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Bone quality of the newest bone formed after two years of teriparatide therapy in patients who were previously treatment-naïve or on long-term alendronate therapy

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Abstract

Summary

The results of the present study, involving analysis of biopsies from patients who received teriparatide for 2 years and were previously either treatment-naïve or on long-term alendronate therapy, suggest that prior alendronate use does not blunt the favorable effects of teriparatide on bone quality.

Introduction

Examine the effect of 2 years of teriparatide (TPTD) treatment on mineral and organic matrix properties of the newest formed bone in patients who were previously treatment-naïve (TN) or on long-term alendronate (ALN) therapy.

Methods

Raman and Fourier transform infrared microspectroscopic analyses were used to determine the mineral/matrix (M/M) ratio, the relative proteoglycan (PG) content, and the mineral maturity/crystallinity (MMC; determined by three methods: carbonate content, full width at half height of the v 1 PO4 band [FWHH], and wavelength at maxima of the v 1 PO4 band), as well as collagen maturity (ratio of pyridinoline/divalent cross-links), in paired iliac crest biopsies at trabecular, endosteal, and osteonal surfaces of newly formed bone in postmenopausal osteoporotic women who were previously either TN (n = 16) or receiving long-term ALN treatment (n = 24).

Results

Trabecular M/M ratio increased and matrix content decreased significantly in the ALN pretreated group. Collagen maturity decreased in both patient groups. Endosteal M/M ratio increased significantly in the TN group. Trabecular M/M ratio was higher at endpoint in the ALN pretreated group than in the TN group. Overall, no changes from baseline were observed in PG content, except that PG content was higher in the ALN pretreated group than in the TN group at endosteal surfaces at endpoint. The ability of TPTD treatment to reduce MMC in both patient groups and at the different bone surfaces depended on the measurement tool (relative carbonate content or wavelength at maxima of the v 1 PO4 band). None of the changes in MMC were different between the two patient groups.

Conclusions

The results suggest some favorable impact of TPTD on bone mineral and organic matrix properties of in situ forming bone in terms of increased initial mineralization and decreased MMC and collagen maturity. Moreover, prior long-term ALN administration may have only limited influence on these properties in bone newly formed after 2 years of TPTD treatment.

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Acknowledgments

The authors thank Eli Lilly & Co, for provision of the biopsy samples and financial support for the present study. This study was also supported by the Allgemeine Unfallversicherungsanstalt (AUVA), research funds of the Austrian workers compensation board, and the Wiener Gebietskrankenkasse (WGKK), Viennese sickness insurance funds.

Conflicts of interest

Drs Birgit Hofstetter, Sonja Gamsjaeger, Franz Varga, Harald Dobnig, Jan J. Stepan, Klaus Klasuhofer, and Eleftherios P. Paschalis declare no conflict of interest. This study was partially supported by Eli Lilly and Co. Helmut Petto and Imre Pavo are full time employees of Eli Lilly and Co.

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Authors and Affiliations

  1. Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK, AUVA Trauma Centre Meidling, 1st Medical Department, Hanusch Hospital, Vienna, Austria

    B. Hofstetter, S. Gamsjaeger, F. Varga, K. Klaushofer & E. P. Paschalis

  2. Department of Internal Medicine, Division of Endocrinology and Nuclear Medicine, Medical University of Graz, Graz, Austria

    H. Dobnig

  3. Institute of Rheumatology, Faculty of Medicine, Charles University, Prague, Czech Republic

    J. J. Stepan

  4. Lilly Research Laboratories, Indianapolis, IN, USA

    H. Petto & I. Pavo

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  1. B. Hofstetter
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  2. S. Gamsjaeger
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  3. F. Varga
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  4. H. Dobnig
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  5. J. J. Stepan
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  6. H. Petto
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  7. I. Pavo
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  9. E. P. Paschalis
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Correspondence to E. P. Paschalis.

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Drs Helmut Petto and Imre Pavo are full time employees of Eli Lilly and Co.

Appendix I

Appendix I

To further verify observations concerning the reduced amount of organic matrix produced in ALN pretreated patients after 2 years of TPTD therapy (Fig. 1c), exploratory cell culture experiments were performed as follows:

MC3T3-E1 cells (kindly donated by Dr. Kumegawa, Meikai University, Japan) were cultured in α-minimum essential medium (MEM) (Biochrom AG, Germany) supplemented with 5 % fetal calf serum (Sigma), 10 μg/ml gentamycin and 50 μg/ml ascorbic acid. All cultures were maintained at 37 °C and 5 % CO2 in a humidified atmosphere. Cells were sub-cultured twice a week using 0.001 % pronase E (Roche) and 0.02 % EDTA in Ca2+ and Mg2+ free phosphate-buffered saline (PBS). To prevent phenotypic drift during repeated sub-cultures, the cells were not used more than 4 weeks after thawing. After seeding the cells for 1 day (at a density of 20,000 cells/cm2), cells were treated with ALN (supplied by Procter & Gamble Pharmaceuticals Clinical Supplies, Norwich, NY) at 10 −6 and 10 −8 M for up to 7 days, with the medium changed every 3 days. Total RNA was isolated according to the single-step method by acid guanidinium thiocyanate-phenol-chloroform extraction [56], with RNeasy columns (Qiagen Sciences, Germany) used for further RNA cleaning. First-strand cDNA was synthesized using 1 μg of total RNA in 20 μl of reverse transcription (RT) reaction mixture containing 0.2 μg of pd(N)6 random hexamer (GE Healthcare), 2 mM of each deoxynucleotide triphosphate (GE Healthcare), first-strand buffer [50 mM Tris/HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2] (Invitrogen), 10 mM dithiothreitol (Hersteller), and 100 units of SuperScriptTM II RNase H RT (Hersteller). The RT reaction was performed at 42 °C for 50 min followed by a 15 min inactivation step at 70 °C. Quantitative RT polymerase chain reaction (PCR) was performed in triplicate using the Rotogene 6000 System (Corbett Research), according to the manufacturer’s instructions. Following this, mRNA expression of COL (Col1a1) was measured, using inventoried TaqMan gene expression assays (Applied Biosystems) normalized to 18S rRNA (Applied Biosystems). Real-time PCR was performed in a mixture consisting of 4 μl cDNA, 1 μl TaqMan probe, and 5 μl of the PCR 2× master mix that was based on AmpliTaq Gold DNA polymerase (Applied Biosystems). Quantification of the mRNA expression was performed with the modified 2-ΔΔC T method [57]. Experiments were performed six times. The results are presented below as the fraction difference from control cultures, per the formula (treated-control)/treated and indicate that COL (Col1a1) mRNA expression is down-regulated at both time points and with both ALN concentrations.

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Hofstetter, B., Gamsjaeger, S., Varga, F. et al. Bone quality of the newest bone formed after two years of teriparatide therapy in patients who were previously treatment-naïve or on long-term alendronate therapy. Osteoporos Int 25, 2709–2719 (2014). https://doi.org/10.1007/s00198-014-2814-2

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