Adequate vascularization in biomaterials is essential for tissue regeneration and repair. Current models do not allow easy analysis of vascularization of implants in vivo, leaving it a highly desirable goal. A tool that allows monitoring of perfusion capacity of such biomaterials noninvasively in a cheap, efficient, and reliable in vivo model would hence add great benefit to research in this field. We established, for the first time, an in vivo magnetic resonance imaging (MRI) method to quantify the perfusion capacity of a model biomaterial, DegraPol(®) foam scaffold, placed on the embryonic avian chorioallantoic membrane (CAM) in ovo. Perfusion capacity was assessed through changes in the longitudinal relaxation rate before and after injection of a paramagnetic MRI contrast agent, Gd-DOTA (Dotarem(®); Guerbet S.A.). Relaxation rate changes were compared in three different regions of the scaffold, that is, at the interface to the CAM, in the middle and on the surface of the scaffold (p<0.05). The highest relaxation rate changes, and hence perfusion capacities, were measured in the interface region where the scaffold was attached to the CAM, whereas the surface of the scaffold showed the lowest relaxation rate changes. A strong positive correlation was obtained between relaxation rate changes and histologically determined vessel density (R(2) = 0.983), which corroborates our MRI findings. As a proof-of-principle, we measured the perfusion capacity in different scaffold materials, silk fibroin either with or without human dental pulp stem cells. For these, three to four times larger perfusion capacities were obtained compared to DegraPol; demonstrating that our method is sensitive to reveal such differences. In summary, we present a novel in vivo method for analyzing the perfusion capacity in three-dimensional-biomaterials grown on the CAM, enabling the determination of the perfusion capacity of a large variety of bioengineered materials.
Recently, a tumor model based on the chorioallantoic membrane (cAM) was characterized structurally with Magnetic Resonance imaging (MRi). Yet, capability of MRi to assess vascular functional reserve and potential of oxygenation-sensitive MRi remain largely unexplored in this model. for this purpose, we compared MC-38 colon and A549 lung adenocarcinoma cell grafts grown on the CAM, using quantitative T1 and T2* MRi readouts as imaging markers. these are associated with vascular functionality and oxygenation status when compared between periods of air and carbogen exposure. our data show that in A549 lung adenocarcinoma cell grafts T2* values increased significantly upon carbogen exposure (p < 0.004, Wilcoxon test; no change in T1), while MC-38 grafts displayed no changes in T1 and T2*), indicating that the grafts differ in their vascular response. Heterogeneity with regard to T1 and T2* distribution within the grafts was noted. MC-38 grafts displayed larger T1 and T2* in the graft centre, while in A549 they were distributed more towards the graft surface. Finally, qualitative assessment of gadolinium-enhancement suggests that A549 grafts display more prominent enhancement compared to MC-38 grafts. Furthermore, MC-38 grafts had 65% larger volumes than A549 grafts. Histology revealed distinct underlying phenotypes of the two tumor grafts, pertaining to the proliferative status (Ki-67) and cellularity (H&E). In sum, a functional gas challenge with carbogen is feasible through gas exchange on the CAM, and it affects MRI signals associated with vascular reactivity and oxygenation status of the tumor graft planted on the CAM. Different grafts based on A549 lung adenocarcinoma and MC-38 colon carcinoma cell lines, respectively, display distinct phenotypes that can be distinguished and characterized non-invasively in ovo using MRi in the living chicken embryo. The chorioallantoic membrane (CAM) of the developing chicken embryo is an established model that is used in biomedical research in a multitude of different applications 1. For instance, it is employed in screening biomaterials 2-4 , testing microsurgical procedures 5 , drug delivery systems and biosensors 6,7 , and in toxicity and pharmacokinetic studies 8,9. Recently, the CAM model was used to asses perfusion capacities of on-planted biomaterials with Magnetic Resonance Imaging (MRI) as a non-destructive imaging readout 10. The CAM serves as a support for the respiratory capillaries outside the embryo. It is highly vascularized and allows for gas exchange between the embryo and its environment. This renders the CAM a suitable model to study angiogenesis 11-14. Notably, as a natural immunodeficient host with a rich vascular network, the CAM is particularly capable to sustain grafted tissues and implants for tissue engineering applications 15. Most importantly, it provides an advantageous environment for tumor formation and is therefore often used to study tumor development, metastasis and progression in xenotransplanted tumors 16. Another advantage is the easy acces...
MRI has recently been presented as a nondestructive in vivo readout to report perfusion capacity in biomaterials planted on the CAM in the living chick embryo in ovo. Perfusion capacity was assessed through changes in T1 relaxation pre-and post-injection of a paramagnetic contrast agent, Gd-DOTA (Dotarem®). Hence local contrast agent concentration was dependent on perfusion, vascular permeability, and extravascular compartment size. In the present study we, therefore, explore intravascular SPIO particles of the FeraSpin® series to deliver a more direct measure of vascularization in a 3D polymer DegraPol® scaffold. Furthermore, we present contrast enhancement upon SPIOs of different particle size, namely FeraSpin® series XS, M, XXL and Endorem® for comparison, and hence different efficiency on T1 and T2, and study respective dose-effects. 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 at the interface to the biomaterial. Furthermore, the effect upon T2 appears stronger than in T1 with all SPIOs investigated and at blood concentrations between 0.46 mM to 4.65 mM. Comparison of different concentrations shows larger T1 enhancement at the highest dose, as expected. Vessel structures in and around the scaffold as seen in MRI were corroborated by histology. Different particle sizes show reduced T1 effect with larger particles, yet the effect on T2 was less apparent. In sum, SPIO-enhanced MRI provides measures for vascularization nondestructively in biomaterials connected to the CAM, based on intravascular contrast enhancement in T1 and T2, in ovo in the living chick embryo. Small SPIOs provide the best efficiency for that purpose, and contrast enhancement is most prominent in T2. Triple Blind Peer Review The handling editor, the reviewers, and the authors are all blinded during the review process. Full Open AccessSupported by the Velux Foundation, the University of Zurich, and the EPFL School of Life Sciences. AbstractMRI has recently been presented as a nondestructive in vivo readout to report perfusion capacity in biomaterials planted on the CAM in the living chick embryo in ovo. Perfusion capacity was assessed through changes in T1 relaxation pre-and post-injection of a paramagnetic contrast agent, Gd-DOTA (Dotarem ® ). Hence local contrast agent concentration was dependent on perfusion, vascular permeability, and extravascular compartment size. In the present study we, therefore, explore intravascular SPIO particles of the FeraSpin ® series to deliver a more direct measure of vascularization in a 3D polymer DegraPol ® scaffold. Furthermore, we present contrast enhancement upon SPIOs of different particle size, namely FeraSpin ® series XS, M, XXL and Endorem ® for comparison, and hence different efficiency on T1 and T2, and study respective doseef...
The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.
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