Supplementary Data

Cartilage structural and mechanical properties evaluated with water-jet indentationUltrasound imaging and water-jet ultrasound indentation test were performed to quantify the thickness and stiffness of emu femoral head cartilage using our established protocol [1]. Briefly, 12 weeks post-treatment, emus were sacrificed and the femora were collected. Both the collapsed and the non-collapsed articular cartilage of the femoral heads were identified for water-jet indentation test to measure the thickness of the cartilage by a high frequency ultrasound imaging system (Vevo 770, VisualSonics Inc., Toronto, Canada) with a 40 MHz transducer of an axial resolution of 30 μm. Contact gel was applied to the femoral head, and then the ultrasound probe was placed onto the top of the sample at the test sites to obtain the B-mode ultrasound images of the femoral head cartilage (Fig. S3). Three typical images were obtained and then the distance from the cartilage surface to the subchondral bone was measured and averaged as the thickness of the articular cartilage of the femoral head. Subsequently, a water-jet ultrasound indentation probe (Fig. S4) was used to measure the stiffness of the cartilage. The probe utilized the water-jet as the media to compress the tissue, and the deformation was measured by the ultrasound probe installed at the tip of the probe [2, 3]. During the test, the sample was fixed with a testing fixator and immersed into water. Cyclic water-jet indentation was performed on the cartilage and the data were then collected and saved for offline processing. A stiffness coefficient was defined as following to indicate the elasticity of the cartilage:

SC= F / AD / L0

which F is the indentation force, A is the indentation area, D is the deformation and L0 is the initial thickness of the cartilage. Three repeated tests were performed at each test site and the calculated parameters were averaged to obtain the stiffness of the tested cartilage.

Reference:[1] Lu MH, Zheng YP, Lu HB, Huang QH, Qin L. Evaluation of bone-tendon junction healing using water jet ultrasound indentation method. Ultrasound in medicine & biology. 2009;35:1783-93.[2] Lu MH, Zheng YP, Huang QH. A novel noncontact ultrasound indentation system for measurement of tissue material properties using water jet compression. Ultrasound in medicine & biology. 2005;31:817-26.[3] Lu MH, Zheng YP, Huang QH, Ling C, Wang Q, Bridal L, et al. Noncontact evaluation of articular cartilage degeneration using a novel ultrasound water jet indentation system. Annals of biomedical engineering. 2009;37:164-75.

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Supplementary Tables and Figures

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Table S1. Primers used for real-time PCR

GenePrimers (5’-3’)

Forward ReverseRabbit-RUNX2 TGACCGCAGACATAATCCAT GCCACTTTCGGAACAGAGAT

Rabbit-BMP-2 TCGAGAACAGATGCAGGAAG GGAATTTCGAGTTGGCTGTT

Rabbit-OC CAGCGAGGTAGTGAAGAGA CTGAAAGCCGATGTGGT

Rabbit-C/EBP-β CTACTACGAGGCGGACTGCT GTACGGGCTGAAGTCGATG

Rabbit-PPAR-γ GCCATATTCACGATGCTGTC GAACTTCATGGCGAAGTCAA

Rabbit-COL1 CAGCAAGAACCCCAAGGAGA CTTCGCTGCCGTACTCGAA

Rabbit-aP2 CGATAAACTGGTGGTGGAATGC

CCCGGGCTTATGCTCTTTCA

Rabbit-CXCR4 TACACCGAGGAAATGGGCTCA AGATGATGGAGTAGATGGTGGG

Rabbit-VCAM1 GCTGCTCAGATTGGAGACTCA CGCTCAGAGGGCTGTCTATC

Rabbit-SDF1 GAGAGCCACATCGCCAGAG TTTCGGGTCAATGCACACTTG

Rabbit-GAPDH TCTGGCAAAGTGGATGTTGT GTGGGTGGAATCATACTGGA

Fig. S1. Experimental emus were allowed to walk freely in Shenzhen Emu Institute.

Fig. S2. A specific posture fixture was designed for in vivo MRI imaging of emu hips to obtain highly reproducible images. (A) Emu was maintained at left lateral lying position, which could be fitted to the MRI examination bed. (B) Bilateral lower limbs were kept parallel by inserting a piece of shaped foam board, which reduced variation in positioning during the repeated in vivo radiographic examinations, between the two legs on the custom-made posture fixture. (C) Emu was in place for MRI scanning.

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Fig. S3. Ultrasound B-mode images of the cartilages. Cartilage thickness measurement using water indentation ultrasound compared between (A) the intact region and (B) the collapsed region where edema was also visualized in the subchondral bone of the collapsed region.

Fig. S4. Water-jet ultrasound indentation setup of the ultrasound probe and specimen. (A) Water-jet indentation probe for stiffness measurement; (B) the testing fixator for holding the indentation probe and specimen in place.

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Fig. S5. Representative FEA of an emu proximal femur with boundary and loading

conditions.

Fig. S6. Representative photographs of FEA of empty control, P/T and P/T/I groups. Element Strain Energy Density (ESEDEN), which was presented using colourimetric scale with red being the highest and dark blue being the lowest, was higher at the stress stimulated region at femoral head (red arrow). The average of ESEDEN was highest in empty control group while lowest in P/T/I group.

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