Abnormal osteogenesis and adipogenesis with decreased β-catenin signaling in the necrotic femoral heads of GONFH patients. Necrotic (n=15) and Non-necrotic (n=10) femoral head samples were obtained from the patients with GONFH or femoral neck fracture, respectively. (A) Gross anatomy analysis of human necrotic and non-necrotic femoral head samples. Black asterisks: subchondral collapsed region. Black arrows: liquefied necrotic region. (B) μCT images of human necrotic and non-necrotic femoral heads. Red and Yellow boxed area: 3D images of subchondral collapsed region and liquefied necrotic region, respectively. White boxed areas: 3D images of corresponding regions in the non-necrotic femoral heads. (C) Quantitative analysis of BV/TV, Tb.N, Tb.Th and Tb.Sp on the necrotic regions. (D) ABH staining of human necrotic and non-necrotic femoral heads. a,c: high magnification images of bone marrow; b,d: high magnification images of bone trabeculae; Yellow arrows: subchondral bone destruction; black arrows: empty lacunae of osteocyte; black triangles: fat droplet. (E) Histomorphological quantitative analysis of trabecular bone area and fat droplet area. (F) Histomorphological quantitative analysis of empty lacunae rate. (G) TUNEL staining of osteocytes in human necrotic and non-necrotic femoral heads. (H) IHC staining of Runx2 and FABP4 expressions in human necrotic and non-necrotic femoral heads. (I) Western blot of β-catenin, Osterix, PPAR-γ and CEBP/α in human necrotic and non-necrotic femoral heads.

Systemic injection of Wnt agonist 1 alleviates abnormal osteogenesis and adipogenesis in rat GONFH femoral heads. The rat model of GONFH was established by a co-induction of lipopolysaccharide and methylprednisolone (MPS). The femoral head samples were harvested after GONFH rats were intravenously injected with Wnt agonist 1 for 6 weeks. (A) Gross anatomy analysis and μCT images of femoral heads in sham, model and Wnt agonist 1 treated groups. (B) μCT quantitative analysis of BV/TV, Tb.N, Tb.Th and Tb.Sp in each group. (C) ABH staining of femoral heads in each group. a-c, high magnification images of the representative region; black arrows: empty lacunae of osteocyte; black triangles: fat droplet. (D) Histomorphological quantitative analysis of trabecular bone area and fat droplet area in each group. (E) Histomorphological quantitative analysis of empty lacunae rate in each group. (F) TUNEL staining of osteocytes in the femoral heads of GONFH rats. (G, H) IHC staining and quantitative analysis of β-catenin, ALP and FABP4 in rat femoral heads of each group.

Activation of β-catenin redirected Dex-induced imbalanced osteogenic/adipogenic differentiation of BMSCs. Primary BMSCs were extracted from the proximal femur of 4-week-old SD rats and cultivated to passage 3 for subsequent experiments. (A) Flow cytometry analyzing the surface markers of rat BMSCs. (B) ALP staining and oil red O staining of BMSCs at the increasing concentrations of dexamethasone (Dex). (C, D) Western blot and quantitative analysis of β-catenin, Runx2, ALP, PPAR-γ and CEBP/α in BMSCs at the increasing concentrations of Dex. (E) ALP staining and oil red O staining of rat BMSCs at the condition with or without 104 nM Dex and 20 μM SKL2001 (an agonist of β-catenin). (F, G) Western blot and quantitative analysis of β-catenin, Runx2, ALP, PPAR-γ and CEBP/α in rat BMSCs at the condition with or without 104 nM Dex and 20 μM SKL2001.

Fate mapping of Col2+ cells and Osx+ cells in the femoral head. TomatoCol2ER mice and TomatoOsxER mice were continuously received 5 dose of TM injections (1 mg/10 g body weight) at the age of 2 weeks for fate mapping analysis. (A) Distributions of Col2+ and Osx+ cells in the femoral head of 1-month-old TomatoCol2ER mice and TomatoOsxER mice. White arrowheads: chondrocyte; Green arrowheads: osteoblast; Yellow arrowheads: osteocyte; Red arrowheads: bone marrow stromal cell; GP: growth plate; SOC: second ossification center; BM: bone marrow. (B) Lineage trace of Col2+ cells in the femoral head for 9 months. a: high magnification image of bone marrow region. (C) Poor Cre-recombinase efficiency in the femoral head of TomatoCol2ER mice with TM induction at the age of 1 month. (D, E) Femoral heads were harvested from 3-month-old β-cateninCol2ER mice and β-cateninOsxER mice to detect expression of β-catenin. IHC staining and quantitative analysis of β-catenin in the femoral heads of 3-month-old β-cateninCol2ER mice and β-cateninOsxER mice.

