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

Steroid-Associated Hip Joint Collapse in Bipedal Emus

<div><p>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.</p></div

Histomorphometrical analysis of emu femoral head.

<p>In SAON group there were significantly higher adipocyte fraction (A), higher adipocyte size (B), thinner subchondral plate (C), thinner cartilage (D), thinner safranin O staining (E), smaller % Ob. Pm (F) and significant more blood vessels per slides at collapsed region or the corresponding region of the non-collapsed femoral head (G). Mean ± SD, n = 5 (10 bilateral hips) for SAON group and n = 3 (6 bilateral hips) for the control group, *: <i>p</i><0.05).</p

Representative histological images.

<p>H&E stained femoral heads of the control group (A1), and SAON group (A2) (dotted circle: collapsed region). Subchondral bone in the control group (B1), and in SAON group (B2) with empty lacunae (black arrows) in bone matrix, increased size and number of fat cells in bone marrow (blue arrows) and decreased number of mononuclear cells (yellow arrows) compressed blood vessel (red arrows) with effused blood cells (red arrowheads) when compared with that of the control group. Representative histological images at different regions of the femoral head: B: beneath the articular cartilage; C: central of the femoral head of normal group (B1, C1) and SAON group (B2, C2); and D: near to the femoral neck; E: fractured subchondral trabecular bone from a collapsed femoral head of SAON group, showing typical histological evidences of osteonecrosis, i.e., numerous empty lacunae or pyknotic nuclei of osteocytes (black arrows) and scattered bone marrow tissue (brown arrows). Safranin O stained femoral head in the control group (F1) and in SAON group (F2). Normal cartilage in the control group (F1) and pathological changes of articular cartilage in SAON group (F2). (10× for A1, A2, F1, F2; 200× for B1, B2, C1, C2, D, E, G1, G2).</p

Representative/MMA-casted SEM images of the subchondral bone.

<p>A: in normal control, osteocytes are found on the solid bone matrix without obvious removal after acid processing; B: in the collapsed femur head region, osteocytes are presented without bone matrix surrounding, i.e. bone matrix removed after acid processing. (1500×).</p

Hematological evaluation of emus in SAON group.

<p>(A) Time course changes in percentage of neutrophils (Neut %) in blood: Neut% was significantly increased at 2 weeks post SAON-induction (*: <i>p</i><0.05, compared with baseline, n = 5). (B) Time course changes in serum parameters related to lipid metabolism: TC significantly increased at each time point post SAON-induction. LDL and the ratio between LDL and HDL also significantly increased at each time point post SAON-induction (*: <i>p</i><0.05, compared with baseline, n = 5). (C) Time course changes in serum parameters related to coagulation: no significant difference was found in coagulation-related parameters after induction as compared with baseline.</p

Representative T2-weighted coronal MRI images of an emu in SAON group.

<p>(A) baseline before SAON-induction; (B) at week 2 post SAON-induction, showing edema (dotted circle) in the proximal femur; and (C) at week 12 post SAON-induction, showing decreased edema (arrow) in the proximal femur. No collapse was found in the first 12 weeks after LPS-MPS induction.</p

High-speed video gait analysis for the included ankle joint angle during a gait stride cycle compared between a representative normal emu and an emu with SAON-induced hip joint collapse (confirmed after its sacrifice).

<p>The major difference was shown in the ankle joint during takeoff at 67.0% stride.</p