Low bone mass and impaired fracture healing in mouse models of Trisomy21 (Down syndrome)

Abstract

Individuals with Down syndrome (DS), the result of trisomy of human chromosome Hsa21 (Ts21), present with an array of skeletal abnormalities typified by altered craniofacial features, short stature and low bone mineral density (BMD). While bone deficits progress with age in both sexes, low bone mass is more pronounced in DS men than women and osteopenia appears earlier. In the current study, the reproductive hormone status (FSH, LH, testosterone) of 17 DS patients (males, ages range 19-52 years) was measured. Although testosterone was consistently low, the hypothalamic-pituitary-gonadal axis was intact with corresponding rises in FSH and LH. To provide further insight into the heterogeneity of the bone mass in DS, the skeletal phenotypes of three of the most used murine DS models, Ts65Dn (Ts65), TC1, and Dp16(Yey1) (Dp16) were characterized and contrasted. Evaluation of the bone phenotype of both male and female 3-month-old Dp16 mice demonstrated sexual dimorphism, with low bone mass apparent in males, as it is in Ts65, but not in female Dp16. In contrast, male TC1 mice had no apparent bone phenotype. To determine whether low bone mass in DS impacted fracture healing, fractures of the middle phalanx (P2) digits were generated in both male and female Dp16 mice at 15 weeks of age, an age where the sexually dimorphic low BMD persisted. Fracture healing was assessed via in vivo microCT over (13 weeks) 93 days post fracture (DPF). At 93 DPF, 0 % of DS male (n = 12) or female (n = 8) fractures healed, compared to 50 % of the male (n = 28) or female (n = 8) WT littermate fractures. MicroCT revealed periosteal unbridged mineralized callus formation across the fracture gap in Dp16 mice, which was confirmed by subsequent histology. These studies provide the first direct evidence of significantly impaired fracture healing in the setting of DS.

Keywords: Down syndrome; Fracture healing; Genetic mouse models; Osteopenia; Skeletal abnormalities.

Fig. 1.

DS patient clinical parameters. (A) Distribution of male DS Bone Mineral Density (BMD) Z-scores at femoral neck (FemN) and posterior-anterior (PA) spine. Z score ranges 0 to −1.5; −1.5 to −2.5; and < −2.5 are indicated by hashed lines; black; gray shading respectively. The percentage of patients in each range is plotted (n = 17) (B) Serum measurements of individual testosterone (ng/ml) (C) Luteinizing hormone (LH) (mIU/ml), (D) Follicle stimulating hormone (FSH) (mIU/ml) levels in male DS patients. Normal range for each hormone indicated by shading. Horizontal line shows median hormone level for each measurement.

Fig. 2.

Bone volume and bone formation measurements in DS mouse tibiae. (A, D) BV/TV and bone formation rate per bone surface (BFR/BS) in male Ts65Dn; (B) BV/TV in male and female Dp16 DS mice and (E) bone formation rate per bone surface (BFR/BS) in male Dp16 DS mice. (C, F) BV/TV and bone formation rate per bone surface (BFR/BS) in male TC1 DS mice. *p < 0.05 between genotypes. MicroCT measurements shown for each individual animal; BFR/BS measured from fluorochrome labeling and dynamic histomorphometry from each animal, and calculated according to Dempster [39].

Fig. 3.

