Tissue-engineered biomaterials in regenerative medicine provide a matrix for cells to attach and proliferate, stimulate angiogenesis and sustain long-term function and survival of the implant. The chorioallantoic membrane (CAM) of the chick embryo is a model for studying vascularization in vivo and effects of the scaffolds size, pore size and pore interconnectivity on its vascularization can be studied. We recently presented MRI as a nondestructive in vivo readout to report perfusion capacity in biomaterials planted on the CAM in the living chick embryo in ovo [1]. Perfusion capacity was assessed in various scaffold materials through changes in T1 relaxation pre and post injection of a paramagnetic contrast agent, Gd-DOTA (Dotarem®, Guerbet S.A.). Hence local contrast agent concentration was dependent on perfusion, vascular permeability and extravascular compartment size. In the present study we explore intravascular SPIO particles of 30-40 nm size (FeraSpin series M, Viscover™, Miltenyi Biotec, Germany) that stay in the vasculature to deliver a more direct measure of vascularization. Fertilized Lohman white LSL chick eggs were incubated at 37°C and 65% relative humidity. After 3.5 days a hole was excised into the eggshell and on incubation day (ID) 7 Matricel® collagen scaffolds were placed onto the CAM (Fig. 1). MRI was conducted on ID 14 in 2 samples. The chick embryos were sedated with 0.3 mg/kg medetomidine (diluted 1:100, volume 0.3 ml) dripped onto the CAM, and antagonized after completion of the MRI. T1- and T2-weighted MR images were acquired from a sagittal slice through the scaffold with a FOV 55 x 20 mm, spatial resolution 200 x 200 um2, slice thickness 1 mm, total scan time 13 min with a RARE sequence of variable TR and TE for quantitative T1 and T2 mapping (TR 200/400/800/1500/3000/ 4500ms, TE 9.3/27.9/46.5/65.1/83.7 ms, RARE-factor 2) pre and 8 and 140 min post i.v. injection of SPIOs at a dose of 40 umol/kg Fe (blood concentration 0.22 mM) in sample 1 and 80 umol/kg Fe (0.44 mM in blood) in the sample 2. Furthermore, in a ‘dose escalation’ test, sample 2 received a second 80 umol/kg Fe dose 3h after the first dose. In accordance with our previous study MR images were obtained from one sagittal slice positioned through the scaffold on the CAM in two chick embryos in ovo. Our medetomidine anesthesia protocol optimized for this special application [2] offered proper sedation of the chick embryo throughout the MRI acquisition so that MR images devoid of motion artifacts were obtained and the scaffold was clearly and reproducibly depicted in all MRI sessions (Fig. 2). No signal change was observed within the egg yolk, consistent with the SPIO remaining in the vasculature. Consequently, T1 positive signal enhancement (reduction in T1) and T2 negative contrast (reduction in T2) were observed only in the vasculature and hence were restricted mainly to the surface of the CAM (Fig. 2, arrowheads). Effect upon T2 was stronger than in T1. Interestingly, no change in contrast was observed inside the scaffold, which might relate to our observation that the slightly viscous contrast agent did not distribute easily but rather slowly within the finer segments of the vasculature, like the vessels penetrating the scaffold. Consistent with this notion is the observation that T1 as well as T2 reduction was more prominently seen at 140 min than 8 min post injection of the SPIOs. Dose escalation from a second SPIO injection did not result in notable further contrast enhancement, possibly for the same reason.This initial experiment demonstrates that SPIO-enhanced MRI is feasible in and well-tolerated by the chick embryo. For studying vascularization of tissue-engineered scaffolds, however, smaller sized SPIOs (e.g. XS series with 10-20 nm size) may be even better suited for future experiments due to their stronger (positive) T1 effect. With our next experiments we aim to provide measures of vascularization non-destructively based on intravascular contrast in biomaterials connected to the CAM, in ovo on the living chick embryo.