Bone 49 (2011) 1160–1165

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Original Full Length Article

Effects of one year daily teriparatide treatment on trabecular bone materialproperties in postmenopausal osteoporotic women previously treated withalendronate or risedronate☆

Sonja Gamsjaeger a, Birgit Buchinger a, Ruth Zoehrer a, Roger Phipps b,1,Klaus Klaushofer a, Eleftherios P. Paschalis a,⁎a Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 1st Medical Department, Hanusch Hospital, Heinrich Collin Str. 30, A-1140Vienna, Austriab Procter & Gamble Pharmaceuticals, Mason, Ohio, USA

☆ Conflict of interest: Dr. Phipps has been an employeceuticals. Dr Paschalis has received research grants fromticals, and the Alliance for Better Bone Health. None oconflict of interest.⁎ Corresponding author at: Ludwig Boltzmann Institu

pital, Heinrich Collin Str. 30, A-1140 Vienna, Austria.E-mail address: eleftherios.paschalis@osteologie.at (

1 Current address: Husson University School of Pharm

8756-3282/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.bone.2011.08.015

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 May 2011Revised 9 August 2011Accepted 11 August 2011Available online 27 August 2011

Edited by: David Burr

Keywords:TeriparatideAlendronateRisedronateBone material propertiesFourier transform infrared imagingRaman MicrospectroscopyMineral maturityCollagen cross-links

In the present work we examined the effect of teriparatide administration following bisphosphonate treat-ment on bone compositional properties by Raman and Fourier Transform Infrared Imaging (FTIR) microspec-troscopic analysis. Thirty two paired iliac crest biopsies (before and after 1 year teriparatide) from sixteenosteoporotic women previously treated with either Alendronate (ALN) or Risedronate (RIS) and subsequent-ly treated 12 months with teriparatide (TPTD) were analyzed at anatomical areas of similar tissue age in boneforming areas (within the fluorescent double labels). The outcomes that were monitored and reported weremineral to matrix ratio (corresponding to ash weight), mineral maturity (indicative of the mineral crystallitechemistry and stoichiometry, and having a direct bearing on crystallite shape and size), relative proteoglycancontent (regulating mineralization commencement), and the ratio of two of the major enzymatic collagencross-links (pyridinoline/divalent). Significant differences in mineral/matrix, mineral maturity/crystallinity,and collagen cross-link ratio bone quality indices after TPTD treatment were observed, indicating a specificresponse of these patients to TPTD treatment. Moreover differences between ALN and RIS treated patientsat baseline in the collagen cross-link ratio were observed. Since tissue areas of similar tissue age were ana-lyzed, these differences may not be attributed to differences in bone turnover.

e of Procter & Gamble Pharma-Procter & Gamble Pharmaceu-f the other authors have any

te of Osteology, Hanusch Hos-

E.P. Paschalis).acy, Bangor, Maine, USA.

rights reserved.

© 2011 Elsevier Inc. All rights reserved.

Introduction

Osteoporosis is characterized by an imbalance in bone turnover,resulting in a net bone loss. Therapies used in management of osteopo-rosis fall under two categories, antiresorptives (bisphosphonates, estro-gen, RANKL inhibitors) and anabolics (parathyroid hormone or PTH).Bisphosphonates are by far the most widely used first line therapy. De-spite their excellent overall safety record, there are concerns that pro-longed osteoclast suppression is detrimental [1–4]. Treatment withthe anabolic agent teriparatide (TPTD; human parathyroid hormone[1–34]) increases bone mineral density (BMD as measured by DXA)and bone formation (via markers of bone formation), and decreasesfracture risk [5–7]. Thus TPTD therapy after a defined period of

bisphosphonate therapy is a therapeutic option. An issue is whetherthe bisphosphonate therapy interferes with the efficacy of subsequentTPTD therapy. TPTD increases bone formation in part via increased dif-ferentiation of osteoblast precursors through canonical wingless (Wnt)signaling [8–11]. Bisphosphonates, through reduction of osteoclast ac-tivity also reduce bone formation, but primarily by reduced remodelingsite activation rather than by reduced osteoblast-team activity.

