Scaffold material and preparation
DegraPol® is a biocompatible and biodegradable material based on poly-hydroxy-butyrate as a crystalline segment and -caprolactone as a soft segment. Originally, its synthesis aimed at generating a suitable scaffold material for bone tissue engineering, but has recently also been shown to be a beneficial scaffold material for cartilage, nerves and as a growth factor delivery device for tendon regeneration.
DegraPol® foams were kindly provided by ab medica, Italy. An Optimaix-3D™ scaffold (Matricel GmbH, Germany) was used in one egg in a preparatory experiment.
After soaking in cyclohexane (Fluka, puriss.) and freezing at -20°C overnight, foams were cut into equal pieces of about 8×4×3 mm3, dried and sterilized with ethylen oxide before placing onto the CAM.
Fertilized Lowman white LSL chick eggs (Animalco AG Geflügelzucht, Staufen, Switzerland) were incubated at 37°C and 65% relative humidity. On incubation day (ID) 3.5 a circular window was excised into the eggshell after removing 2 mL albumen so that the developing CAM detached from the eggshell. On ID 7, Optimaix-3D™ (in one egg) and DegraPol® foam scaffolds (in 11 eggs) were planted on top of the CAM, one or two scaffolds into each egg, in the middle of 1 cm diameter plastic rings to flatten the CAM surface. Eggs were then incubated until ID 14.
SPIO-enhanced MRI to assess vascularization of the 3D DegraPol® scaffold
On ID 14, vascularization of the scaffold by capillaries of the chick embryo’s chorioallantoic membrane was studied in situ on the CAM (“in ovo”) of the living chicken embryo (“in vivo”) using Magnetic Resonance Imaging (MRI). For the MRI examination, the eggs were placed onto a custom-built sliding bed and enveloped by warm water tubing (37°C) to maintain the temperature of the chick embryo in a physiological range. Chick embryos were sedated with 0.3 mg/kg medetomidine (diluted 1:100, volume 0.3 mL) dripped onto the CAM in 3 doses 30 min prior start of MRI examinations and immediately before pre- and post-contrast-enhanced MRI.
MRI was performed on a 4.7 T/16 cm Bruker PharmaScan small animal scanner (Bruker BioSpin, Ettlingen, Germany), equipped with an actively decoupled two-coil system, consisting of a 72 mm bird cage resonator for excitation and a 20 mm single loop surface coil for reception. The surface coil was fixed onto a Petri dish cover plate that covered the eggshell window directly above the scaffold for optimal sensitivity.
Anatomical reference images were acquired in coronal, transversal and sagittal slice orientations with a routine FLASH sequence. T1- and T2-weighted MR images were then obtained from one sagittal slice placed through the scaffold with a RARE sequence of variable TR and TE for quantitative T1 and T2 mapping with TR 200/400/800/1500/3000/4500 ms, TE 9.3/27.9/46.5/65.1/83.7 ms, field of view 55×22 mm, image matrix 275×100, spatial resolution 200×200 um2, slice thickness 1 mm, RARE-factor 2, 2 averages, total scan time 13 min.
T1 and T2 maps were collected before and after injection of SPIOs. Intravenous injection was performed under a surgical microscope with a 300 uL insulin syringe and 30G needle into medium size vein. Quantitative T1 and T2 maps were computed using ParaVision® 5.1 software package (Bruker BioSpin, Ettlingen, Germany).
Different SPIO preparations and doses of the FeraSpin® series (Viscover, Miltenyi Biotec, Germany) were investigated in the present study, and Endorem® (Guerbet SA, France) was used in 2 eggs for comparison. Different SPIO particle sizes affect contrast efficiency with respect to T1 and T2 relaxation as well as blood circulation time and liver uptake.
In a preparatory experiment tolerance of high-dose iron administration into the chick embryo was tested in one egg planted with a Optimaix-3D™ scaffold and was studied prior and 8 and 140 min post-injection of SPIOs of the FeraSpin® series M (particle size 30–40 nm, 10 mM) a 0.46 mM dose in blood upon injection. In a ‘dose escalation’ test it additionally received a second 0.46 mM Fe dose 3 hrs after the first.
Throughout the paper blood concentrations upon injection are given at the corresponding places. Doses were varied by adaption of injection volume between 50 and 100 uL, and iron concentration in contrast agent preparation by dilution of contrast agent in saline. The following SPIOs were explored in the present study: FeraSpin® XXL (particle size 60–70 nm, 10 mM), FeraSpin® M (particle size 30–40 nm, 10 mM), FeraSpin® XS (particle size 10–20 nm, prepared to a custom-tailored 200 mM Fe molarity by the manufacturer). For comparison, Endorem® (particle size 120–180 nm, 200 mM) was used in 2 eggs at concentrations of 0.46 mM and 4.65 mM.
After completion of the MRI measurements the scaffold-CAM complex was fixed overnight using 4% formalin solution in PBS, then excised, embedded in paraffin, cross-sectioned into 5 µm slices and stained with H&E and Haemalaun Sudan Red.
Analysis of contrast enhancement
Histograms of color-coded quantitative T1 and T2 maps obtained pre- and post-injection of the contrast agent were used to compute contrast enhancement within the scaffold. We analyzed, using a freely downloadable software package (http://arohatgi.info/WebPlotDigitizer/citation.html ) in the blue channel, decrease in T1 and T2 values, respectively, which translates into increased distribution in the blue values with contrast enhancement. Results were then explored with respect to SPIO particle size and SPIO concentration of the contrast agent.