The Regenerative Capacity of Tissue-Engineered Amniotic Membranes.

Scaffolds can be introduced as a source of tissue in reconstructive surgery and can help to improve wound healing. Amniotic membranes (AMs) as scaffolds for tissue engineering have emerged as promising biomaterials for surgical reconstruction due to their regenerative capacity, biocompatibility, gradual degradability, and availability. They also promote fetal-like scarless healing and provide a bioactive matrix that stimulates cell adhesion, migration, and proliferation. The aim of this study was to create a tissue-engineered AM-based implant for the repair of vesicovaginal fistula (VVF), a defect between the bladder and vagina caused by prolonged obstructed labor. Layers of AMs (with or without cross-linking) and electrospun poly-4-hydroxybutyrate (P4HB) (a synthetic, degradable polymer) scaffold were joined together by fibrin glue to produce a multilayer scaffold. Human vaginal fibroblasts were seeded on the different constructs and cultured for 28 days. Cell proliferation, cell morphology, collagen deposition, and metabolism measured by matrix metalloproteinase (MMP) activity were evaluated. Vaginal fibroblasts proliferated and were metabolically active on the different constructs, producing a distributed layer of collagen and proMMP-2. Cell proliferation and the amount of produced collagen were similar across different groups, indicating that the different AM-based constructs support vaginal fibroblast function. Cell morphology and collagen images showed slightly better alignment and organization on the un-cross-linked constructs compared to the cross-linked constructs. It was concluded that the regenerative capacity of AM does not seem to be affected by mechanical reinforcement with cross-linking or the addition of P4HB and fibrin glue. An AM-based implant for surgical repair of internal organs requiring load-bearing functionality can be directly translated to other types of surgical reconstruction of internal organs.

High heparin content surface-modified polyurethane discs promote rapid and stable angiogenesis in full thickness skin defects through VEGF immobilization.

Three-dimensional scaffolds have the capacity to serve as an architectural framework to guide and promote tissue regeneration. Parameters such as the type of material, growth factors, and pore dimensions are therefore critical in the scaffold's success. In this study, heparin has been covalently bound to the surface of macroporous polyurethane (PU) discs via two different loading methods to determine if the amount of heparin content had an influence on the therapeutic affinity loading and release of (VEGF165 ) in full thickness skin defects. PU discs (5.4 mm diameter, 300 µm thickness, and interconnected pore size of 150 µm) were produced with either a low (2.5 mg/g) or high (6.6 mg/g) heparin content (LC and HC respectively), and were implanted into the modified dorsal skin chamber (MDSC) of C57BL/6 J mice with and without VEGF. Both low- and high-content discs with immobilized VEGF165 (LCV and HCV, respectively) presented accelerated neovascularization and tissue repair in comparison to heparin discs alone. However, the highest angiogenetic peak was on day 7 with subsequent stabilization for HCV, whereas other groups displayed a delayed peak on day 14. We therefore attribute the superior performance of HCV due to its ability to hold more VEGF165, based on its increased heparin surface coverage, as also demonstrated in VEGF elution dynamics. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2543-2550, 2017.

Improved vascularization of porous scaffolds through growth factor delivery from heparinized polyethylene glycol hydrogels.

Surface modification with heparin has previously been shown to increase vascularization of porous scaffolds. In order to determine its efficacy with sustained release, heparin (Hep) was covalently incorporated into degradable (Type D) and non-degradable (Type N) polyethylene glycol (PEG) hydrogels. After in vitro characterization of their physicochemical properties, growth factor (GF) loaded, heparinised Type D gels were formed within the pores of porous polyurethane disks, which were then implanted and evaluated in a subcutaneous model. Type N gels formed faster (3.1±0.1 vs. 7.2±0.2min), were stiffer (10.0±0.5kPa vs. 7.1±1.2kPa) and more stable than degradable gels (>6month stability vs. disintegration ⩽22d in vitro; all p<0.001). Sustained release of covalently incorporated (CI) heparin from Type N (56days; first order kinetics) and Type D (21days; zero order kinetics) was achieved, as opposed to non-covalently incorporated (NI) heparin that eluted in a burst release within the first 2days. While Type D gels initially impeded tissue ingrowth into the porous scaffolds, they were completely degraded and replaced by ingrown tissue after 28days in vivo. At the latter timepoint disks containing gels without Hep or with non-covalently incorporated Hep were less vascularized than empty (no gel) controls. In contrast, the incorporation of covalently heparinized (no GF) and GF containing gels (no Hep) resulted in a 50% and 42% (p<0.05) improvement in vascularization, while an increase of 119% (p<0.001) was achieved with a combination of covalently attached Hep and GF. These gels thus provide a sustained release system for heparin and GF that extends the duration of their action to local tissue ingrowth. STATEMENT OF SIGNIFICANCE: The paper describes the modification and covalent incorporation of heparin into degradable and non-degradable polyethylene glycol hydrogels in a way that provides for the hydrolytic cleavage of the linker for the release of the heparin in original and active form, and in an extended (21-56d) controlled (zero and first order respectively) manner. The successful use of these gels as growth-factor containing and releasing matrices for the improvement of in vivo vascularization holds promise for many potential uses in tissue engineering and regenerative medicine applications, such as vascular grafts and myocardial infarction therapy, where the antithrombotic and/or growth factor binding/potentiating properties are required.

