ASAIO Journal 2015

Tissue Engineering/Biomaterials

Effects of Fabrication on Early Patency and Regeneration of Small Intestinal Submucosa Vascular Grafts Diana M. Sánchez-Palencia,*† Javier Navarro,* Juan C. Araque,‡ Juan B. Umaña,§ Alvaro F. Guerrero,§ Lina M. Quijano,* Rocío D. P. López,§¶ Néstor F. Sandoval,‡ and Juan C. Briceno*†║

Small intestinal submucosa grafts for vascular regeneration have produced variable patency (0–100%) that has been concurrent with variability in fabrication techniques. We hypothesized that 1) preservation (P) or removal (R) of the stratum compactum layer of the intestine and 2) a dehydrated (D) or hydrated (H) state of the graft, affect early patency and tissue regeneration. We combined both parameters through a 22 factorial experimental design into four groups (PD, RD, PH, RH), and compared them in an in vivo early response predictive model (swine, ID 4.5 mm, 7d, n = 4). Patency, thrombogenicity, vascularization, fibroblast infiltration, macrophage polarization profile, endothelialization, and biaxial mechanics were assessed. PD grafts remained patent (4/4) but had scarce vascularization and fibroblast infiltration. RD and RH had extensive vascularization and fibroblast infiltration, however, RD had sustained patency (4/4) and the highest number of regeneration-associated phenotype macrophages (M2), whereas RH had lower patency (3/4) and less M2 macrophages. PH had a modest cellular infiltration, but the lowest patency (2/4) and a dominant adverse macrophage phenotype. Elasticity of R grafts evolved toward that of native carotids (particularly RD), while P grafts kept their initial stiffness. We concluded that fabrication parameters drastically affected early patency and regeneration, with RD providing the best results. ASAIO Journal 2015; 61:596–604.

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RS, regeneration score.

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EARLY VASCULAR REGENERATION WITH SIS

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Figure 2. Explanted SIS grafts with adjacent native vessels after 7 days of implantation in a porcine carotid artery model. Notice the presence of white, shiny areas suggesting the ongoing development of a vascular wall. The PH graft provides an example of an occluded graft, where a thrombus was found in the distal carotid artery (yellow arrow). SIS, small intestinal submucosa.

encircled five rods within the tissue holder. The loops around the rods assured that forces were evenly distributed in the lines and along the sides of the samples. The sides of the

samples were aligned to the longitudinal (X1) and circumferential (X2) directions of the graft. Samples, hooks, and pulleys were immersed in a water bath at 37°C to maintain

Figure 3. Explanted SIS grafts stained with H&E, MT, CCR7 antibody or CD206 antibody (lumen on the top left, 40×). PD grafts had the lowest cellular infiltration, while R grafts showed strong cellular infiltration related to regeneration. Fibrin meshes were observed on the surface of some grafts, with a laminar and nonocclusive character in RD and RH grafts, while occlusive in PH samples (H&E and MT, black arrows). The overall inflammatory reaction comprised mainly the presence of mononuclear cells (H&E, white arrow heads) and only a few giant cells (less than 5) in all the observed samples. Vascularization was fairly observed in all grafts but PD, and in an extensive amount in RH grafts (H&E, black arrow heads). Fibroblasts were also commonly observed in all grafts with the exception of PD (H&E, white arrows). CCR7+ macrophages were observed in a higher amount in PH grafts, whereas the CD206+ phenotype was largely observed in RD grafts (grey arrows). Bar = 100 μm. H&E, hematoxilin and eosin; MT, Masson’s thrichrome.

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Figure 4. Histological scoring according to the criteria listed in Table 1. PD scaffolds had significantly higher scores in thrombogenicity and inflammatory reaction than the RD counterparts, but lower scores in vascularity and fibroblast count when compared with the other groups. Vascularity was lowest in PD scaffolds, higher in RD and PH, and highest in RH. Fibroblast count was similar among RD, PH, and RH groups. Mean ± SEM; *p < 0.05; **p < 0.01; #p < 0.0001 vs. PD, p < 0.05 vs. RD, p < 0.01 vs. RH.

hydration and physiological temperature of all samples during testing. Tests were displacement-controlled up to a maximum motor displacement of 6.5 mm. Loads were monitored with two load cells (1 mN/0.1 g resolution) and strains were digitally calculated from the displacement of five black markers affixed to the surface of the sample. Data was transformed into stresses and stretches (stretch = (final length)/ (initial length)) for analysis. An anisotropy ratio (AR) defined as (X2 maximum stretch)/(X1 maximum stretch)24 was also calculated to assess changes in the preferential fiber orientations over time.

Results Patency Outcome and Macroscopic Appearance Assessment of outflow through the explanted samples indicated patency rates of 100% (4/4) for PD grafts, 100% (4/4) for RD grafts, 50% (2/4) for PH grafts, and 75% (3/4) for RH grafts. Patent grafts had white, glistening areas distributed throughout the luminal surface (Figure 2, PD, RD, and RH). Occluded specimens had dark and stiff thrombi present mainly at the distal anastomoses (Figure 2, PH). Macroscopically, PD grafts sharply kept their tubular shape but were stiff and fragile. One PD specimen had laminar (nonocclusive) thrombi close to the anastomoses and along the length of the graft. RD samples had a soft feeling. A laminar (non occlusive) thrombus was observed in one RD specimen and a slight stenosis at the distal anastomoses was observed in two specimens. The two patent PH grafts had a minor stenosis and one of the occluded specimens was surrounded by inflammatory exudate. One of the patent RH grafts had a minor stenosis caused by tissue ingrowth and a laminar thrombus.