There is data suggesting a potential interference of prior bispho-sphonate therapy with subsequent TPTD treatment, and that this maydiffer among bisphosphonates. The effects of TPTD on BMDwere some-what retarded for patients previously treated with the bisphosphonateAlendronate (ALN) compared to previously untreated patients [12], al-though TPTD still had beneficial effects on bone mass and bone forma-tion markers regardless of previous bisphosphonate treatment [13,14].Moreover, based on results of finite element analysis after 12 monthsTPTD therapy in patients previously treated with either Risedronate(RIS) or ALN differences in biomechanical performance were predicted[14,15]. A recent study reported that one year daily TPTD treatment ex-erts a significant effect on the Bone Mineral Density Distribution(BMDD) as determined by quantitative backscattered electron imaginganalysis (qBEI), independent of prior bisphosphonate (ALN or RIS) use

1161S. Gamsjaeger et al. / Bone 49 (2011) 1160–1165

[16]. The level of suppression of bone remodeling varies among thebisphosphonates at clinical dose levels [17], and this may be a factorinfluencing the efficacy of subsequent TPTD therapy.

The purpose of the present study was to investigate TPTD effectson bone composition (specifically mineral to matrix ratio, mineralcrystallite maturity/crystallinity, relative proteoglycan content, andratio of pyridinoline to divalent collagen cross-links) as a function ofprior bisphosphonate (ALN or RIS) therapy, independent of boneturnover. The specific hypothesis tested was that previous bispho-sphonate use does not interfere with the compositional propertiesof bone formed under TPTD administration for 1 year. To achievethis, tissue age needs to be taken into account, and we used Ramanmicrospectroscopy and Fourier Transform Infrared Imaging (FTIRI)to examine paired iliac crest bone biopsies collected during a study,in which postmenopausal women with osteoporosis were treatedfor 12 months with TPTD after at least 2 years treatment with RIS orALN (the OPTAMISE study). Both spectroscopic techniques are capa-ble of providing information on bone composition in tissue sectionswith a spatial resolution of ~1 μm (Raman) or 10 μm (FTIRI). Boththese techniques have been validated for provision of informationon mineral and organic matrix (proteoglycans) characteristics [18–20], while the latter is also validated for information pertaining to col-lagen cross-links [21,22]. We compared the two bisphosphonates atbaseline prior to TPTD treatment, and also after 12 months TPTDtreatment. Analysis was focused on formation sites, between fluores-cent double labels. This anatomical area selection criterion ensuresthat the results are independent of tissue age and thus bone turnover.

Materials and methods

Specimens

Details of the patients have been published elsewhere [14]. Postmen-opausal women with osteoporosis who had received ALN (either10 mg/day or 70 mg/week) or RIS (5 mg/day or 30–35 mg/week) for at

Table 1Descriptive statistics by previous bisphosphonate treatment — biopsy subjects.

Variable Statistic All subjects

Age (years) Mean 68.3Standard Deviation 7Minimum 52Median 68Maximum 79Number of subjects 16

Height (cm) Mean 158.8Standard Deviation 6.43Minimum 148Median 150Maximum 171Number of subjects 16

Weight (Kg) Mean 60.31Standard Deviation 7.29Minimum 46.1Median 60.2Maximum 72.5Number of subjects 16

BMI (kg/m2) Mean 23.95Standard Deviation 2.933Minimum 17.8Median 23.9Maximum 29.1Number of subjects 16

Bisphosphonate Duration(months)

Mean 40.9Standard Deviation 12Minimum 25Median 39Maximum 71Number of subjects 16

least 24 months (mean duration of therapy 37.2 months for ALN and38.0 months RIS) stopped their bisphosphonate therapy and were thentreated with TPTD (20 μg/day subcutaneously) for 12 months. Detailson the subjects that agreed to a biopsy are shown in Table 1.

