Bone biopsies were obtained from the Hamburg Bone Register at the University Medical Center Hamburg-Eppendorf. The Hamburg Bone Register is a collection of diseased human bone samples representing patients covering various bone pathologies; the samples are prepared and archived in an undecalcified state. Documentation is available for each biopsy regarding the details of the patients, including their history, treatment course, and clinical complications. This resource enabled tracking and analysis of the following case of osteoporosis in a female patient over a six-year time period. The patient characteristics for this study are as follows. The patient was postmenopausal, 75 years old and had multiple vertebral fractures (Th 6-8; LS 2-4) before initiating the BP treatment. During the BP treatment, new or progressive vertebral fractures were detected with an additional reduction in height accompanied by an unchanged low BMD; thus, treatment was switched from BP to SR. The study was approved by the institutional review boards of the University of California, Berkeley & San Francisco and IDG Immanuel Diakonie Group Berlin/Brandenburg. Informed written consent was given by the patient for the study and publication of the data. Five consecutive iliac crest biopsies were obtained from the patient over a period of six years; at the same time as the biopsies, the associated hip T-Scores (comparison of the patient's BMD value to the mean BMD of the young adult healthy population) and serum biomarkers were determined. Both the presence and concentration of type I collagen's crosslink peptide sequence were measured by the biomarker for carboxy-terminal collagen crosslinks (CTX). The portion of this peptide sequence that is cleaved by osteoclasts during bone resorption provides bone turnover information, where its serum levels are a proportional measure of osteoclastic activity. Serum bone-specific alkaline phosphatase (bAP) was measured as a biochemical indicator of bone turnover. The bone-specific isoform of alkaline phosphatase is a tetrameric glycoprotein found on the surface of osteoblasts and can be used as an index of active bone formation. Calcium and phosphorus blood samples were analyzed to differentiate between osteoporosis and other diseases associated with fracture risk and low bone mineral density.
All of the bone biopsies were performed at the same clinic by one of the authors (JS, Immanuel-Hospital Berlin-Wannsee, Berlin, Germany), who has experience with over 5000 iliac crest biopsies. All of the biopsies were taken during the BP and SR treatment periods (Fig. 1), see the timeline in figure 1. The side of the pelvis that the biopsy was extracted from was alternated at each visit; biopsies taken from the same side were extracted at a similar site but at a different biopsy angle. Therefore, biopsies 1,3,5 and biopsies 2,4 were taken in an alternating caudocranial or craniocaudal direction to avoid regional overlaps and possible interference from regional acceleratory phenomenon (RAP) or other bone healing effects. JS used an 8-gauge Jamshidi needle with an inner diameter of 3 mm to obtain the biopsies. The biopsies meet the standard required for histomorphometry because the length of the biopsies averages 2 cm (0.79 in.). Thus, the bone volume of Jamshidi biopsies for histomorphometry equates to the bone volume of transiliacal Bordier biopsies without the limiting factor of bilateral subcortical bone heterogeneity.
Undecalcified and polymethylmethacrylate (PMMA) embedded biopsies were scanned with a micro-computed tomography system (microCT 40, Scanco Medical AG, Basserdorf, Switzerland) with a 12-microm isotropic nominal resolution. Application of the threshold algorithm is based on previous studies and has been described in detail. The bone volume fraction (BV/TV), trabecular thickness (Tb.Th.), trabecular separation (Tb.Sp.), trabecular number (Tb.N.), connectivity density (Conn.D), structure model index (SMI) and trabecular bone pattern factor (TBPf) were measured from the segmented images as described elsewhere. Conn.D, SMI and TBPf are closely related to each other and provide information about the characteristics of the cancellous bone structure. Calculation of the Euler characteristics can be applied to networks to provide information regarding the number of connected entities within the structure. In networks of cancellous bone, the connectivity density (Conn.D) can be calculated by dividing the connectivity estimate by the volume of the sample. Basically, Conn.D provides an estimation of the number of trabecular connections per unit volume. The structure model index (SMI) determines the geometry of the intertrabecular network. It is based on the change in surface area with an infinitesimal increase in volume. Quantification of the SMI gives an impression of whether the trabecular structure consists of a plate-like (SMI   0) or rod-like (SMI   3) configuration. In cortical bone, the SMI may shift towards negative values if many concave surfaces are present. The TBPf describes the ratio of intertrabecular connectivity, which was first developed to analyze histological sections in 2D. The concept of TBPf is based on varying values for the convexity and/or concavity of a structure. Due to dilation, there will always be increases in bone area but increases in bone perimeter only occur with convex surfaces. Trabecular bone with many concave structures has a negative TBPf, whereas many convex structures results in a positive value (osteopenia/osteoporosis). A low TBPf value reflects a cancellous morphology that has adapted from a rod-like configuration to a plate-like configuration. Excellent correlation has been reported between the SMI and 2D-TBPf. The bone tissue density was determined from the original microCT grayscale images. The binary image map from the segmentation step was eroded by two voxels to remove partial volume components and to mask the grayscale data. The mean linear attenuation was calculated as the sum of the masked image divided by the number of bone voxels in the eroded mask. This value was converted to mineral density (MD) in mg HA/cm3 based on a phantom-derived linear calibration.
