7 days after culture. B ALP activity at 7 days after culture. Enhanced ALP activity in op-MSCs compared to that in hp-MSCs was observed. There were also donor-specific variations. PC positive control, NC negative control, ALN alendronate, IBN ibandronate, ZON zoledronate

Fig. 4

Effect of bisphosphonate treatment on osteogenic gene expression in differentiating hBMSCs. A At 7 days after treatment. B At 21 days after treatment. Although ALP and BSP gene expression levels were dependent on individual donor, the expression level of BSP gene, a late osteoblastic differentiation marker, in op-MSCs was increased significant earlier compare to hp-MSCs. PC positive control, NC negative control, ALN alendronate, IBN ibandronate, ZON zoledronate

Fig. 5

Comparative statistical effect of bisphosphonate treatment in op-MSCs and hp-MSCs. A ALP activity. B Calcium concentration. PC positive control, NC negative control, ALN alendronate, IBN ibandronate, ZON zoledronate