Osteocytes' response to PTH(1-34) regulates perilacunar tissue composition
Gardinier JD, Al-Omaishi S, Morris MD, and Kohn DH. Osteocytes' response to PTH(1-34) regulates perilacunar tissue composition. J Orthop Res 2017; 35
Journal of orthopaedic research
INTRODUCTION: The mechanical strength of bone is highly regulated by chemical composition and heterogeneity . Unlike osteoblasts, osteocytes are ideally located throughout cortical bone to augment the intrinsic properties by modifying the perilacunar tissue through 'osteocytic osteolysis' . Several studies have identified PTH as potential mechanism to modify the intrinsic properties of bone through osteocytes' remodeling their lacuna structure [2-5]; however, the manner in which perilacuar remodeling influences the tissue composition and mechanical properties of cortical bone remain unclear. Therefore, the purpose of this study was to establish how PTH influences perilacunar composition alongside whole bone mechanical properties. We hypothesize that bone strength gained through PTH(1-34) treatment is associated with an increase in perilacunar density. METHODS: 16 week old male C57Bl/6J mice were divided into two weight-matched groups and received daily subcutaneous injections of PTH(1-34) (40μg/kg) or vehicle control (50-l of 0.9% saline) for 21 consecutive days. On day 22, each mouse was sacrificed and the tibea were removed. The left tibia was used for ex-vivo micro-CT scans to measure the cross-sectional geometry and mechanical testing under four-point bending. The right tibia was embedded in PMMA, sectioned at the mid-diaphysis, polished to a thickness of 200 μm, and then imaged using a locally constructed Raman microprobe with a line focused 785-nm diode laser (Invictus, Kaiser Optical Systems) and 40X/0.75DIC objective (Plan Fluor, Nikon Instruments). Within the medial cortex of each tibia, 3 to 4 lacunae were identified and Raman spectroscopic signatures were taken across the perilacunar region (within 0-5 μm from the lacunae wall) and non-perilacunar region (within 5-10 μm from the lacunae wall). The carbonate-to-phosphate ratio (CPR) was calculated based on the carbonate (1071 cm-1) and phosphate (959 cm-1) band intensities. The mineral-to-matrix ratio (MMR) was calculated based on the phosphate and hydroxyproline intensities (851 cm-1 + 873 cm-1). Peak fitting was implemented using GRAMS software. The MMR and CPR for each group were averaged across 7 samples, each sample being the average taken from 3 to 4 imaged lacunae. Two-way ANOVA with a post-hoc Tukey-Kramer test was used to establish interactions between groups. RESULTS SECTION: Based on Raman spectroscopy, 3-weeks of PTH(1-34) treatment caused a significant decrease in the MMR of the perilacunar region compared to the non-perilacuar region (Figure 1A). In contrast, the MMR for vehicle treated controls displayed no significant difference between perilacunar and non-perilacunar regions. The CPR was significantly decreased across both regions following PTH(1-34) treatment compared to vehicle (Figure 1B). The cross-sectional area of the lacunae was also greater following PTH(1-34) treatment compared to vehicle (Figure 1C). Based on a linear regression across both groups, the increase in lacuna area correlated with the decrease in perilacunar MMR (p-value = 0.04). Whole bone mechanics indicated that PTH(1-34) compared to vehicle treatment decreased yield-displacement (237 ± 28 mm vs. 269 ± 40 mm) and yieldstrain (19,072 ± 1,016 vs. 21,910 ± 3,172 ). At the same time, PTH(1-34) compared to vehicle treatment increased stiffness (94 ± 13 N/mm vs. 77 ± 16 N/mm) and modulus (12 ± 0.9 GPa vs. 9.4 ± 1.5 GPa). Based on micro-CT analysis, PTH(1-34) treatment increased cortical area compared to vehicle treatment (0.78 ± 0.06 mm2 vs. 0.73 ± 0.06 mm2), but not the moment of inertia (0.092 ± 0.01 mm4 vs. 0.088 ± 0.01 mm4). DISCUSSION: Osteocytes' response to PTH has a large influence over the mineral composition localized around their lacuna structure. Contrary to expectation, perilacunar remodeling following PTH treatment included a reduction in mineral density localized around their lacuna structure along-side a reduction in carbonate substitution throughout entire tissue. Although perilacunar MMR and carbonate-substitution were expected to ncrease given the corresponding increase in mechanical strength, the reduction in perilacunar MMR can allow bone to absorb more energy under repeatative loading. As a result, we anticipate that the reduction in MMR would explain the increase in fatigue properties reported following PTH(1-34) treatment of bovine samples . The reduction in perilacunar mineral density was also considered a function of osteocytes' enlarging their lacuna space through tissue resorption. Lactation studies have also demonstrated osteocytes' ability to remodel the lacunae structure upon activation of the PTH/PTH-related protein receptor . In addition, osteocytes' response to PTH has shown increase their expression of catabolic factors, such as tartrate resistant acid phosphatase and cathepsin-K [3-5]. (Figure Presented).