Trabecular reorganization in consecutive iliac crest biopsies when switching from bisphosphonate to strontium ranelate treatment

Abstract

Background: Several agents are available to treat osteoporosis while addressing patient-specific medical needs. Individuals' residual risk to severe fracture may require changes in treatment strategy. Data at osseous cellular and microstructural levels due to a therapy switch between agents with different modes of action are rare. Our study on a series of five consecutively taken bone biopsies from an osteoporotic individual over a six-year period analyzes changes in cellular characteristics, bone microstructure and mineralization caused by a therapy switch from an antiresorptive (bisphosphonate) to a dual action bone agent (strontium ranelate).

Methodology/principal findings: Biopsies were progressively taken from the iliac crest of a female patient. Four biopsies were taken during bisphosphonate therapy and one biopsy was taken after one year of strontium ranelate (SR) treatment. Furthermore, serum bone markers and dual x-ray absorptiometry measurements were acquired. Undecalcified histology was used to assess osteoid parameters and bone turnover. Structural indices and degree of mineralization were determined using microcomputed tomography, quantitative backscattered electron imaging, and combined energy dispersive x-ray/µ-x-ray-fluorescence microanalysis.

Conclusions/significance: Microstructural data revealed a notable increase in bone volume fraction after one year of SR treatment compared to the bisphosphonate treatment period. Indices of connectivity density, structure model index and trabecular bone pattern factor were predominantly enhanced indicating that the architectural transformation from trabecular rods to plates was responsible for the bone volume increase and less due to changes in trabecular thickness and number. Administration of SR following bisphosphonates led to a maintained mineralization profile with an uptake of strontium on the bone surface level. Reactivated osteoclasts designed tunneling, hook-like intratrabecular resorption sites. The appearance of tunneling resorption lacunae and the formation of both mini-modeling units and osteon-like structures within increased plate-like cancellous bone mass provides additional information on the mechanisms of strontium ranelate following bisphosphonate treatment, which may deserve special attention when monitoring a treatment switch.

Figure 1. Timeline of treatment and biopsy obtainment.

We investigated 5 serial iliac crest biopsies from a 75-year-old female patient over a period of 6 years. The patient was diagnosed with osteoporosis with multiple vertebral fractures and was first treated with bisphosphonates (BP) for 26 months (biopsy 1). The BP treatment was halted for 12 months until a second biopsy was obtained (biopsy 2). The BP treatment was then continued and another biopsy was taken after 13 months (biopsy 3). The antiresorptive therapy was stopped after an additional 51 months of treatment and another biopsy was taken (biopsy 4). Next, strontium ranelate was administered for 12 months (2g/d) in total before another iliac crest biopsy was conducted. BP and SR were administered according to the manufacturer's instructions and the patient additionally received a Vitamin D3 metabolite during osteoporosis treatment.

Figure 2. Clinical data.

A) C-terminal cross-linking telopeptides of type I collagen (CTX) showed very low levels under antiresorptive therapy and rose to normal levels after SR treatment was initiated. B) The bone specific alkaline phosphatase (bAP) level varied under BP treatment from 25.9 to 13.5 µg/l. After the treatment was switched to SR, the bAP level rose again to 21.7 µg/l. C) DXA measurements showed only marginal changes in bone mineral density at the femoral neck following both the BP and SR treatments.

Figure 3. µCT reconstructions of biopsies and structural indices.