Biopsies were collected at the end of bisphosphonate treatmentbefore initiation of TPTD treatment (baseline or BSN), and after12 months TPTD treatment (treatment or TR). For the baseline biop-sies, subjects continued to take their bisphosphonate throughoutthe labeling period, and did not discontinue their bisphosphonateuntil the day before TPTD therapy was scheduled to begin. A 3-daybone label (tetracycline) tablet pack (1 tablet to be taken 4 timesper day for 3 days) was supplied to the subject. This 3-day coursewas followed by 14 days without labeling medication, and then an-other 3-day course of tetracycline. During the entire labeling period,subjects discontinued any supplemental calcium and/or vitamin Dtablets. Biopsies were taken 5 to 14 days after the second bone labelcourse. Paired transiliac crest biopsies (N=8 per group) wereobtained at the end of the bisphosphonate treatment (ALN-BSN)and (RIS-BSN), and after 12 months TPTD treatment [prior ALN after12 months of TPTD treatment (ALN-TR), prior RIS after 12 months ofTPTD treatment (RIS-TR)]. Upon excision, biopsies were fixed in alco-hol, dehydrated through a series of acetones and embedded in poly-methyl methacrylate (PMMA).

Anatomical area selection

The biopsies analyzed in the present study were double tetracyclinelabeled, and both the Raman and FTIR microspectroscopic measure-ments were obtained in the area between the two labels (Fig. 1). Thisensured that bone of similar tissue age was analyzed, and thus the re-sults are independent of bone turnover considerations. Moreover, thelabels that were chosen for analysis had a distance between the secondlabel and themineralizing front of less than 4 μm(in the case of ALN-TR,and RIS-TR) so as to ensure that the bone analyzed was formed duringthe TPTD treatment. For each biopsy, three trabeculae with double

Prior-Risedronate Prior-Alendronate Probability

69.5 67 0.46854.66 9.09

61 5270 6575 798 8

160.4 157.1 0.28826.94 5.85

151 148159 159171 165

8 861.5 59.13 0.65246.172 8.519

54.5 46.160.1 60.470.2 72.58 8

23.94 23.96 0.56782.663 3.368

20.9 17.823.9 24.229.1 29.18 8

40.0 41.9 0.394211.08 13.5525 3139 3757 718 8

5 µm

Fig. 1. Typical trabecular surface with double label evident, as seen through the Ramanmicroscope. The areas of analysis are indicated by the crosses.

Table 2Spectroscopic analysis outcomes reported as mean and (SD). Results of two-way re-peated measures ANOVA test. Two-sided pb0.05 was considered statisticallysignificant.

ALN-BSN RIS-BSN ALN-TR RIS-TR

v1PO4/Amide I 11.54 (3.62) 12.15 (4.08) 16.76 (8.18) 15.42 (5.69)v2PO4/Amide III 0.027 (0.015) 0.028 (0.016) 0.072 (0.049) 0.081 (0.045)FWHH 24.00 (2.62) 23.48 (2.43) 27.00 (4.98) 27.63 (3.69)PG/v2PO4 1.14 (1.00) 0.98 (0.59) 0.78 (0.85) 0.54 (0.49)Pyr/divalent 4.83 (0.34) 3.95 (0.19) 4.215 (0.23) 3.78 (0.22)

Interaction term Factor1 year of TPTD

Factorprior bisphosphonate

v1PO4/Amide I ns ⁎ nsv2PO4/Amide III ns ⁎⁎⁎ nsFWHH ns ⁎⁎ nsPG/v2PO4 ns ns nsPyr/divalent ⁎ ⁎⁎ ⁎⁎⁎

ns not significant.⁎ pb0.05.

⁎⁎ pb0.01.⁎⁎⁎ pb0.001.

