Supplementary Materials forsections. Images were obtained using an environmental scanning electron...
Transcript of Supplementary Materials forsections. Images were obtained using an environmental scanning electron...
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: [email protected]
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.