Dual electrospinning with sacrificial fibers for engineered porosity and enhancement of tissue ingrowth.

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 (Ey ) of the constructs increased linearly with increasing PU fiber fraction (UTS: r2 = 0.97, p < 0.0001, Ey : r2 = 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.

Covalent incorporation and controlled release of active dexamethasone from injectable polyethylene glycol hydrogels.

Dexamethasone (Dex) is used in a wide range of applications, but may have undesirable systemic side effects. A number of techniques have thus been developed to deliver the substance locally. In this study, dexamethasone was acrylated, pegylated, and tethered to hydrolytically degradable (acrylate based) and nondegradable (vinyl sulfone based) polyethylene glycol hydrogels by nucleophilic addition. Hydrogel swelling, drug elution and drug activity were followed over an extended period in vitro. Nondegradable gels were stable for more than a year, while degradable gels showed increasing swelling ratios due to degradation that resulted in disintegration after ~12 days. Near-linear (zero order) release could be achieved in some cases with the degradable gels, while release from the nondegradable gels approximated first order initial release kinetics. Significantly delayed release was observed in all cases where the Dex was linked to the gels, when compared with controls where the drug was merely physically incorporated. Eluates from the gels containing the tethered drug showed high levels of activity for extended time periods, while the activity of the eluates from gels containing nonbound dexamethasone decreased rapidly within the first few days. Dexamethasone can thus be incorporated using nucleophilic addition chemistry to produce gels that are capable of sustained release of the active drug. The methodology is applicable to a variety of drugs that contain hydroxyl groups.

Covalent surface heparinization potentiates porous polyurethane scaffold vascularization.

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.

The effects of cross-link density and chemistry on the calcification potential of diamine-extended glutaraldehyde-fixed bioprosthetic heart-valve materials.

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.

Bioprosthetic tissue preservation by filling with a poly(acrylamide) hydrogel.

Glutaraldehyde (GA) fixation has been used for more than 40 years as the preferred treatment to suppress immunogenicity and increase durability of bioprosthetic tissues (BPT) used in heart valve prostheses. This fixative and its reaction products have, however, been implicated in the calcific degeneration and long-term failure of these devices. The current study investigates stabilization of BPT and the mitigation/prevention of calcification by filling aortic wall samples with a synthetic poly(acrylamide) (pAAm) hydrogel, with and without pre-treatment with GA. Histological and gravimetric analysis showed full penetration of the acrylamide (AAm) into the fresh tissue, while only partial filling could be achieved with GA pre-fixed tissue. The observed decrease in amino-group content (0.157+/-0.012-0.123+/-0.021 micromol/mg, p<0.03) and corresponding increase in shrinkage temperature (67.2+/-0.8-78.1+/-1.8 degrees C, p<0.0001) when fresh tissue was filled, indicate the participation of tissue-amines in a process that leads to BPT crosslinking. These effects were much less pronounced when the tissue was pre-fixed with GA. Filling increased the tensile stiffness of fresh tissue (to levels half that of 0.2% GA fixed tissue), but decreased the stiffness of GA pre-fixed tissue. When compared to standard 0.2% GA fixed samples, fresh tissue filled with AAm showed 88% (p<0.0001) less calcification while exhibiting similar resistance toward degradation by protease. Filling did not result in significant decreases in calcification when the tissue was pre-fixed with GA.