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tetracycline labels were analyzed, and in each of these regions threemeasurements were obtained, for a total of nine measurements. Foreach patient, these nine values were averaged and the resultant valuewas treated as a single statistical unit.

Raman analysis

For Raman microspectroscopic analyses, a Senterra (Bruker OptikGmbH) instrument was employed. A continuous laser beam was fo-cused onto the sample through a Raman fluorescence microscope(Olympus BX51, objective 50×) with an excitation of 785 nm(100 mW), a lateral resolution of ~0.6 μm, and equipped with a Olym-pus Model U-LH100HG fluorescence source with a UV fluorescencefilter (Chroma UV/Violet 110113v3). All data analysis was donewith the Opus Ident software package (OPUS 6.5, Bruker OptikGmbH). Once acquired, the Raman spectra were baseline corrected(rubber band, 5 iterations) so as to account for fluorescence [23],and the following Raman parameters were calculated as publishedelsewhere [19,24,25]: (1) the mineral/matrix ratio was expressed asthe integrated areas of the v1PO4 (930–980 cm−1) to the amide I(1620–1700 cm−1) bands, and of the v2PO4 (410–460 cm−1) to theamide III (1215–1300 cm−1) bands [26–28]. The former is dependenton the amount of mineral and matrix as well as the organization of la-mellar bone while the latter is dependent solely on the amount ofmineral and matrix present; (2) the relative proteoglycan contentwas expressed as the proteoglycan/mineral ratio (the ratio of the in-tegrated areas of the proteoglycan/CH3 [1365–1390 cm−1] band [rep-resentative of mucopolysaccharides] [18,19] to the v2PO4 [410–460 cm−1] band); (3) the maturity/crystallinity of the bone mineralapatite crystallites was approximated from the full width at halfheight (FWHH) of the v1PO4 (930–980 cm−1) band [26]. It has beenpreviously shown that FWHH is inversely proportional to mineralmaturity/crystallinity [20,26].

FTIRI analysis

Two thin tissue sections (~5 μm) were taken from each biopsyblock. Spectra were obtained on a Bruker Equinox 55 (Bruker Optics)spectrometer interfaced to a Mercury Cadmium Telluride (MCT) de-tector, and equipped with a Hg fluorescence lamp (A674-HG, BrukerOptics). Each spectrum analyzed an area of ~10×10 μm. Spectral res-olution was 4 cm−1. Background spectra were collected under identi-cal conditions from the same BaF2 windows used to place the sectionsin, at the beginning and end of each experiment to ensure instrumentstability. The spectrometer was continuously powered to minimize

warm-up instabilities and purged with dry-air (Bruker Optics) tominimize the water vapor and CO2 interference.

After acquisition, spectra were transferred to an off-line computer(Dell Precision 650), andwere zero-corrected for the baseline in the spec-tral area of Amide I &II (~1490–1700 cm−1) using Grams/32 (GalacticSoftware), water vapor and polymethylmethacrylate (PMMA) spectralinterferences corrected for as previously described [22,29], followed bycurvefitting of the spectra and calculation of the relative area ratio ofthe underlying bands at 1660 and 1690 cm−1. This ratio has previouslybeen shown to correspond to the relative ratio of pyridinoline and diva-lent collagen cross-links, due to the perturbation that the cross-linksexert on the molecular vibrations of the carbonyl groups present in thecollagen chains [21,22]. For each biopsy, three trabeculae with evidentfluorescent double labels were analyzed, and in each of these regionsthree measurements were obtained, for a total of nine measurements.For each patient, these nine values were averaged and the resultantvalue was treated as a single statistical unit.

