Bisphosphonates: restrictions for vasculogenesis and angiogenesis: inhibition of cell function of endothelial progenitor cells and mature endothelial cells in vitro. Clin Oral Invest 2011;15:105-11

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原文題目(出處): Bisphosphonates: restrictions for vasculogenesis and angiogenesis: inhibition of cell function of endothelial progenitor cells and mature endothelial cells in vitro. Clin Oral Invest 2011;15:105-11

原文作者姓名: Thomas Ziebart, Andreas Pabst, Marcus Oliver Klein, Peer Kämmerer, Leonie Gauss, Dan Brüllmann, Bilal Al-Nawas

& Christian Walter

通訊作者學校: Klinik für Mund-, Kiefer-und Gesichtschirurgie, Johannes Gutenberg–Universität Mainz, Augustusplatz, Mainz, Germany

報告者姓名(組別): R2 許逸忠

報告日期: 2011/11/22



 Bisphosphonate-associated osteonecrosis of the jaws (BP-ONJ) is one of the main side effects

 The exact mechanism of action and etiopathology is still unknown.

 In addition to inhibition of bone remodelling, an anti-angiogenetic effect has become the focus of research

 The aim of these study was to investigate the effect of different bisphosphonates on human umbilicord vein endothelial cells (HUVEC) and endothelial progenitor cells (EPC), which play an important role in angiogenesis.

 Using varying concentrations, the impact of one non-nitrogen containing bisphosphonate (clodronate) and three nitrogencontaining bisphosphonates (ibandronate, pamidronate and zoledronate) on HUVEC and EPC was analysed.

 The biologic behaviour of HUVEC after incubation with different bisphosphonates was measured in a Boyden migration assay as well as in a 3D angiogenesis assay. The number of apoptotic cells was measured by Tunnel assay.

 To underline the importance of neoangiogenesis in the context of BP-ONJ, we measured the EPC number after incubation with different bisphosphonates in vitro

 HUVEC and EPC were significantly influenced by bisphosphonates at different concentrations compared with the non-treated control groups.

 The nitrogen-containing bisphosphonates pamidronate and zoledronate had the greatest impact on the cells, whereas clodronate followed by ibandronate was less distinct on cell function.

 These results underline the hypothesis that inhibited angiogenesis induced by bisphosphonates might be of relevance in the development and maintenance of BP-ONJ.


 The benefit of bisphosphonates in the treatment of malignant bone neoplasias like multiple myeloma, bone metastases or metabolic bone diseases like Paget’s disease and severe osteoporosis is without controversy.

 Since 2003, a new specific side effect, bisphosphonate-associated osteonecrosis of the jaws (BP-ONJ), has increasingly become the focus of clinical and preclinical investigations.

 BP-ONJ is defined as exposed necrotic bone in the maxillofacial region for a


period of at least 8 weeks in connection with current or previous bisphosphonate therapy and a lack of head and neck radiation in the patient’s history.

 Nitrogencontaining bisphosphonates such as pamidronate and zoledronate are more often associated with BP-ONJ compared to the less potent ibandronate and the nonnitrogen- containing clodronate.

 The reduced bone remodelling in BPONJ patients is attributed to bisphosphonate-induced inhibition of osteogenic cells and osteoclasts in a dose-dependent manner. Furthermore, bisphosphonates have been proven to reduce viability of oral keratinocytes, which corresponds with impaired mucosal wound healing

 Bone tissue is particularly vascularised, and endothelial cells have been proven to play an essential role during bone remodeling

 the term angiogenesis refers to sprouting from preexisting vessels of rather mature endothelial cells. Of great scientific interest is the fact that bone-marrow-derived, circulating endothelial progenitor cells (EPC) bear the potential of creation of primordial vessels, called vasculogenesis

 EPC have the potential to differentiate into mature endothelial cells.

Furthermore, EPC show strong paracrine effects by production of several cytokines and growth factors which increase neovascularisation

 However, histopathological analysis of BP-ONJ bone specimens revealed markedly reduced density of vessels or even avascular necrosis.

 The focus of the present study was to monitor the influence of four differently potent bisphosphonates on EPC viability, migration and apoptosis rate.

Furthermore, mature endothelial cells (human umbilical vein endothelial cells, HUVEC) were investigated accordingly to monitor possible influence of the maturation stage of the endothelial lineage.

Materials and methods -Cell culture-

 HUVEC were cultured in an endothelial basal medium (EBM) supplemented with 1 μg/mL hydrocortisone, 12 μg/mL bovine brain extract, 50 μg/mL gentamicin, 50 ng/mL amphotericin-B, 10 ng/mL epidermal growth factor and 10% foetal calf serum (FCS) until the third passage

-EPC culture assay-

 Mononuclear cells (MNCs) were isolated by density gradient centrifugation with Biocoll (Biochrom KG, Berlin, Germany) from peripheral blood of healthy human volunteers

 Immediately following isolation, total MNCs (8×106 cells/mL medium) were plated on 25 cm2 culture flasks coated with human fibronectin (Sigma, Steinheim, Germany) and maintained in EBM supplemented with EGM SingleQuots, VEGF (100 ng/mL), and 20% FCS.