Deletion of β-catenin in Col2+ cells leads to a GNOFH-like phenotype. Femoral heads were harvested from 3-month-old β-cateninCol2ER mice and Cre-negative littermates with 5 continuous dosage of TM injections (1 mg/10 g body weight) at the age of 2 weeks. (A) Gross anatomy analysis of femoral heads in β-cateninCol2ER mice and Cre-negative littermates. (B) Representative μCT images and quantitative analysis of BV/TV, Tb. N, Tb. Th and Tb. Sp in the femoral heads of β-cateninCol2ER mice. (C) ABH staining of femoral heads in β-cateninCol2ER mice. a-d: high magnification images of representative subchondral bone region; Black triangle arrowheads: fat droplet; Black arrows: empty lacunae of osteocyte. (D) Histomorphological quantitative analysis of trabecular bone area and fat droplet area in the femoral heads of β-cateninCol2ER mice. (E) Histomorphological quantitative analysis of empty lacunae rate in the femoral heads of β-cateninCol2ER mice. (F) TUNEL staining of osteocytes in the femoral heads of β-cateninCol2ER mice. (G) IHC staining and quantitative analysis of Runx2, ALP, PPAR-γ and CEBP/α in the femoral heads of β-cateninCol2ER mice.

Loss-of-function of β-catenin in Osx+ cells causes bone loss in the femoral head. Femoral heads were harvested from 3-month-old β-cateninOsxER mice and Cre-negative littermates with 5 continuous dosage of TM injections (1 mg/10 g body weight) at the age of 2 weeks. (A) Gross anatomy analysis of femoral heads in β-cateninOsxER mice and Cre-negative littermates. (B) μCT images and quantitative analysis of BV/TV, Tb. N, Tb. Th and Tb. Sp in the femoral heads of β-cateninOsxER mice. (C) ABH staining of femoral heads in β-cateninOsxER mice. (D) Histomorphological quantitative analysis of trabecular bone area and fat droplet area in the femoral heads of β-cateninOsxER mice. (E) Histomorphological quantitative analysis of empty lacunae rate in the femoral heads of β-cateninOsxER mice.

Older β-cateninCol2ER mice display subchondral bone destruction and collapse tendency in femoral heads. Femoral heads were harvested from 6-month-old β-cateninCol2ER mice and Cre-negative littermates with 5 continuous dosage of TM injections (1 mg/10 g body weight) at the age of 2 weeks. (A) Gross anatomy analysis of femoral heads in 6-month-old β-cateninCol2ER mice. (B) Representative μCT images of femoral heads in 6-month-old β-cateninCol2ER mice. Red dotted lines: integral deformity. Red arrows: local collapse. Yellow arrows: subchondral bone destruction. (C) Quantitative analysis of BV/TV in the femoral heads in 6-month-old β-cateninCol2ER mice. (D) Quantitative analysis of subchondral bone defect area on the femoral head surface in older β-cateninCol2ER mice. (E) ABH staining of femoral heads in 6-month-old β-cateninCol2ER mice. a-c: high magnification images of representative subchondral bone region. Red dotted lines: integral deformity. Red arrows: local collapse. Black arrows: empty lacunae of osteoctye. Black triangles: fat droplet. (F) Histomorphological quantitative analysis of empty lacunae rate. (G) Loading-bearing stiffness of the femoral heads in 6-month-old β-cateninCol2ER mice.

Systemic injection of Wnt agonist 1 can not alleviate the GONFH-like phenotype in β-cateninCol2ER mice. β-cateninCol2ER mice with 5 continuous dosage of TM injections (1 mg/10 g body weight) were treated with Wnt agonist 1 three times once week until sacrifice at the age of 3 months. (A) Representative μCT images of femoral heads in Wnt agonist 1 treated β-cateninCol2ER mice. (B) Quantitative analysis of BV/TV, Tb.N, Tb.Th and Tb.Sp in the femoral heads of Wnt agonist 1 treated β-cateninCol2ER mice. (C) ABH staining of the femoral heads in Wnt agonist 1 treated β-cateninCol2ER mice. a-b: high magnification images of subchondral bone region. Black triangles: fat droplet. (D) Histomorphological quantitative analysis of trabecular bone area and fat droplet area in the femoral heads of Wnt agonist 1 treated β-cateninCol2ER mice. (E) Histomorphological quantitative analysis of empty lacunae rate in the femoral heads of Wnt agonist 1 treated β-cateninCol2ER mice. (F, G) IHC staining and quantitative analysis of ALP and FABP4 in the femoral heads of Wnt agonist 1 treated β-cateninCol2ER mice.