Steroid-associated hip joint collapse in bipedal emus

In this study we established a bipedal animal model of steroid-associated hip joint collapse in emus for testing potential treatment protocols to be developed for prevention of steroid-associated joint collapse in preclinical settings. Five adult male emus were treated with a steroid-associated osteonecrosis (SAON) induction protocol using combination of pulsed lipopolysaccharide (LPS) and methylprednisolone (MPS). Additional three emus were used as normal control. Post-induction, emu gait was observed, magnetic resonance imaging (MRI) was performed, and blood was collected for routine examination, including testing blood coagulation and lipid metabolism. Emus were sacrificed at week 24 post-induction, bilateral femora were collected for micro-computed tomography (micro-CT) and histological analysis. Asymmetric limping gait and abnormal MRI signals were found in steroid-treated emus. SAON was found in all emus with a joint collapse incidence of 70%. The percentage of neutrophils (Neut %) and parameters on lipid metabolism significantly increased after induction. Micro-CT revealed structure deterioration of subchondral trabecular bone. Histomorphometry showed larger fat cell fraction and size, thinning of subchondral plate and cartilage layer, smaller osteoblast perimeter percentage and less blood vessels distributed at collapsed region in SAON group as compared with the normal controls. Scanning electron microscope (SEM) showed poor mineral matrix and more osteo-lacunae outline in the collapsed region in SAON group. The combination of pulsed LPS and MPS developed in the current study was safe and effective to induce SAON and deterioration of subchondral bone in bipedal emus with subsequent femoral head collapse, a typical clinical feature observed in patients under pulsed steroid treatment. In conclusion, bipedal emus could be used as an effective preclinical experimental model to evaluate potential treatment protocols to be developed for prevention of ON-induced hip joint collapse in patients

Icaritin did not affect MSCs proliferation at a wide range of doses.

<p>The cells were incubated with Icaritin (10∧-10 M to 10∧-4 M) for 1, 2, 3 and 4 days, then MTT assay was performed to test the proliferation ability. DMSO served as control.</p

Icaritin upregulated osteoblastic marker genes expression during osteogenic differentiation of MSCs.

<p>MSCs cultured in OS medium with or without Icaritin (10∧-6 M) for 3, 6 and 12 days respectively, before the RNA was extracted and Real-time PCR was performed (* p<0.05).</p

Hematology/cytology/MRI data analysis.

<p>There was no significant change from baseline in ALT (A) and AST (B) when compared to the CON group. Significantly increased TM (C) from baseline in the CON group was attenuated in the L-EF group or prevented in both the M-EF and H-EF group at week 1 post induction, and adipocyte positive colonies (D) in the CON group were attenuated in the L-EF group or prevented in both the M-EF and H-EF group after induction. In addition, the significantly decreased PEP (E) from baseline in the CON group was attenuated in the L-EF group or prevented in both the M-EF and H-EF group at week 1 post induction. Note: * P<0.05 for comparison with CON; # P<0.05 for comparison with baseline. • CON group; ⧫ L-EF group; ▪ M-EF group; ▴ H-EF group.</p

Seven major flavonoid compounds are identified in <i>Epimedium</i>-derived Flavonoids (EF).

<p>The common structure is 8-prenylkaempferol. R1 and R2 are substituted by glucose, rhamnose, or xylose, and R3 is replaced by methyl.</p

A total ion chromatogram in full scan mode generated by HPLC/UV/MS/MS.

<p>(A)∼(B) Compared with the blank sera, a peak shown in 38.1 min in the sera from L-EF, M-EF and H-EF group. (C) HPLC profile of standard Icaritin. (D) 391 (<i>m/z</i> [M+Na]⁺) for Icaritin selected for the subsequent selected ion chromatography (SIC), with a peak at 38.1 min. (E) The +MS showed the mass weight by 391 ion (<i>m/z</i> [M+Na]⁺) and the absence of 56 exhibited the existence of prenyl in the +MS² chromatography. (F) <i>Epimedium</i>-derived flavonoids with common stem nuclei intestinally metabolized to Icaritin.</p

Icaritin increased but not induced alkaline phosphatase (ALP) activity during osteogenic differentiation of MSCs.

<p>(A) MSCs treated with Icaritin (10∧-8 M to 10∧-5 M) in absence of OS or in presence of OS for 3, 7 and 10 days respectively, then the cells were lysed and ALP activity assay was performed (OS: osteogenic supplements, ** p<0.01versus control; # p<0.05 and ## p<0.01 versus other group). (B) MSCs were treated the same as in (A) for 10 days, then ALP was stained with BCIP/NBT kit.</p

Icaritin promoted but not induced mineralization in osteogenic differentiation of MSC.

<p>(A) MSCs treated with Icaritin (10∧-8 M to 10∧-5 M) in absence of OS or in presence of OS for 16 days, then the calcium deposits were stained by Alizarin Red S (ARS). (B) The Alizarin Red S in (A) was eluted by 10% (wt/vol) cetylpyridinium chloride, and the concentrations were determined by absorbance measurement at 562 nm (** p<0.05).</p

Supernate soluble TM in endotoxin induction group (A) and optical density in destained oil red O staining in steroid induction group (B) was significantly higher than the corresponding control group, respectively, whereas Icaritin dose-dependently lowered supernate soluble TM (A) and optical density in destained oil red O staining (B) when compared to the corresponding induction group, respectively.

<p>However, no differences were found between the seven parent flavonoids in EF at the concentration of 10⁻¹⁴ M and corresponding induction group both in supernate soluble TM (C) and optical density in destained oil red O staining (D). Note: * P<0.05 for comparison with the induction group. # P<0.05 for comparison with the low dose group.</p

Characterization of human fetal bone marrow derived mesenchymal stem cells.

<p>MSCs were collected and wished with PBS, then incubated with fluochrome-conjugated primary antibodies against CD34, CD44, CD45, CD73, CD90, CD105, and corresponding isotype control. The stained cells were immediately subjected to flow cytometric analysis using LSRFortessa Flow Cytometry.</p