-Apoptosis assay-

 HUVECs (5×104) in EBM-2 medium were seeded in sixwell plates; after 24 h, HUVECs were incubated with bisphosphonates (clodronate, ibandronate, pamidronate and zoledronate) at increasing bisphosphonate concentrations (0, 5, 50, 100, 200 and 500 μmol) for 24 h

-Migration assay-

 To examine the effect of bisphosphonates on HUVEC migration, we used a


 HUVECs were stimulated to migrate from the upper to lower chambers by the addition of 10 ng/mL VEGF to the lower chambers. After 12 h, cells were stained with fluorescent dye calcein- AM.

-In vitro angiogenesis assay-

 Different concentrations of bisphosphonates were added to the test medium.

The sprouting colonies were photo-documented over 48 h. For measurement of the number, length and area of the sprouts, we utilised a newly designed automatic analyzing programme (CellAnalyser).

-Statistical analysis-

 Continuous variables are expressed as mean ± SEM. Comparisons between groups were analysed by t test (twosided) or ANOVA (post hoc test: Tukey) for experiments with more than two subgroups (SPSS software). Values of p < 0.05 were considered as statistically significant.

Results -EPC count-

 EPC count was reduced in a dose-dependent manner for all investigated bisphosphonates after 48 h.

 Cell count : Zoledronate had the greatest impact.

-HUVEC viability-

 HUVEC viability was significantly reduced for pamidronate (p<0.001) and zoledronate (p<0.001) after 48 h.

-HUVEC migration-

 The effect of pamidronate and zoledronate was significantly stronger compared to the other bisphosphonates


-Angiogenesis assay-

 ibandronate and pamidronate showed markedly reduced sprouts after 48 h.

Incubation with zoledronate basically showed no sprouts after 24 h.

  pamidronate and zoledronate had a significantly negative impact at 48 h


 The development of blood vessels is essential for healing and regeneration processes, especially in the avascular necrosis of BP-ONJ

 EPC are important for vasculogenesis, a mechanism of new vessel formation

 Nitrogen-containing bisphosphonates have an influence on the migration ability of HUVEC

 HUVEC treated with pamidronate and zoledronate had a higher rate of apoptosis compared to the groups treated with clodronate or ibandronate.

 In the 3D angiogenesis model, pamidronate and zoledronate had significantly reduced numbers and areas of sprouts.

 EPC cultures treated with bisphosphonates, especially zoledronate, had significantly reduced cell numbers.


 The biological ability, cell function and viability of HUVEC and EPC is influenced by the nitrogen-containing bisphosphonates ibandronate, pamidronate and zoledronate. Interestingly, clodronate has only a minor effect.

 In vitro studies on mature endothelial cells have shown impairment of cell proliferation , promotion of programmed cell death and inhibition of capillary tube formation by bisphosphonates

 Non-nitrogen-containing bisphosphonates such as clodronate are built into non-hydrolysable analogues of adenosine triphosphate (ATP) that inhibit many different ATP-dependent intracellular enzymes. Therefore, very high concentrations are needed to completely inhibit special pathways.

 In contrast, the nitrogencontaining bisphosphonates, e.g. pamidronate and zoledronate, inhibit a key enzyme of the mevalonate pathway: the farnesyl pyrophosphate synthase. This pathway is important for the production of small Gproteins, which are important for intracellular structure and mechanisms such as intracellular transport

 This may contribute to the greater impact of nitrogen-containing bisphosphonates

 With the natural limitations of in vitro studies, these data support the theory of the anti-angiogenetic component (angiogenesis and vasculogenesis) in the development and maintenance of BP-ONJ.

 Therefore, these findings may also explain the higher occurrence of BP-ONJ in patients receiving nitrogen-containing bisphosphonates as compared to patients receiving non-nitrogen-containing bisphosphonates like clodronate.

題號 題目

1 對於osteonecrosis of the jaw,何項錯誤?

(A) IV較口服容易得到ONJ

(B) 服用corticosteroid亦是危險因子

(C) 若牙科治療有牽涉到齒槽骨則會提高風險,抽菸喝酒則較無相


(D) 對於ONJ的發生理論有許多,抑制血管再生乃為其中一項

答案(C) 出處:Bisphosphonates and Osteonecrosis of the Jaw, 2011 Nov 8.

題號 題目

2 關於雙磷酸鹽類何項錯誤?

(A) 雙磷酸鹽類可抑制osteoclast的作用

(B) 雙磷酸鹽類可以治療一些cancer,如multiple myeloma,bone metastasis

(C) 雙磷酸鹽類對胃刺激性大,需伴隨飯後食用

(D) 不含氮基的雙磷酸鹽類相對需較大濃度才有效用

答案(C) 出處:Bisphosphonates and Osteonecrosis of the Jaw, 2011 Nov 8.




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