Age-Dependent Responses in 3D Architectural Developmental Patterns of the Rat Tibia to Daily Parathyroid Hormone Administration

Huh, B; Luo, S; Altman, A; Chandra, A; Lan, S; Qin, L; Liu, X

University of Pennsylvania, Philadelphia, PA, USA

Introduction: Daily parathyroid hormone (PTH) injection is the only FDA-approved anabolic treatment for osteoporosis. Past clinical data clearly demonstrate PTH's efficacy in improving bone mass and microstructure. Rodent models have been used to investigate the cellular and structural mechanisms of PTH's anabolic effect on bone. It has been reported that the magnitude and rate of responsiveness of rat bone to PTH are greater in young rats than mature rats. However, the exact mechanism is not clear. The goal of this study was to investigate the age-dependent differences in temporal and spatial responses of trabecular bone to intermittent PTH, which might lead to a better understanding of PTH's anabolic action and its treatment efficiency. We hypothesized that PTH has a direct effect on the primary spongiosa in growing rats while in contrast the responses of the mature rat skeleton are due to modified local bone remodeling activities. To address this hypothesis, we used in vivo µCT and 3D image registration techniques to monitor the immediate responses in rat tibial trabecular bone to intermittent PTH.

Methods: Saline (Veh) or PTH (80 μg/kg) was injected daily to 1-month (1mo) and 3-month-old (3mo) rats for 12 days (Veh n=6 and PTH n=8 for both age groups). Rats were anesthetized and scanned at the right tibia by an in vivo µCT system (vivaCT 40, Scanco Medical; 10.5 µm voxel size). A 4.2 mm region below the growth plate was acquired. The Veh group was scanned at day 0 and 12. The PTH group was scanned every 4 days for 12 days because PTH exerts a protective effect against radiation to allow multiple scans. This was further confirmed in the current study by a right-left tibia comparison at day 12.
For the 3mo groups, a 2.5 mm region of trabecular bone in the secondary spongiosa (II SP) at day 12 was chosen and then the corresponding volume of interest (VOI) was accurately located in the II SP of day 0, 4 and 8 scans (Fig. 1) by using a mutual information-based 3D image registration (ITK, NLM). Based on the registered scans of 1mo rats, we found that the average rate of new bone generation from the growth plate was ~0.25 mm/day. The region of II SP scanned at day 0 was pushed away from the growth plate toward the mid shaft region and moved out of the scanned region at day 12. Therefore, for the 1mo PTH group, a 1.5 mm bone segment in the II SP of each scan of day 4, 8 and 12 was separately selected and compared to the registered corresponding VOI that was located in the primary spongiosa (I SP) of each 4-day-prior scan (Fig. 2).
For each registered image, bone volume fraction (BV/TV), trabecular thickness (Tb.Th*), spacing (Tb.Sp*), number (Tb.N*), and structure model index (SMI) were evaluated by 3D standard microstructural analysis. Student's t-tests were performed with p<0.05 indicating significant difference.

Results: 1mo groups: PTH As shown in Fig 2, periosteal resorption (red) and endocortical formation (green) occurred in 4 days while the major trabecular pattern remained. Rapid bone growth caused I SP to translate into II SP toward diaphysis, resulting in decreases in both periosteal and endosteal perimeters (Fig 2C). First, longitudinal comparisons were made between the trabecular bone of the II SP (day 4, 8 and 12) and the registered corresponding VOI located in the I SP of the 4-day-prior scans (day 0, 4, and 8, respectively). Surprisingly, we found decreases of 21-31% in Tb.N* but increases of 20-24% in Tb.Th* in the II SP at day 4, 8 and 12 compared to the corresponding VOI 4 days earlier (Fig. 3). There were also decreases in BV/TV of 13% at day 4 and 22% at day 8 compared to the same VOI at day 0 and day 4. Next, we performed comparisons for the I SP between scans at day 0, 4, 8 and 12. There were increases of 52%, 20% and 18% in BV/TV, Tb.N* and Tb.Th*, and 23% and 24% decreases in Tb.Sp* and SMI, respectiviely, in the I SP at day 8 vs. day 0. Consistently, the corresponding bone volume in the II SP at day 12, which was translated from the I SP at day 8, had 69%, 37% and 20% increases in BV/TV, Tb.N* and Tb.Th*, and 32% and 20% decreases in Tb.Sp and SMI, respectively, than the II SP at day 4 (translated from I SP at day 0). Veh No change was found between scans at day 0 and 12. At day 12, BV/TV, Tb.N*, and Tb.Th* of the PTH group were 186%, 57% and 37% greater and Tb.Sp* and SMI 39% and 32% less than the Veh group, respectively.
3mo groups: Minimal growth was observed in the adult group thus longitudinal comparisons of the same VOI in the II SP between scans of day 0, 4, 8 and 12 were performed. PTH BV/TV gradually increased by 7%, 18% and 32% at day 4, 8 and 12 compared to day 0, primarily caused by increased Tb.Th* by 7%, 20%, and 35% at day 4, 8 and 12. SMI decreased by 13%, 28% and 52% at day 4, 8 and 12 vs. day 0, suggesting a more plate-like structure by PTH treatment. In contrast, Tb.N* and Tb.Sp* remained constant from day 0 to 12. Veh Compared to day 0, Tb.Th* had 8% increases at day 12; none of the other parameters were significantly different. At day 12, Tb.Th* was 26% greater in PTH vs. Veh group. No differences in the other parameters were found between the PTH and Veh groups.

Discussion: By using a novel in vivo imaging technique we demonstrated distinct structural mechanisms of PTH's effect on young vs. adult rat skeleton to intermittent PTH. To our knowledge, this is the first study that aims to capture the immediate response in trabecular bone structure to PTH with the highest possible spatial resolution (10.5 µm) and temporal resolution (4 days). Due to the longitudinal study design, we were able to detect a significant response in bone volume and microstructure starting as early as day 4 and a continuous anabolic effect until day 12, which corresponds to a 1-year clinical treatment in human.
The mechanisms of the anabolic responses of trabecular bone to PTH were distinct between young and mature rats. In young rats, PTH directly improved the bone quality of the I SP that was continuously generated from the growth plate at a daily rate of 0.25 mm. Although trabecular volume and number decreased during the translation from the I SP into II SP, the fast growth rate in young rats mediated the translation of improved bone volume and structure of the I SP into the II SP. In adult rats, minimal translation from the I SP to II SP was observed due to the lower growth rate. In contrast, PTH increased trabecular bone mass in adult rats by thickening the existing trabeculae. This was possibly due to an elevated local bone formation.

Significance: In addition to the anabolic effect on local bone remodeling, PTH may have a direct effect on the growth plate and I SP that lead to improved II SP of young rats, making it a treatment candidate for promoting bone health in the growing skeleton.

Fig. 1 Registered tibial bone structure of a PTH-treated 3 mo rat.
Fig 2. Registered tibia bone structure of PTH-treated 1 mo rat at (A) day 4 and (B) day 8; and (C) overlapped 2D registered bone structure of day 4 (Red) and day 8 (Green). Yellow: common bone area.
Fig. 3 Changes in I SP and II SP of PTH-treated 1 mo rat. * indicates significant difference between unregistered I SP ( II SP) ; # indicates difference between the I SP and the corresponding II SP 4 days later.