www.sciencetranslationalmedicine.org/cgi/content/full/4/141/141ra93/DC1

Supplementary Materials for

A Tissue Engineering Solution for Segmental Defect Regeneration in Load-Bearing Long Bones

Johannes C. Reichert, Amaia Cipitria, Devakara R. Epari, Siamak Saifzadeh, Pushpanjali Krishnakanth, Arne Berner, Maria A. Woodruff, Hanna Schell, Manav Mehta, Michael

A. Schuetz, Georg N. Duda, Dietmar W. Hutmacher*

*To whom correspondence should be addressed. E-mail: dietmar.hutmacher@qut.edu.au

Published 4 July 2012, Sci. Transl. Med. 4, 141ra93 (2012) DOI: 10.1126/scitranslmed.3003720

This PDF file includes:

Methods Fig. S1. Scaffold preparation and transplantation. Fig. S2. White blood cell infiltrate 12 months after reconstruction with scaffold/rhBMP-7. Fig. S3. Histology of bone defects after 3 and 12 months. Fig. S4. Images of histological sections stained with Safranin-Orange/von Kossa. Legends for Movies S1 to S7

Other Supplementary Material for this manuscript includes the following: (available at www.sciencetranslationalmedicine.org/cgi/content/full/4/141/141ra93/DC1)

Movie S1 (.wmv format). ABG, 3 months. Movie S2 (.wmv format). ABG, 12 months. Movie S3 (.wmv format). Empty, 3 months. Movie S4 (.wmv format). Scaffold only, 3 months. Movie S5 (.wmv format). Scaffold only, 12 months. Movie S6 (.wmv format). Scaffold and rhBMP-7, 3 months. Movie S7 (.wmv format). Scaffold and rhBMP-7, 12 months.

SUPPLEMENTARY METHODS

Histology

For histological processing, 3-mm–thick slices were cut and dehydrated in a

graded series of ethanol prior to embedding in poly(methyl methacrylate)

(PMMA, Technovit 9100 NEU, Heraeus Kulzer). Sections of 6-μm thickness were

prepared (Leica SM 2500S) and stained with Safranin-Orange/von Kossa and

Movat’s pentachrome. Samples were visualized using a light microscope (Leica

DMRB and AxioCam MRc, Zeiss).

Computed tomography

After sacrifice, a clinical CT scanner (Philips Brilliant CT 64 channels) was used

to scan operated and contralateral tibiae. Slice thickness was 0.5 mm and

exposure settings were 120 kV/100–76 mA·s. Image data saved in the DICOM

(digital imaging and communication in medicine) format.

For quantitative analysis, the datasets were cropped to image stacks with

equal bounding box dimensions using AMIRA 5.2.2 (Visage Imaging GmbH). Next,

cortical bone and callus tissue were segmented with a threshold of 300 HU

(Hounsfield units) and a 3D surface was generated and saved as a binary file

(STL binary Little Endian format). These .stl files were loaded into

Rapidform2006 (Inus Technology) and a minimum of four corresponding

reference points were selected on each intact and operated tibia and the tibiae

were aligned. An in-house MATLAB program (MATLAB 7.6.0, MathWorks, Inc.)

routine was used to determine the transformation matrix to align the image data

stacks of intact and defect tibia and to calculate the amount of newly formed

bone within the total defect and the proximal, medial and distal third.

Backscattered electron imaging

Backscattered electron imaging was performed on PMMA-embedded transversal

sections. Images were obtained using an environmental scanning electron

microscope (FEI FEG-ESEM Quanta 600, FEI Company) in the backscattered

electron mode operated at an accelerating voltage of 10 kV under low vacuum

(0.8 Torr). The sample detector was set at 9.8 mm working distance and a spot

size of 4.0.

Radiographic analysis

After surgery (t = 0 days) and after 6, 12, 24, and 48 weeks, conventional x-ray

analysis (3.2 mA·s, 65 kV) in two standard planes (anterior-posterior and

medial-lateral) was performed.

SUPPLEMENTARY FIGURES

Figure S1: Scaffold preparation and transplantation. (A and B) Cylindrical

mPCL-TCP scaffolds were produced via fused deposition modeling with an outer

diameter of 20 mm, inner diameter of 8 mm, and a height of 30 mm, as shown in

the 3D µCT reconstructions. Scaffold parameters include a porosity of 70%, fully

interconnected pores of dimensions determined by the filament diameter of 300

μm, the filament separation of 1200 μm and the 0/90° lay down pattern. Top

view (A) and lateral view (B). Scale bars, 1 cm. (C) Prior to transplantation, the

scaffolds were surface treated with NaOH to render the scaffolds more

hydrophilic as demonstrated in the scanning electron microscopy images prior

to (inset) and after treatment. (D to G) To load the scaffolds with rhBMP-7, the

lyophilized protein was mixed with 3.5 ml of sterile saline (D, E) and transferred

to the inner duct of the scaffold and onto the contact interfaces between bone

and scaffold (F, G). (H) The constructs were then transplanted into segmental

tibial defects in sheep.

Figure S2. White blood cell infiltrate 12 months after reconstruction with

scaffold/rhBMP-7. Images are representative of n = 1; such an infiltrate was not

observed in the other animals. (A to C) Movat’s Pentachrome staining shows

complete defect bridging. The marked area in (A) is magnified in (B), the marked

area in (B) is magnified in (C). Around the remnants of the collagen carrier (light

yellow, black arrowheads), white blood cells, such as granulocytes (white

arrowheads) and monocytes/macrophages (yellow arrowheads), can be

identified (C).

Figure S3: Histology of bone defects after 3 and 12 months. Representative

histology is shown in Fig. 2. Safranin-Orange/von Kossa–stained histology

sections of the samples under investigation (orientation: top = proximal; left =

medial).

Figure S4: Images of histological sections stained with Safranin-Orange/von

Kossa. Images represent high-resolution version of those shown in Fig. 2 and fig.

S3.

SUPPLEMENTARY MOVIES

Movie 1: ABG, 3 months. Animated three-dimensional µCT reconstruction of a representative defect augmented with ABG 3 months after surgery. Movie 2: ABG, 12 months. Animated three-dimensional µCT reconstruction of a representative defect augmented with ABG 12 months after surgery. Movie 3: Empty, 3 months. Animated three-dimensional µCT reconstruction of a representative empty control defect months after surgery. Movie 4: Scaffold only, 3 months. Animated three-dimensional µCT reconstruction of a representative defect augmented with a mPCL-TCP scaffold 3 months after surgery.

Movie 5: Scaffold only, 12 months. Animated three-dimensional µCT reconstruction of a representative defect augmented with a mPCL-TCP scaffold 12 months after surgery. Movie 6: Scaffold and rhBMP-7, 3 months. Animated three-dimensional µCT reconstruction of a representative defect augmented with scaffold + rhBMP-7 3 months after surgery. Movie 7: Scaffold and rhBMP-7, 12 months. Animated three-dimensional µCT reconstruction of a representative defect augmented with scaffold + rhBMP-7 12 months after surgery.