Dp16 DS ex vivo bone marrow cell differentiation. (A-C) Murine bone marrow cells from adult (3–4 months) WT (circles) and Dp16 DS (squares) male and female mice were cultured towards osteoblastogenesis. (A) Recruitment of cells into the osteoblast lineage was measured on d 10. The number of colonies staining positive for alkaline phosphatase (AP+) were counted and expressed as a percentage of the total number of colonies per well. (B) The total number of mesenchymal progenitors was determined in d 10 AP-stained and Fast green counter-stained dishes and both the number of AP+ colonies and the Fast green non-AP-stained colonies combined to express as total colonies per well. Number of mesenchymal progenitors was increased in both male and female Dp16 mice. (C) Mineralized osteoblastic colony-forming units (CFU-OB) were determined on d 28 by alizarin red staining, and the number of CFUOB normalized to the micrograms of protein content in each well. A significant increase in osteoblast differentiation capacity was observed in female Dp16 mice, but not male Dp16 mice. Data are representative of at least two similar experiments. p < 0.05 (*), p < 0.002 (**), or p < 0.001 (***) compared to WT gender-matched control. (D) Primary murine bone marrow cells were cultured for the development of osteoclasts. Cells were stained for TRAP activity, and the number of TRAP+ multinucleated cells (MNC) with 3 or more nuclei per well was counted. Data are representative of at least two similar experiments harvested on d 8–12. *p < 0.05.

Fig. 4.

Time course of P2 Fracture Healing by in vivo microCT in WT and Dp16 DS mice. (A) MicroCT reconstruction of an intact P2 with the fracture plane (dashed line). In vivo microCT reconstructions of representative (B) male and (C) female WT and DS digits across the time course of fracture healing up to 93 days post-fracture (DPF). Normal fracture healing occurred in WT, with mineralization of the soft callus visible by 21 DPF, and fracture bridging by 42 DPF, when remodeling begins. Both male and female DS digits began mineralization by 21 DPF but did not successfully bridge by 42 DPF when WT digits were completely bridged and resulting in non-union fractures by 93 DPF.

Fig. 5.

Computed Radiographic Assessment of P2 (CRAPP) Method for Fracture Scoring of Dp16 DS P2 Fractures. (A) In vivo microCT reconstructions of murine P2 digits demonstrating the use of CRAPP to quantify fracture healing. Fractures were scored from a range of I-IV, with I representing a non-union and IV representing a complete union, or healed fracture. Any fracture observed between two scores was assigned the lower score. (B) Fractures were scored using the CRAPP method, demonstrating a normal range of healing in both male and female WT mice. None of the DS fractures healed (score of IV), and most did not surpass a score of I in both males and females.

Fig. 6.

A: Male P2 fracture histology at 93 DPF. Safranin-O/Fast Green staining of male WT (i, ii) and Dp16 DS (iii) digits and the corresponding microCT reconstruction at 93DPF. Orientation is proximal (P) to distal (D). Bone is shown in green and cartilage proteoglycan is shown in red. (i) Bridged WT fracture, original fracture plane is not visible and the bony callus has been remodeled to lamellar cortical bone (CB) with a bone marrow (BM) cavity. (ii) WT non-union fracture shows extensive cortical bone (CB) proximal to the fracture gap, as well as intense red unmineralized cartilaginous proteoglycan-rich matrix surrounding chondrocytes in the fracture gap. (iii) DS non-union at 93DPF showing robust periosteal woven bone (WB) formation proximal to the fracture gap and the absence of red cartilaginous proteoglycan staining in the evident fracture gap (*). Scale bar is 100 μm. B: Female P2 fracture histology at 93 DPF. Safranin-O/Fast Green staining of female WT (i, ii) and Dp16 DS (iii) digits and the corresponding microCT reconstruction at 93DPF. Orientation is proximal (P) to distal (D). Bone is shown in green and cartilage proteoglycan is shown in red. (i) Bridged WT female fracture, original fracture plane is not visible and the bony callus has been remodeled to lamellar cortical bone (CB) with a bone marrow (BM) cavity. (ii) WT female non-union fracture shows extensive cortical bone (CB) proximal to the fracture gap, as well as intense red unmineralized cartilaginous proteoglycan-rich matrix surrounding chondrocytes in the fracture gap. (iii) Female DS non-union at 93DPF showing robust periosteal woven bone (WB) formation proximal to the fracture gap and the absence of red cartilaginous proteoglycan staining in the evident fracture gap (*). Scale bar is 100um.