Developing a pro-angiogenic placenta derived amniochorionic scaffold with two exposed basement membranes as substrates for cultivating endothelial cells

Abstract

Decellularized and de-epithelialized placenta membranes have widely been used as scaffolds and grafts in tissue engineering and regenerative medicine. Exceptional pro-angiogenic and biomechanical properties and low immunogenicity have made the amniochorionic membrane a unique substrate which provides an enriched niche for cellular growth. Herein, an optimized combination of enzymatic solutions (based on streptokinase) with mechanical scrapping is used to remove the amniotic epithelium and chorion trophoblastic layer, which resulted in exposing the basement membranes of both sides without their separation and subsequent damages to the in-between spongy layer. Biomechanical and biodegradability properties, endothelial proliferation capacity, and in vivo pro-angiogenic capabilities of the substrate were also evaluated. Histological staining, immunohistochemistry (IHC) staining for collagen IV, and scanning electron microscope demonstrated that the underlying amniotic and chorionic basement membranes remained intact while the epithelial and trophoblastic layers were entirely removed without considerable damage to basement membranes. The biomechanical evaluation showed that the scaffold is suturable. Proliferation assay, real-time polymerase chain reaction for endothelial adhesion molecules, and IHC demonstrated that both side basement membranes could support the growth of endothelial cells without altering endothelial characteristics. The dorsal skinfold chamber animal model indicated that both side basement membranes could promote angiogenesis. This bi-sided substrate with two exposed surfaces for cultivating various cells would have potential applications in the skin, cardiac, vascularized composite allografts, and microvascular tissue engineering.

Figure 1

(a) Histological features of fresh amniochorionic membrane (fACM); (b) histological features of dbACM; (c) macroscopic features of fACM; macroscopic features of dbACM.

Figure 2

Histological features of fACM and dbACM; (a) H&E staining and (b) Mason’s trichrome staining of fACM; (c) IHC staining for collagen type IV in fACM; (d) H&E staining dbACM; (e) Mason’s trichrome staining of dbACM; (f) IHC staining for collagen type IV in dbACM; (black arrow: epithelial cells, black brace: trophoblasts, green arrow: amniotic basement membrane, blue arrow: chorionic basement membrane).

Figure 3

In vitro biodegradation test of fACM, dbACM, and cross-linked dbACM using crude collagenase with a concentration of 0.01%; values are expressed as the mean ± S.E.M (n = 3) results were analyzed using One-Way ANOVA followed by Tukey's post-Hoc test; analysis was conducted using GraphPad Prism version 9.

Figure 4

The results of MTT assays after culturing HUVECs on both amniotic and chorionic sides of dbACM and their comparison to the control group (HUVECs on culture plate); (****P < 0.0001, values are expressed as the mean ± S.E.M (n = 3) results were analyzed using One-Way ANOVA followed by Tukey's post-Hoc test); analysis was conducted using GraphPad Prism version 9.

Figure 5

SEM images of dbACM before and after culturing HUVECs; (a) epithelial cells of amniotic membrane; (b) de-epithelialized amniotic membrane (basement membrane under the epithelial layer); (c) HUVECs on the amniotic side basement membrane of dbACM; (d) trophoblastic layer of chorion; (e) basement membrane under the trophoblastic layer after removing trophoblasts; (f) HUVECs on the chorionic side basement membrane of dbACM.

Figure 6

IHC for vWF after culturing HUVECs on both sides of dbACM; (a) amniotic side basement membrane of dbACM; (b) chorionic side basement membrane of dbACM.

Figure 7

Evaluation of gene expression of CD31 and VE-cadherin in HUVECs cultured on amniotic and chorionic side of dbACM in days 6th and 12th. Values are expressed as the mean ± S.E.M (n = 5) results were analyzed using One-Way ANOVA followed by Tukey's post-Hoc test; Analysis was conducted using GraphPad Prism version 9.

Figure 8

Result of dorsal skinfold chamber after 10 days of culture; (a) dorsal skinfold chamber mounted on rats; (b) dorsal skinfold chamber equipment; (c) skinfold chamber window after 10 days (angiogenesis induction is shown by the borders of dbACM implantation site with arrows); (d) control group (bared skin); (e) amniotic side down dbACM; (f) chorionic side down dbACM; (d’,e’,f’) ImageJ skeletonize picture of the same image as control, amniotic side down dbACM, and chorionic side down dbACM, respectively; result of ImageJ analysis: (g) total length of vessels in regions of interest; (h) number of branches in regions of interest; ****significance (p value < 0.001); values are expressed as the mean ± S.E.M; analysis was conducted using GraphPad Prism version 9; images were analyzed using ImageJ software with the Fiji plugin package (v.1.53c), which is based on ImageJ2 core.