Low bone turnover and low BMD in Down syndrome: effect of intermittent PTH treatment

Abstract

Trisomy 21 affects virtually every organ system and results in the complex clinical presentation of Down syndrome (DS). Patterns of differences are now being recognized as patients' age and these patterns bring about new opportunities for disease prevention and treatment. Low bone mineral density (BMD) has been reported in many studies of males and females with DS yet the specific effects of trisomy 21 on the skeleton remain poorly defined. Therefore we determined the bone phenotype and measured bone turnover markers in the murine DS model Ts65Dn. Male Ts65Dn DS mice are infertile and display a profound low bone mass phenotype that deteriorates with age. The low bone mass was correlated with significantly decreased osteoblast and osteoclast development, decreased bone biochemical markers, a diminished bone formation rate and reduced mechanical strength. The low bone mass observed in 3 month old Ts65Dn mice was significantly increased after 4 weeks of intermittent PTH treatment. These studies provide novel insight into the cause of the profound bone fragility in DS and identify PTH as a potential anabolic agent in the adult low bone mass DS population.

Figure 1. Trisomy effects on the proximal tibia and femur or 3-month and 24-month Ts65Dn mice.

(A) Micro CT reconstructions of metaphyseal tibial cancellous bone in 3-month (left column) and 24-month (right column) old WT (top) and Ts65Dn (bottom) mice. The low bone volume is evident at 3-months in the Ts65Dn mice vs. WT. (B). Micro CT analysis of % bone volume/tissue volume (BV/TV), trabecular number (Tb.N.), trabecular thickness (Tb.Th.), trabecular separation (Tb.Sp) in Ts65Dn 3-month and 24-month old WT and Ts65Dn proximal tibia. (C). Decreased cortical thickness at the femoral midshaft by micro CT in 24 month old WT and Ts65Dn mice. Representative MicroCT reconstructions of the midshaft and measurement of cortical thickness in both WT and Ts65Dn are shown. *, p<0.05 vs. WT.

Figure 2. Bone Formation and bone turnover is decreased in Ts65Dn Mice.

(A). Representative double-fluorochrome-labeled regions of trabecular bone in the proximal tibia of 3 month old WT (top) and Ts65Dn (bottom). Arrowheads indicate the distance between the two labels used to calculate the bone formation rate normalized to bone surface (BFR/BS) (um³/um²/day). (B). Tibial sections (N = 6 per group) were used to quantify BFR/BS, percent osteoblast surface/bone surface (Ob.S./BS), percent osteoclast surface/bone surface (Oc.S./BS) and number of osteoclasts/bone surface (N.Oc./BS.) (C). Serum obtained from WT and Ts65Dn mice at 3- and 24-months of age was analyzed for bone resorption, tartrate resistant acid phosphatase 5b (TRAP 5b) and bone formation, procollagen-1 N-terminal peptide (P1NP) markers by ELISA. *, p<0.05 vs. respective WT vehicle control.

Figure 3. Osteoclast and osteoblast formation is significantly decreased in 3 month old Ts65Dn Mice.

(A) Ex vivo recruitment into the osteoblast lineage was measured at culture day 10 by staining for alkaline phosphatase (AP) and counting the number of AP+ colony forming units (CFU-F) per well. (B) Ex vivo osteoblast differentiation was assessed at culture day 28 by staining mineralized bone nodules containing differentiated colonies of osteoblasts (CFU-OB) with Alizarin Red and the number of CFU-OB per well enumerated. (C) Ex Vivo osteoclast differentiation was assessed by staining on day 14 for tartrate resistant acid phosphatase (TRAP) and the number of TRAP+-multinucleated cells per well counted. *, p<0.05 vs. WT vehicle control.

Figure 4. Efficacy of intermittent PTH in WT vs. Ts65Dn mice.

Male mice (N = 6–8 per group) were given daily injections of vehicle or 30 or 80 ug/kg PTH(1–34) for 4 weeks. (A) BMD was measured at the beginning and end of the experiment. (open squares, WT; closed circles, Ts65Dn). Dotted lines show BMD of vehicle treated animals, dashed lines, 30 ug/kg PTH; solid lines, 80 ug/kg PTH (B) % bone volume/tissue volume (BV/TV), trabecular number (Tb.N.) and trabecular thickness (Tb.Th.) were determined in the tibia (open bars WT; solid bars Ts65Dn). Representative superior view of a transverse micro-CT images of the trabecular bone from the proximal tibia of representative animals in each group are shown. (C) Micro CT measurements of the effects of PTH treatment on cortical thickness, periosteal perimeter and endocortical perimeter in the distal tibia were performed (open bars WT; solid bars Ts65Dn). *, p<0.05 vs. respective vehicle control; #, p<0.05 vs. WT vehicle.

Figure 5. Histomorphometric measurement of bone formation and bone resorption following intermittent PTH in WT and Ts65Dn mice.

Male mice (N = 6–8 per group) were given daily injections of vehicle or 30 or 80 ug/kg PTH(1–34) for 4 weeks. (A) Mineral apposition rate (MAR) (um³/um²/day), (B) Bone formation rate/bone surface (BFR/BS) (um³/um²/day), (C) Number of osteoblasts/bone perimeter (N.Ob./B.Pm), (D) Number of osteoclasts/bone perimeter (N.Oc./B.Pm.) were measured in WT and Ts65Dn mice vehicle or PTH treated (30 or 80 ug/kg/day) (open bars, WT; closed bars, Ts65Dn). *, p<0.05 vs. respective vehicle control; #, p<0.05 vs. WT vehicle.