Fig 1.
Evaluation of systemic iron accumulation in blood serum and bone.
In blood serum: a) Total iron concentration; b) Transferrin saturation; c) Total concentration of serum ferritin. In bone: d) Detection of iron deposits (stained in blue) in femur bone trabeculae and bone marrow through Perls staining. Each group analyzed was composed by n = 6 animals for [C+I], [KO] and [KO+I], and by n = 5 for [C]. Statistical comparison was performed with T-test (Welch’s correction) between control and iron-enriched diets for both WT ([C] and [C+I]]) and Hfe-KO mice ([KO] and [KO+I])). Group deviations are expressed as standard error to the mean (SEM). Statistical significance: (*)-p<0.05; (**)-p<0.01; (****)-p<0.0001.
Fig 2.
Gene expression analysis of iron metabolism, oxidative stress and inflammation markers in bone tissue.
a) Iron metabolism: ferroportin (Slc40a1), ferritin heavy polypeptide 1 (Fth1) and transferrin receptor 1 (Tfrc). b) Oxidative stress: catalase (Cat). c) Inflammation response: Tumor necrosis factor (Tnf) and Interleukin 6 (Il6). Relative expression was obtained through ΔΔCt method and was normalized with Gapdh. Each group analyzed was composed by n = 6 animals for [C+I], [KO] and [KO+I], and by n = 5 for [C]. Statistical comparison was performed with T-test (Welch’s correction) between control and iron-enriched diets for both WT ([C] and [C+I]]) and Hfe-KO mice ([KO] and [KO+I])). Group deviations are expressed as standard error to the mean (SEM). Statistical significance: (*)-p<0.05; (**)-p<0.01; (***)-p<0.001.
Fig 3.
Evaluation of bone microarchitecture status by micro-Ct.
Analysis of bone status was performed by determining microarchitecture parameters of bone volume fraction (BV/TV), bone volume (BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and structural model index (SMI). Each group analyzed was composed by n = 6 animals for [C+I], [KO] and [KO+I], and n = 5 for [C]. Statistical comparison was performed with T-test (Welch’s correction) between control and iron-enriched diets for both WT ([C] and [C+I]]) and Hfe-KO mice ([KO] and [KO+I])). Group deviations are expressed as standard error to the mean (SEM). Statistical significance: (*)-p<0.05; (**)-p<0.01; (****)-p<0.0001.
Fig 4.
Micro-CT scans showing bone status from femur and tibia.
Evaluation of microarchitecture parameters following three-dimensional reconstructions from micro-CT scans of femur and tibia as described previously. Three-dimensional reconstructions were obtained from femur and joint scans and from tibia trabecular bone scans using CTvox software (v3.1.1, Bruker, Belgium). Images scale bars equivalence: Femur and joint- 150μm, Tibia trabeculae- 100μm.
Fig 5.
Impact of iron overload on bone formation.
a) Evaluation of osteoid surface (OS/BS) and number of osteoblast (N.Ob/BS). b) Gene expression of bone formation markers: osteocalcin (Bglap2), lactoferrin (Ltf), sclerostin (Sost) and runt related transcription factor 2 (Runx2). Relative expression was obtained by ΔΔCt method and was normalized with Gapdh. Each group analyzed was composed by n = 6 animals for [C+I], [KO] and [KO+I], and by n = 5, for [C]. Statistical comparison was performed with T-test (Welch’s correction) between control and iron-enriched diets for both WT ([C] and [C+I]]) and Hfe-KO mice ([KO] and [KO+I])). Group deviations are expressed as standard error to the mean (SEM). Statistical significance: (*)-p<0.05; (**)-p<0.01; (***)-p<0.001.
Fig 6.
Trap positive cells per bone surface and correlation with trabecular bone microarchitecture.
a) Number of trap positive cells per trabeculae surface and relative expression of acid phosphatase 5, tartrate resistant (Acp5) obtained by ΔΔCt method normalized with Gapdh. Each group analyzed was composed by n = 6 animals for [C+I], [KO] and [KO+I], and by n = 5, for [C]. b) Correlation between number of osteoclasts per trabeculae surface with Tb.N, Tb.Sp and SMI. Correlation was determined through Pearson’s test, with significance of p<0.05. Dashed line identifies confidence interval (95%). Statistical comparison with T-test (Welch’s correction) between control and iron-enriched diets for both WT ([C] and [C+I]]) and Hfe-KO mice ([KO] and [KO+I])). Group deviations are expressed as standard error to the mean (SEM). Statistical significance: (*)-p<0.05.
Fig 7.
Gene expression of markers associated with regulation of bone metabolism.
Relative expression: a) bone morphogenic protein 6 (Bmp6); b) Bone morphogenic protein 2 (Bmp2); c) Sp7 transcription factor 7 (Sp7); d) Parathyroid hormone 1 receptor (Pthr). Expression was determined by ΔΔCt method and normalized with Gapdh. Each group was composed by n = 6 animals ([C+I], [KO] and [KO+I]) except for [C] (n = 5). Comparison among groups was done by unpaired t-student test with Welch's correction and significance of p<0.05. Group deviations are expressed as standard error to the mean (SEM). Statistical significance: (*) = p<0.05.