Statistical analysis

Data are reported asmean and standarddeviation (SD) for the variousgroups. The outcomes were analyzed statistically so as to address threedifferent questions: (1) Unpaired t tests were used to analyze the differ-ences between the study groups at baseline, ALN-BSN vs RIS-BSN, andafter 12 months of teriparatide treatment, ALN-TR vs RIS-TR; (2) pairedt tests were used to analyze changes from baseline within each studygroup (ALN-BSN vs ALN-TR and RIS-BSN vs RIS-TR) for information onthe teriparatide effects in the prior ALN and prior RIS groups separately;and (3) two-way repeated measures (rm) ANOVA with Bonferronipost-hoc test was used for analyzing teriparatide effects between thetwo study groups (prior ALN versus prior RIS). Two-way repeated mea-sures ANOVA tested three hypotheses: (i) Therewas no significant differ-ence between prior bisphosphonate treatment at baseline, (ii) there wasno significant difference betweengroups after 1 year of teriparatide treat-ment, and (iii) there was no interaction between the factor prior bispho-sphonate and the factor 1 year of teriparatide, indicating that changesfrombaselinewere not significantly different between the 2 prior bispho-sphonate groups. Differences were considered significant at pb0.05 on atwo-tailed test.

Results

The results of the two way repeated measures ANOVA analysis areshown in Table 2. All monitored outcomes were influenced by the1 year TPTD treatment with the exception of the relative proteoglycan

Fig. 3. Mineral maturity/crystallinity as determined from Raman analysis. A significantdecrease in mineral maturity/crystallinity after TPTD treatment in both ALN and RISgroups compared to their baseline values is evident. p-values for significant differencesare listed. Error bars represent SD (not SEM).

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content. The pyr/divalent collagen cross-link ratio was influenced bythe prior bisphosphonate used, and the 1-year TPTD administration,but since the interaction term is statistically significant, it is difficult tointerpret.

Raman microspectroscopic analysis

Both ways of calculating this ratio based on Raman analysis indicat-ed significant increases in mineral/matrix ratio within the double tetra-cycline labels in both ALN and RIS groups after 12 month teriparatidetreatment compared to respective baselines (ALN-TR vs ALN-BSN andRIS-TR vs RIS-BSN) (Fig. 2). There were no differences between ALNand RIS groups either at baseline or after TPTD treatment.

TPTD significantly decreased mineral maturity/crystallinity in bothALN and RIS groups compared to the respective baselines. There wereno differences between ALN and RIS groups at baseline or after TPTDtreatment (Fig. 3).

TPTD treatment decreased the relative proteoglycan content, but thedecrease was statistically significant only in the RIS group (RIS-TR vsRIS-BSN) (Fig. 4).

FTIR microspectroscopic analysis

Pyr/divalent collagen cross-link ratio was significantly lower atbaseline in RIS group compared to ALN group (RIS-BSN vs ALN-BSN).

Fig. 2. Mineral/matrix ratio as deduced by Raman microspectroscopic analysis, basedon the utilization of either the v1PO4 and amide I (a), or the v2PO4 and amide III(b) Raman bands. Mean values and SD (bars) for each group are shown. Both methodsindicated that there is a significant increase in this ratio within the double labels, afterTPTD treatment (ALN-TR and RIS-TR) compared to their respective baseline values(ALN-BSN, RIS-BSN). p-values for significant differences are listed.

Fig. 4. Raman microspectroscopic analysis between labels showed a significant de-crease in the relative proteoglycan content of the RIS-TR group compared to RIS-BSN.p-values for significant differences are listed. Error bars represent SD (not SEM).

TPTD significantly reduced the cross link ratio in the ALN group (ALN-TR vs ALN-BSN) but not in the RIS group (Fig. 5). At end-point (after1 year TPTD therapy), the Pyr/divalent collagen cross-link ratiowas sig-nificantly lower in the prior-RIS compared to prior-ALN treated group.

Fig. 5. FTIRI analysis indicated that the pyr/divalent collagen cross-link ratio was signif-icantly lower in RIS-BSN compared to ALN-BSN; ALN-TR was also significantly lowercompared to ALN-BSN, while at end-point, RIS-TR was significantly lower comparedto ALN-TR. p-values for significant differences are listed. Error bars represent SD (notSEM).