A) µCT-3D reconstruction of the complete bone core (biopsy 1). Rod-like structures with a microcallus formation (*→) are evident in the trabecular architecture. Some rods are likely to be perforated (→). B) µCT reconstruction of biopsy 2. Bone core with a combination of plate- and rod-like architecture. Note the thinning of the connecting rods (→) and reticulate perforation sites on a plate (*→). C) µCT reconstruction of biopsy 3. The trabecular architecture of this bone core presents an irregular composition of fine rod-like elements with some small plates. Several dead-ending rods are visible (→). D) µCT reconstruction of biopsy 4. The trabecular architecture in this iliac crest sample is almost completely deteriorated. Another trabecular fracture of a rod leading to microcallus formation is visible (→). Microcallus formations are bulky nodes with an irregular oftentimes clefted structure. E) µCT reconstruction of the biopsy after one year of SR treatment showing thick plate-like structures containing multiple intratrabecular ‘tunnels’. Although a very dense architecture is evident in the structure, big gaps of lost connectivity are also visible. Bulky microcallus formation (→). F–L) Changes in the trabecular network emphasized by structural indices evaluated through assessment of 3D µCT reconstructions. The dots represent mean values, and the error bars represent standard deviations of the mean. M) Mean density measured by µCT showed already high values during BP treatment that can be traced back to low bone turnover. The mean density after 12 months of SR remained almost unchanged suggesting maintained mineralization during a normal bone turnover situation.

Figure 4. Reorganization pattern, µCT mean density and 2D histomorphometry.

A) µCT reconstruction of biopsy 5. Note the coarse structure with surface irregularities and the occurrence of an intratrabecular ‘tunnel’. The area with the dark-colored background demonstrates the 2D section plane as it can be seen on the histology section in panel B. B) Congruent histology section following the µCT slice in panel A. Note the irregular trabecular structure with varying trabecular diameter. The thickened trabecular architecture indicates intratrabecular resorption sites filled with fibrovascular tissue (mineralized bone  =  light purple; Giemsa, 40x). C) A high magnification of the µCT reconstruction of biopsy 5 indicates the irregular trabecular surface with multiple hook-like, tunneling and sometimes perforating resorption lacunae. Virtual serial sections can show that all intratrabecular ‘tunnels’ are connected at some point and have openings to the trabecular surface. Small humps due to bone formation (mini-modeling) arise from the trabecular surface (→). D) Axial µCT section of biopsy 5: Distribution of local mineral densities (mg HA/cm³) represented by different gray levels. Due to bone formation, a low mineral content is detected in the proximity of new osteons, whereas the interstitial regions show a higher mineral content due to an older tissue age. E) Two (re-)modeling sites with distinct osteoid seems are visible. The osteoid seam on the trabecular surface at the bottom (white arrow) is covered with some cubic-shaped osteoblasts that become flattened to the lower image border. Non-active endosteal appostion is evident on the top of the image (black arrow) without prior resorption (modeling) as flat osteoblasts or lining cells cover osteoid (mineralized bone  =  green, osteoid  =  red; Goldner-Masson, 400x). F–I) Osteoid and cellular indices determined by 2D histomorphometry point to a low bone turnover situation at the time of biopsies 1, 2 and 4. Normal bone turnover, leading to an increase in osteoid apposition and thus increasing osteoid surface and volume appeared only after 50 months of BP treatment and after the treatment was switched to strontium ranelate. Osteoid indices did not indicate any signs of hyperosteoidosis considering the reference ranges reported by Lips et al. .

Figure 5. Bone mineral density distribution and microanalysis.

A) Histology of the cancellous bone area after strontium ranelate treatment. Intratrabecular cavities and thin osteoid seams are visible (mineralized bone  =  green, Goldner-Masson, 40x). B) Quantitative backscattered image of the selected area in panel A. Intratrabecular resoption sites showed osteon-like structures (→). The gray level distribution reflects the mineralization density, which varied between new and old bone. Newly formed osteon-like structures showed dark-gray pixels in comparison to older bright-gray bone packets. C) Microanalysis mapping showed that increased strontium deposition (red dots) appeared predominantly around newly formed osteon-like structures (→) as well as in modeling sites on endosteal surfaces (→). D) Combined EDX/µXRF microanalysis spectra demonstrated a deposition of 1.63 Wt% strontium (peak) in the mineralized tissue after SR treatment.