Engineered microvessels with strong alignment and high lumen density via cell-induced fibrin gel compaction and interstitial flow

Abstract

The development of engineered microvessels with clinically relevant characteristics is a critical step toward the creation of engineered myocardium. Alignment is one such characteristic that must be achieved, as it both mimics native capillary beds and provides natural inlet and outlet sides for perfusion. A second characteristic that is currently deficient is cross-sectional lumen density, typically under 100 lumens/mm²; the equivalent value for human myocardium is 2000 lumens/mm². Therefore, this study examined the effects of gel compaction and interstitial flow on microvessel alignment and lumen density. Strong microvessel alignment was achieved via mechanically constrained cell-induced fibrin gel compaction following vasculogenesis, and high lumen density (650 lumens/mm²) was achieved by the subsequent application of low levels of interstitial flow. Low interstitial flow also conferred microvessel barrier function.

FIG. 1.

(A–D) Schematics of interstitial flow chamber design. (A) The chamber bottom snapped into the well for casting, at which point the well measured 20 mm (l)×4.8 mm (w)×4 mm (h). Flared glass capillary tubes (1 mm outer diameter, 0.58 mm inner diameter) were inserted into the small holes in the sides of the well. The chamber top was not used for casting. During culture, the chamber bottom was removed to allow nutrient transport from either side. The chamber bottom and top were used to enclose the gel during interstitial flow. The pieces were tightened together using a screw at each corner. (B) Top view schematic of a construct at casting. (C) Side view schematic of a construct at casting. The gel (pink) filled the entire well and covered the glass capillary tubes. (D) Top view schematic of a construct after compaction, prior to embedding with agarose gel. The construct was only adherent to the glass capillary tubes. (E) A compacted construct after 8 days of culture. Scale bar=5 mm. Color images available online at www.liebertpub.com/tea

FIG. 2.

(A, B) Representative alignment maps of uncompacted (day 5; A) and compacted (day 8; B) constructs, in which the red lines indicate the local direction and strength of alignment. Scale bars=1 mm. The black lines in (B) indicate the approximate locations of inlet, middle, and outlet cross sections. (C–J) Representative longitudinal sections stained for CD31 (red) of control and flow constructs. Pericytes (PCs) were green fluorescent protein (GFP)-labeled (green), and nuclei were stained with Hoechst 33342 (blue). The axial direction is vertical. The arrows in (C, D) indicate some of the microvessels with lumens. Scale bars=100 μm. (K) Birefringence (a measure of fibril alignment), quantified from polarimetry, increased with compaction, but did not vary between the static and flow conditions, suggesting that flow did not have an effect on fibril alignment. (L) Microvessel anisotropy index, a measure of microvessel alignment obtained from images, also increased with compaction but did not vary between the static and flow conditions. ^$p<0.05 in comparison to day 5 control. ⁺p<0.05 in comparison to day 8 control. Color images available online at www.liebertpub.com/tea

FIG. 3.

(A–H) Representative cross sections stained for CD31 (red) from the middle region of constructs from all days and flow conditions studied. PCs were GFP-labeled (green), and nuclei were stained with Hoechst 33342 (blue). Scale bars=50 μm. (I–N) Quantification of images. (I) The fraction of the section stained positively for CD31 remained constant across all compacted conditions. (J) The number of lumens per square millimeter was increased by low flow relative to time-matched controls but not by high flow. (K) The mean diameter of lumens decreased with compaction but was independent of day or flow condition. (L) The cell number per square millimeter, based on Hoechst 33342 staining, was somewhat variable between conditions, but no trends emerged. (M) The number of PCs per square millimeter was reduced with exposure to either flow rate. (N) The fraction of PCs that were recruited to CD31+ microvessels was the same across all conditions. *p<0.05. ^$p<0.05 in comparison with day 5 control. ⁺p<0.05 in comparison with day 8 control. Color images available online at www.liebertpub.com/tea

FIG. 4.

(A–H) Representative cross sections from the middle region of constructs in all day and flow conditions studied, stained for laminin (red). PCs were GFP-labeled (green), and nuclei were stained with Hoechst 33342 (blue). Scale bars=50 μm. Collagen IV staining was similar and therefore is not shown. (I, J) Quantification of the total staining intensity per nonlumen area for laminin (I) and collagen IV (J). The increased staining in control constructs appeared to be due to additional protein in the interstitial space rather than the perivascular region. *p<0.05 for main effect. Color images available online at www.liebertpub.com/tea

FIG. 5.

Quantification of CD31 stained cross sections from various tissue regions. (A) At day 11, lumen density was increased in the middle region over the inlet and outlet regions. However, even when all of the data were combined, the increase in lumen density with low flow remained. (B) Average lumen diameter at day 11 did not vary by tissue region, but was larger in constructs exposed to low flow. (C) No differences in PC recruitment occurred between regions or flow conditions at day 11. (D) At days 5 and 8, no differences in lumen density occurred across tissue regions. *p<0.05 for main effects. ^$p<0.05 for main effects between control and low flow. ⁺p<0.05 for main effects between low and high flow.

FIG. 6.

(A, B) Representative images of sections of control (A) and low flow (B) constructs from day 14 stained for ephrinB2 (red). PCs are green and nuclei are blue. Scale bars=50 μm. (C) Quantification of blood outgrowth endothelial cell ephrinB2 staining, normalized to values of CD31 fraction obtained from staining of nearby sections (data shown in Fig. 3I). *p<0.05 for main effects. ^$p<0.05 in comparison with drugs control. Color images available online at www.liebertpub.com/tea

FIG. 7.

Endothelial barrier function assay. (A–C) Representative images of lumens within control constructs at days 8 (A) and 11 (B), and constructs exposed to low flow for 3 days (from day 8 to 11; C). Scale bar=10 μm. (D–F) Enlarged portions of the lumens showing in (A–C), for easier visualization of the gold nanoparticles (GNP). (G) Quantification of the GNP present within the lumens (mean±SEM); low levels of GNP within lumens of constructs exposed to low flow indicated strong EC barrier function. *p<0.05. Color images available online at www.liebertpub.com/tea