Statistical Analysis Regeneration scores and macrophage phenotype quantitative data are reported as mean ± standard error of the mean. Statistically significant differences were determined with two-way ANOVA and Tukey’s test (p value < 0.05). Differences between parameters (i.e., P vs. R and H vs. D) were analyzed with Student’s t-test. All analyses were performed in GraphPad Prism 6.

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Figure 5. A: Cell count in a 40× magnification field of CCR7+ (M1 phenotype) and CD206+ (M2 phenotype) cells. PH grafts had a higher amount of CCR7+ macrophages over PD and RD grafts. RD grafts had a higher count of CD206+ cells compared with PD. B: CD206+/CCR7+ cells ratio (log scale). Dehydrated scaffolds (PD and RD) had a higher ratio (i.e., stronger M2 phenotype dominance) over the hydrated scaffolds. Note that the ratio value for the PH group was below one. Mean ± SEM; *p < 0.05; #p < 0.0001 for PD vs. PH and RD vs. PH, p < 0.05 for PD vs. RH, p = 0.0002 for RD vs. RH.

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tissue surrounding PD grafts (white arrow heads in Figure 3). PD scaffolds had the best outcome for polymorphonucleocytes, which were predominantly present close to the distal anastomoses. Very few giant cells were found in the observed fields (seven in total across all groups, five in one RD specimen, and two in one RH specimen). Vascularization (black arrow heads in Figure 3) was lowest in PD (RS 1), higher in RD and PH (RS 2), and highest in RH (RS 3). Fibroblast population was lowest in PD (RS 1) and had an RS 3 in all the other groups (white arrows in Figure 3). Capillaries and fibroblasts were observed distributed throughout RD, PH and RH scaffolds. Immunohistochemical Assessment PD scaffolds had a scarce macrophage infiltration both of CCR7+ (M1) and CD206+ (M2) phenotypes (Figure 3, gray arrows, and Figure 5A). CCR7+ macrophages were found in a similar amount in the grafts with a removed dense collagen layer (RD and RH groups, Figure 5A), while PH scaffolds had the highest count of this phenotype. The CD206+ count was highest in RD scaffolds and similar between hydrated scaffolds (PH and RH). PD, RD, and RH scaffolds had an M2 dominant profile, whereas PH had a M1-dominant profile, as indicated by the CD206+/CCR7+ ratio (Figure 5B). The M2 predominance was significantly stronger in D scaffolds than in H scaffolds. Endothelial cells were found on the luminal surface close to the anastomosis in two RD, one PH, and one RH specimen (­Figure 6). No ECs were found in PD specimens. FVIII staining was also found around vasa vasorum and capillaries within the regenerated vascular wall. Biaxial Mechanical Properties

Figure 6. FVIII+ cells in RD (A), RH (B), and PH (C) explanted SIS grafts. Bar = 100 μm. SIS, small intestinal submucosa.

Histological Assessment and Quantification of the Regeneration Overall, the early host response comprised a generally populous infiltration of inflammatory cells and fibroblasts inside the scaffold and tissue growth on the abluminal periphery of all the grafts (Figure 3). Differences in RS between groups were more marked in vascularization and fibroblast population of the scaffold (Figure 4). Laminar thrombi composed of a fibrin mesh, sometimes with entrapped red blood cells, were found covering the luminal surface of a large proportion of the specimens (black arrows in Figure 3), as quantified by the thrombus length criteria (RS 2, Figure 4). Nonetheless, the short thrombi diameters proved their mainly nonocclusive character, especially in PD scaffolds which had the best outcome in thrombogenicity related to early-failure. Overall, the inflammatory response to all the scaffolds was given an RS of 3. Monocytes were observed inside RD, PH, and RH scaffolds and in the

The biaxial mechanical properties of the four SIS materials before implantation (Figure 7A) were previously obtained24 and were used here to analyze the mechanical evolution of the grafts after early in vivo implantation. Before implantation, only the X2 data of PH and RH samples was beyond a 1.024 mm/mm stretch. Grafts in the same hydration state had a similar mechanical biaxial behavior, with H grafts more elastic than D grafts, and X1 was the preferential direction in all scaffolds (AR > 1). After implantation, P grafts were found to be stiffer than R grafts (Figure 7B) and there were no clear differences between the elasticity of H and D groups. PD samples had the stiffest behavior and RD grafts were the most elastic. R grafts were found to have maximal stress-stretch values outside the initial stretch range (shaded areas in Figure 7). The elastic behavior of RD samples was the closest to that of NCA. Anisotropy remained the same in RD grafts, had a marked increase in RH grafts, and, surprisingly, became inverted in P grafts: the behavior in the X1 direction after the 7d period evolved into that for the initial X2 direction and vice versa, making the X2 direction preferential in P scaffolds after implantation and changing their AR to a

Effects of Fabrication on Early Patency and Regeneration of Small Intestinal Submucosa Vascular Grafts.

Small intestinal submucosa grafts for vascular regeneration have produced variable patency (0-100%) that has been concurrent with variability in fabri...
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