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Discussion

The results of the present study indicate that compositional proper-ties of newly formed bone under TPTD administration for 12 monthsare not affected by prior bisphosphonate use.

Bone strength depends on both bone quantity and bone quality,the latter including the structural and material properties. Mineralto matrix ratio, mineral maturity/crystallinity, and collagen cross-links are important indices of the material properties of bone [30].

In the present study, we were specifically interested in the changeof these bone material properties in bone forming areas and thereforefocused our analysis on actively bone forming areas as indicated bythe presence of tetracycline fluorescent double labels. This focuswas also based on other previously published data from this studythat showed teriparatide increased bone formation markers soonerin the prior RIS group vs the prior ALN group [13,14]. This anatomicalarea selection allowed us to examine areas of similar tissue age andthus minimized bone turnover effects on bone material properties.

TPTD increased the new bone (within the double labels) mineral/matrix ratio in prior ALN and prior RIS groups (Figs. 2a and b). UnlikeBMD, which gives the amount of mineral per area per volume ana-lyzed, the mineral to matrix ratio expresses the amount of mineralper amount of matrix (mainly type I collagen) per volume analyzed[21] and reflects bone ash weight [31]. In a recent report it wasshown to be a better predictor of bending stiffness than BMD [32].

In a placebo controlled study, TPTD produced a significant decreasein the mineral/matrix ratio compared to placebo treatment [33]. Thereare several factors that could account for this apparent discrepancy in-cluding: i. very strictly selected anatomical areas (between the doublelabels) analyzed in the present study vs larger areas analyzed in theplacebo-controlled study; ii. comparison within groups (teriparatidetreatment vs baseline) in the present study compared to across groups(teriparatide vs placebo) in the placebo-controlled study, and iii. bispho-sphonates bind on hydroxyapatite surfaces and are potent inhibitors ofboth crystal growth and dissolution [34], thus it is feasible that in thepresent study the baseline values are lower than even in the treat-ment-naïve patients due to the presence of bisphosphonates, and thisinhibition is reversed through the TPTD anabolic effect. Unfortunatelya treatment-naïve group was not available in the present study.

Bone mineral crystallites are poorly crystalline, highly substitutedapatites [21]. Mineral maturity is a description of the chemical make-up of the crystallites with a direct bearing on their size and shape(crystallinity) [21]. Several observations show the importance of themineral maturity/crystallinity in determining bone strength, andthat there can be changes in mineral maturity/crystallinity in diseaseand potentially in response to therapy that are partly independent ofbone turnover [35–38]. In the present study, teriparatide decreasedmineral maturity/crystallinity in prior ALN and RIS groups (Fig. 3).This teriparatide effect is consistent with that expected from freshlyformed bone mineral crystallites [26,39,40].

Raman microspectroscopy allows the monitoring of relative pro-teoglycan content in undemineralized bone tissue. In in vitro studies,it has been reported to correlate with hydroxyapatite crystal forma-tion,with lower proteoglycan concentrations increasing the formationof these crystals to a greater extent than higher ones [41], and it hasalso been suggested that the small cellular/pericellular matrix proteo-glycan decorin may act as an inhibitor of collagen matrix mineraliza-tion, thus modulating the timing of matrix mineralization [42]. Inthe present study, TPTD treatment decreased relative proteoglycancontent normalized for amount of mineral in newly formed bone inboth bisphosphonate groups compared to their respective baselines,although only the decrease in the prior RIS group was statistically sig-nificant. This may contribute to the earlier upregulation of bone for-mation (based on N-terminal propeptide of type1collagen increases)in the patients treated with RIS compared to those treated with ALN[13,14].

