Porous scaffolds play an integral role in many tissue-engineering approaches, and the ability to improve vascularization, without eliciting an excessive inflammatory response, would constitute an important step towards achieving long-term healing and function of devices made from these materials. After having previously optimized the dimensional requirements of the well-defined pores, the present study aimed at a further shift from inflammation to vascularization via surface immobilization with heparin. Porous polyurethane disks were produced to contain well-defined pores (147 +/- 2 microm) with abundant interconnecting windows (67 +/- 2 microm). After heparinization via copolymer grafting and amination to contain 32 microg of heparin, the modification appeared as a uniform layer on all exposed surfaces, with no signs of pore obliteration or significant changes in pore size. After 28 days implantation in a rat subcutaneous model, the scaffolds were assessed for vascularization/arteriolization and inflammation using CD31/actin and ED-1 staining, respectively. Heparinization resulted in a significant increase in vascularization: capillaries increased by 62% in number (66.2 +/- 0.8 to 107.3 +/- 1.4 vessels/mm( 2); p < 0.03) and 56% in total area (0.9 +/- 0.1 to 1.4 +/- 0.02%; p<0.02). Arteriolization also increased in absolute terms (200% in number; p<0.03), but did not change significantly when normalized to capillary number. Heparinization did not significantly affect the inflammatory response at this time-point, as quantified by ED-1 positive macrophage and foreign body giant cell (FBGC) content. Thus, the in vivo vascularization of porous scaffolds could be increased without concomitant increase in the inflammatory response by employing a simple surface modification technique. This could be a valuable tool for in vivo tissue engineering applications where enhanced vascularization is required.

Porosity, pore size and pore interconnectivity are critical factors for cellular infiltration into electrospun scaffolds. This study utilized dual electrospinning with sacrificial fiber extraction to produce scaffolds with engineered porosity and mechanical properties. Subsequently, scaffolds were covalently grafted with heparin, a known anti-coagulant with growth-factor binding properties. We hypothesized that the tissue ingrowth would correlate positively with the porosity of the scaffolds. Pellethane® (PU) was spun simultaneously with poly(ethylene oxide) (PEO, subsequently extracted). Low, medium and high porosity scaffolds and heparinized versions of each were characterized and implanted in vivo for evaluation of cellular infiltration and inflammation subcutaneously in male Wistar rats (7,14 and 28 days, n = 6). Average pore-size for low (76 ± 0.2%), medium (83 ± 0.5%) and high (90 ± 1.0%) porosity scaffolds was 4.0 ± 2.3 µm, 9.9 ± 4.2 µm and 11.1 ± 5.5 µm (p < 0.0001). Heparinization resulted in increased fiber diameter (3.6 ± 1.1 µm vs. 1.8 ± 0.8 µm, p < 0.0001) but influenced neither pore-size (p = 0.67) nor porosity (p = 0.27). Cellular infiltration for low, medium and high porosity scaffolds reached 33 ± 7%, 77 ± 20% and 98 ± 1% of scaffold width, respectively, by day 28 of implantation (p < 0001); heparinization did not affect infiltration (p = 0.89). The ultimate tensile strength (UTS) and Young's modulus (E ) of the constructs increased linearly with increasing PU fiber fraction (UTS: r = 0.97, p < 0.0001, E : r = 0.76, p < 0.0001) and heparinization resulted in decreased strength but increased stiffness compared to non-heparinized scaffolds. Increased PEO to PU fraction in the scaffold resulted in predictable losses to mechanical strength and improvements to cellular infiltration, which could make PEO to PU fraction a useful optimization parameter for small diameter vascular grafts. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1559-1572, 2017.

Despite indications that GA (glutaraldehyde)-crosslinked tissues remain prone to long-term degradation and calcification, it is still the reagent of choice in the fixation of bioprosthetic heart valves. We have shown previously that increased GA concentrations and diamine extension of cross-links with lysine incorporation lead to mitigated in vivo calcification, mainly of porcine aortic-wall tissue. The present study was performed to assess the correlation between the cross-link density of all three commonly used tissue types [PW (porcine aortic wall), PL (porcine aortic leaflet) and BP (bovine pericardium)] and tissue calcification in the subcutaneous rat model after GA treatment with or without lysine. The effect of lysine enhancement, and increased GA concentration in the presence of lysine, resulted in significant increases in tissue cross-linking in all three tissue types. Although increased GA concentration on its own resulted in decreased calcification without an increase in cross-link density, overall positive correlations were found between denaturation temperature and RPD (resistance towards protease degradation) [correlation coefficient (rho) values: rhoPW =0.922, rhoPL =0.783 and rhoBP =0.955], whereas negative correlations existed between RPD and calcification (rhoPW=-0.836, rhoPL=-0.929 and rhoBP=-0.579). The combination of lysine enhancement and an increase in GA concentration from 0.2 to 3% resulted in 79, 44 and 56% decreases in calcification in PW, PL and BP. In the case of BP, a decrease in calcification of 81% could be achieved merely by adding lysine extension to low-concentration (0.2 %) GA cross-linking. Thus it is concluded that the increase in cross-link density achieved by lysine incorporation, and by increased GA concentration in the presence of lysine, results in significant and marked decreases in calcification of all three types of tissues commonly used in bioprosthetic heart valves.

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