The study of abnormal bone development in the Apert syndrome Fgfr2+/S252W mouse using a 3D hydrogel culture model

Abstract

Apert syndrome is caused by mutations in fibroblast growth factor receptor 2 (Fgfr2) and is characterized by craniosynostosis and other skeletal abnormalities. The Apert syndrome Fgfr2+/S252W mouse model exhibits perinatal lethality. A 3D hydrogel culture model, derived from tissue engineering strategies, was used to extend the study of the effect of the Fgfr2+/S252W mutation in differentiating osteoblasts postnatally. We isolated cells from the long bones of Apert Fgfr2+/S252W mice (n=6) and cells from the wild-type sibling mice (n=6) to be used as controls. During monolayer expansion, Fgfr2+/S252W cells demonstrated increased proliferation and ALP activity, as well as altered responses of these cellular functions in the presence of FGF ligands with different binding specificity (FGF2 or FGF10). To better mimic the in vivo disease development scenario, cells were also encapsulated in 3D hydrogels and their phenotype in 3D in vitro culture was compared to that of in vivo tissue specimens. After 4 weeks in 3D culture in osteogenic medium, Fgfr2+/S252W cells expressed 2.8-fold more collagen type I and 3.3-fold more osteocalcin than did wild-type controls (p<0.01). Meanwhile, Fgfr2+/S252W cells showed decreased bone matrix remodeling and expressed 87% less Metalloprotease-13 and 71% less Noggin (p<0.01). The S252W mutation also led to significantly higher production of collagen type I and II in 3D as shown by immunofluorescence staining. In situ hybridization and alizarin red S staining of postnatal day 0 (P0) mouse limb sections demonstrated significantly higher levels of osteopontin expression and mineralization in Fgfr2+/S252W mice. Complementary to in vivo findings, this 3D hydrogel culture system provides an effective in vitro venue to study the pathogenesis of Apert syndrome caused by the analogous mutation in humans.

Fig. 1

Stained whole skeleton of the Fgfr2^+/S252W mice and the bone marker staining of isolated cells in monolayer. (A, B)mutant; (C) wild-type controls. (A)Mouse skeleton of Fgfr2 ^+/S252W mice, with bone stained purple (alizarin red S staining) and cartilage stained blue (alcian blue). Osteoblasts were isolated from the middle shaft of the long bones (arrows). (B, C) Immunofluorescence staining of collagen type I demonstrated similar amounts of deposition in both Fgfr2 ^+/S252W cells and wild-type controls. S252W: Fgfr2^+/S252W;WT: wild-type. Scale bars: B,C, 100 µm.

Fig. 2

Proliferation and ALP activity of isolated Fgfr2 ^+/S252W cells during monolayer expansion and their responses to FGF ligands. (A) Fgfr2^+/S252W cells proliferated significantly faster than the wild-type controls and S252W mutation leads to altered responses in cell proliferation to FGF ligands. (B) Fgfr2^+/S252W cells produced significantly higher amounts of ALP than the wild-type controls and FGF2 inhibited the ALP activity in both Fgfr2 ^+/S252W cells and wild-type controls. (C) FGF10 induced partial inhibition of ALP activity in Fgfr2 ^+/S252W cells, with no significant effects observed on wild-type controls. The same data set of the ALP activity of Fgfr2 ^+/S252W cells and wild-type controls in monolayer culture without growth factors was presented in (B) and (C) for additional comparison. Error bars are the standard deviations of triplicate samples (*: p<0.01 vs. S252W group,**: p<0.01 compared between two indicated groups, †: p<0.01 vs. WT group).

Fig. 3

Proliferation and collagen deposition of Fgfr2 ^+/S252W cells in hydrogel culture. (B, D) mutant cells; (C, E) wild-type controls. (A) Proliferation is reflected by DNA content at day 1 after encapsulation and day 28 at harvest. Error bars are the standard deviations of triplicate samples (*: p<0.01 vs.WT). (B, C) After 28 days of culture under osteogenic conditions, immunofluorescence staining of collagen type I showed increased deposition by mutant cells in hydrogels. (D, E) Strong staining of cartilage specific marker, collagen type II, was observed in the mutant group while only minimal staining was seen in the wild-type controls. Scale bars: B–E, 100 µm.

Fig. 4

Normalized gene expressions of osteogenesis and bone remodeling markers by quantitative PCR of Fgfr2 ^+/S252W cells and wild-type controls in 3D hydrogel after 4 weeks of culture in osteogenic medium. Results are presented as relative fold changes in the Fgfr2^+/S252W group using ΔΔCt method, with normalized mRNA level in the wild-type group as a control. Error bars are the standard deviations of triplicate samples (*: p<0.01 vs. WT).

Fig. 5

In situ hybridization and alizarin red S (ARS) staining in P0 Fgfr2^+/S252W middle shaft of the mouse limb sections. (A, C, E) mutant mice; (B, D, F) control littermates. (A, B) In situ hybridization of osteopontin (OP) showed increased expression in the mutant mice. (C, D) No significant differences were observed in bone sialoprotein (BSP) expression between the mutant and control at P0. (E, F) Increased ARS staining showed more mineralization in the mutant group. Scale bars: A–D, 50 µm; E,F, 500 µm.