In Vivo Modulation of Angiogenesis and Immune Response on a Collagen Matrix via Extracorporeal Shockwaves

Abstract

The effective management of tissue integration and immunological responses to transplants decisively co-determines the success of soft and hard tissue reconstruction. The aim of this in vivo study was to evaluate the eligibility of extracorporeal shock wave therapy (ESWT) with respect to its ability to modulate angiogenesis and immune response to a collagen matrix (CM) for tissue engineering in the chorioallantoic membrane (CAM) assay, which is performed with fertilized chicken eggs. CM were placed on the CAM on embryonic development day (EDD) 7; at EDD-10, ESWT was conducted at 0.12 mJ/mm2 with 500 impulses each. One and four days later, angiogenesis represented by vascularized area, vessel density, and vessel junctions as well as HIF-1α and VEGF gene expression were evaluated. Furthermore, immune response (iNOS2, MMP-9, and MMP-13 via qPCR) was assessed and compared between ESWT- and non-ESWT-groups. At EDD-14, the vascularized area (+115% vs. +26%) and the increase in vessel junctions (+751% vs. +363%) were significantly higher in the ESWT-group. ESWT significantly increased MMP-9 gene expression at EDD-11 and significantly decreased MMP-13 gene expression at EDD-14 as compared to the controls. Using the CAM assay, an enhanced angiogenesis and neovascularization in CM after ESWT were observed. Furthermore, ESWT could reduce the inflammatory activity after a latency of four days.

Does Platelet-Rich Fibrin Enhance the Early Angiogenetic Potential of Different Bone Substitute Materials? An In Vitro and In Vivo Analysis

“…Furthermore, possible implications for angiogenesis were analyzed via the CAM assay in vivo. The assay has been well-described for this indication and widely used in the literature [ 7 , 20 ]. As a major advantage, this approach is in accordance with the concept of the replacement of experimental animals, the reduction of the total number of experimental animals needed, and refined testing protocols (“3R aspects”: replacement, reduction, refinement [ 30 ]).…”

“…In addition, it can respond to osteogenic stimuli and offers significant potential as an in vivo model for xenograft organ culture [ 31 , 32 ]. Evidence has shown that the assay may also be used to evaluate possible inflammatory processes and is therefore of great interest for future studies in the field of tissue engineering, especially with platelet-derived concentrates [ 20 , 33 ]. Here, microscopically and immune-histochemically, all tested BSMs led to a noteworthy decrease in vessel-formation and branching-points in comparison to PRF alone.…”

“…To evaluate the influence of BSM/PRF on angiogenesis in vivo, a chorioallantoic membrane (CAM) assay was used, as previously described [ 7 , 20 ]. The samples, both native (AKM, APKM, XMK1, and XKM2) and with PRF bio-activated BSMs (AKM+, APKM+, XKM1+, and XKM2+), were assessed and compared to the control of the CAM alone (Ctrl.…”

The Chorioallantoic Membrane Assay in Nanotoxicological Research—An Alternative for In Vivo Experimentation

“…The number of vessel branches as well as vessel size and vascular density are frequently used as parameters for quantification [34,37,40,41,[43][44][45]. A detailed methodological description of the monitoring of microvessel density as well as vessel branches and junctions was recently published by Heimes et al [88]. Histological and immunohistochemical analysis of the CAM can also be used for the determination of vessel density [42].…”

Zinc Oxide Nanoparticles Exhibit Favorable Properties to Promote Tissue Integration of Biomaterials

“…Previous studies already proved that the chicken chorioallantoic membrane assay (CAM assay) is well suited for assessing the biocompatibility of nanomaterials [32], their fate within the organism, and hemocompatibility, as well as potential toxic effects [33]. Furthermore, the CAM is an excellent platform with which to study angiogenesis [34] and the angiogenetic activity of biomaterials [35,36].…”