Bone biopsies were sectioned to a 4-microm thickness and stained with Goldner-Masson, Giemsa and Tartrate Resistant Acid Phosphatase (TRAP). Osseous cells and osteoid indices were assessed quantitatively in accordance with the guidelines of the American Society of Bone and Mineral Research using histomorphometry software OSTEO II (Bioquant, Nashville, TN, USA). The ratio of the osteoid surface to the bone surface (OS/BS,%), the ratio of the osteoblast surface to the bone surface (Ob.S/BS,%), the ratio of the number of osteoblasts to the bone surface (N.Ob/BS, #/mm) and the ratio of the number of osteoclasts to the bone surface (N.Oc/BS, #/mm) were determined. Bone turnover was classified by analyzing serum markers (bAP and CTX) and osteoid indices, which were compared to reference values from the literature for naive and treated patients. Furthermore, visible bone remodeling units were evaluated as an additional supportive measurement indicating bone turnover information and were based on the following histological criteria. The total number of osteoblasts and osteoclasts were counted at individual bone remodeling units (magnification 40x) and divided by the total number of assessed fields of view (approx. 4-6). 'Low turnover' is attributed to bone tissue under low endosteal remodeling showing <1 bone remodeling unit with cubic-shaped osteoblasts and a maximum of one osteoclast per field of view (magnification 40x) in the underlying histological section. 'Normal turnover' is attributed to bone tissue with maintained endosteal remodeling showing 1-2 bone remodeling units with cubic-shaped osteoblasts and 2-4 single osteoclasts per field of view (magnification 40x). 'High turnover' is attributed to bone tissue with both increased endosteal remodeling and osteoclastic resorption showing >2 bone remodeling units with prominent seams of osteoblasts and >4 groups of osteoclasts per field of view (magnification 40x) in the underlying histological section. Polarized light was used to observe collagen birefringence and lamellar orientation.
The bone mineral density distribution was measured on plastic-embedded cross-sections of iliac crest biopsies with coplanar, polished and carbon-coated surfaces using quantitative backscattered electron imaging (qBEI). The scanning electron microscope (LEO 435 VP, LEO Electron Microscopy Ltd., Cambridge, England) was operated at 15 kV and 665 pA at a constant working distance (BSE Detector, Type 202, K.E. Developments Ltd., Cambridge, England). The pixel size was 3 im, as recommended by Roschger et al.. Synthetic hydroxyapatite (HA) samples were used to create a calibration curve. These HA samples (DOT Medical Solutions, Rostock, Germany) contained different Ca/P ratios, which were determined with energy dispersive X-ray analysis (DX-4, EDAX, Mahwah, NJ) and qBEI. A highly linear relationship between the gray values of backscattered signal intensities and the calcium content of each sample has been reported previously by other authors, enabling calibration of the method. The generated mineralization profile (gray value histogram) reflects the degree of mineralization in the cross-sectioned bone.
Strontium deposition in mineralized bone tissue was measured using microXRF (Micro-X-ray Fluorescence Analysis; IMOXS, IFG, Germany) in combination with EDX (Energy Dispersive X-ray Analysis); EDAX DX-4, USA). An XRF-module with capillary zoom optics enables x-ray fluorescence analysis in the SEM with a high sensitivity. The advantage of this method is the high spatial resolution. The detection limit ranges between 1 and 0.01 weight percent (wt%) due to electron-induced Bremsstrahlung background. The strontium (Sr) content in the bone biopsies was evaluated in wt% via software that enabled combined EDX/microXRF quantification (IMOXS Quant 2.10, IFG, Berlin, Germany).