Although the analysis of mineral at the microscopic level and thecontribution of aforementioned mineral crystallinity (crystallite size/shape) and maturity (chemical composition) to bone strength hasbeen fairlywell studied [30], thematrix/collagen has received consider-ably less attention. An important observation is that in cases wherebone strength and BMD exhibit divergent trends, the mineral maturity/crystallinity and collagen cross-links ratio correlate with the formerrather than the latter [33,43], further emphasizing the contribution ofthese bone material properties to bone strength. The collagen cross-link ratio was significantly lower in the RIS group at baseline comparedto the ALN group (Fig. 4a). This is in agreement with previously pub-lished reports that showed that this cross-link ratio was unaltered inALN treated patients compared to placebo [44] but was reduced in RIStreated patients compared to placebo [45]. This difference at baselinemay result in part from differences between RIS and ALN in interactionsand effects on bone apatite and osteoblasts [34,46]. ALN and RIS havedifferent binding affinities for apatite crystals, and once adsorbed atpH 7.4 they result in different zeta-potentials (a form of surface doublelayer electric potential) of the apatite crystals. Cells will sense and reactto potential, thus it is plausible that the affinities and zeta-potentialsdifferentially influence osteoblastic function quantitatively and/or qual-itatively. Teriparatide had no significant effect on the cross link ratio inprior RIS subjects, but significantly reduced the ratio in prior ALN sub-jects, an effect that may be related to the baseline cross-link ratio.After 1 year TPTD therapy, the Pyr/divalent crosslink ration was signifi-cantly lower in RIS-TR compared to ALN-TR group, possibly due to thedifferences in actively bone forming areas at baseline.

The TPTD effects on the monitored outcomes were not significant-ly influenced by the type of prior bisphosphonate (Table 2; interac-tion term was not significant by two-way repeated measuresANOVA), suggesting that there is no differential effect of the type ofprevious bisphosphonate in the monitored bone material propertiesresponse to 1 year of TPTD treatment. The exception is the collagencross-link ratio, which was strongly dependent on both prior bispho-sphonate used and TPTD treatment. Nevertheless, the relatively lowsample size of each prior bisphosphonate group should be kept inmind when considering these results.

A limitation of the present study is the lack of a placebo group thusno determination of the direct (without prior bisphosphonate treat-ment) teriparatide effect on the material properties was feasible. An-other is the relatively small sample number within each group. Also,TPTD was administered for 12 months instead of the recommended24months duration. Finally, the tissue analyzed was PMMA-embedded(a process involving tissue dehydration). Nevertheless, the same pro-cessingwas applied to all biopsies, thus any differences seen in the anal-ysis are most likely due to differences among the various groups ofpatients rather than potential embedding processing artifacts. On theother hand, a strength of the study is the analysis of paired biopsies.

In conclusion, the results of the present study have shown a re-sponse to TPTD treatment in newly forming bone composition andmaterial properties in patients who had been treated with bispho-sphonates. TPTD produced significant effects on mineral/matrixratio, mineral maturity/crystallinity and collagen cross-link ratio.TPTD-induced changes in relative proteoglycan content were ob-served only in the prior-RIS treated group. Collagen cross-link ratiowas also significantly different between the two bisphosphonategroups at baseline, and at end-point. In previous analyses from thisstudy, the increases in bone formation markers were similar forboth bisphosphonate groups after some 6–12 months of TPTD treat-ment [13,14]. The data presented here on bone quality indices atbone forming areas are consistent with this. There were no statistical-ly significant differences between the prior ALN and prior-RIS groupsin any of the outcomes after 12 months TPTD treatment, with the no-table exception of collagen cross-link ratio. These results strengthenthe importance of assessing effects on bone formation (both quantita-tive and qualitative) when evaluating the long-term bone effects of

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bisphosphonates, and suggest that prior BP use does not blunt TPTD'seffect on bone material properties.

Acknowledgments

The authors thank G. Dinst and P. Messmer for careful samplepreparation at the bone material laboratory of the Ludwig BoltzmannInstitute of Osteology, Vienna, Austria. This study was supported bythe AUVA (Austrian Social Insurance for Occupational Risk), and theWGKK (Social Health Insurance Vienna). Financial support for thisstudy was provided by The Alliance for Better Bone Health.

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