Multivariate Analysis of Cellular Uptake Characteristics for a (Co)polymer Particle Library.

Controlling cellular responses to nanoparticles so far is predominantly empirical, typically requiring multiple rounds of optimization of particulate carriers. In this study, a systematic model-assisted approach should lead to the identification of key parameters that account for particle properties and their cellular recognition. A copolymer particle library was synthesized by a combinatorial approach in soap free emulsion copolymerization of styrene and methyl methacrylate, leading to a broad compositional as well as constitutional spectrum. The proposed structure-property relationships could be elucidated by multivariate analysis of the obtained experimental data, including physicochemical characteristics such as molar composition, molecular weight, particle diameter, and particle charge as well as the cellular uptake pattern of nanoparticles. It was found that the main contributors for particle size were the polymers' molecular weight and the zeta potential, while particle uptake is mainly directed by the particles' composition. This knowledge and the reported model-assisted procedure to identify relevant parameters affecting particle engulfment of particulate carriers by nonphagocytic and phagocytic cells can be of high relevance for the rational design of pharmaceutical nanocarriers and assessment of biodistribution and nanotoxicity, respectively.

Composition-Dependent Protein-Material Interaction of Poly(Methyl Methacrylate-co-styrene) Nanoparticle Series.

Polymer nanoparticles continue to be of high interest in life science applications. Still, adsorption processes occurring in protein-containing media and their implications for biological responses are not generally predictable. Here, the effect of nanoparticle composition on the adsorption of bovine serum albumin (BSA), fibronectin (FN) and immunoglobulin G (IgG) as structurally and functionally different model proteins was explored by systematically altering the composition of poly(methyl methacrylate-co-styrene) nanoparticles with sizes in a range of about 550 nm. As determined by protein depletion from the suspension medium via a colorimetric assay, BSA and IgG adsorbed at similar quantities, while FN reached larger masses of adsorbed protein (up to 0.4 ± 0.06 µg·cm-2 BSA, 0.42 ± 0.09 µg·cm-2 IgG, 0.72 ± 0.04 µg·cm-2 FN). A higher content of styrene as the more hydrophobic polymer component enhanced protein binding, which suggests a contribution of hydrophobic interactions despite the particles exhibiting strongly negatively charged surfaces with zeta potentials of -44 to -52 mV. The quantities of adsorbed proteins were estimated to correspond to a confluent surface coverage. Overall, this study illustrated how protein binding can be controlled by systematically varying the nanoparticle bulk composition and may serve as a basis for establishing interfaces with a targeted level of protein retention and/or presentation.

22 nm Resolution Achieved by Femtosecond Laser Two-Photon Polymerization of a Hyaluronic Acid Vinyl Ester Hydrogel.

Three-dimensional (3D) bioinspired hydrogels have played an important role in tissue engineering, owing to their advantage of excellent biocompatibility. Here, the two-photon polymerization (TPP) of a 3D hydrogel with high precision has been investigated, using the precursor with hyaluronic acid vinyl ester (HAVE) as the biocompatibility hydrogel monomer, 3,3'-((((1E,1'E)-(2-oxocyclopentane-1,3-diylidene) bis(methanylylidene)) bis(4,1-phenylene)) bis(methylazanediyl))dipropanoate as the water-soluble initiator, and dl-dithiothreitol (DTT) as the click-chemistry cross-linker. The TPP properties of the HAVE precursors have been comprehensively investigated by adjusting the solubility and the formulation of the photoresist. The feature line width of 22 nm has been obtained at a processing laser threshold of 3.67 mW, and the 3D hydrogel scaffold structures have been fabricated. Furthermore, the average value of Young's modulus is 94 kPa for the 3D hydrogel, and cell biocompatibility has been demonstrated. This study would provide high potential for achieving a 3D hydrogel scaffold with highly precise configuration in tissue engineering and biomedicine.

Biodegradable, Self-Reinforcing Vascular Grafts for In Situ Tissue Engineering Approaches.

Clinically available small-diameter synthetic vascular grafts (SDVGs) have unsatisfactory patency rates due to impaired graft healing. Therefore, autologous implants are still the gold standard for small vessel replacement. Bioresorbable SDVGs may be an alternative, but many polymers have inadequate biomechanical properties that lead to graft failure. To overcome these limitations, a new biodegradable SDVG is developed to ensure safe use until adequate new tissue is formed. SDVGs are electrospun using a polymer blend composed of thermoplastic polyurethane (TPU) and a new self-reinforcing TP(U-urea) (TPUU). Biocompatibility is tested in vitro by cell seeding and hemocompatibility tests. In vivo performance is evaluated in rats over a period for up to six months. Autologous rat aortic implants serve as a control group. Scanning electron microscopy, micro-computed tomography (µCT), histology, and gene expression analyses are applied. TPU/TPUU grafts show significant improvement of biomechanical properties after water incubation and exhibit excellent cyto- and hemocompatibility. All grafts remain patent, and biomechanical properties are sufficient despite wall thinning. No inflammation, aneurysms, intimal hyperplasia, or thrombus formation are observed. Evaluation of graft healing shows similar gene expression profiles of TPU/TPUU and autologous conduits. These new biodegradable, self-reinforcing SDVGs may be promising candidates for clinical use in the future.

Synthesis of coumarin-containing poly(2-oxazoline)s and light-induced crosslinking for hydrogel formation.

Herein, we present a new route to synthesize poly(2-oxazoline)s (POx) with coumarin moieties as pendant groups that can be crosslinked by irradiation to form hydrogels. The synthesis of a novel coumarin-containing 2-oxazoline monomer, 2-[(4-methyl-7-coumarinyloxy)methyl]oxazoline is described in four steps and further its subsequent homo- and copolymerization with 2-ethyl-2-oxazoline (EtOx) via cationic ring-opening polymerization (CROP). The received polymers with different coumarin content (8, 4 and 2 mol%) were compared with a known copolymer synthesized via postpolymerization modification of partially hydrolyzed poly(2-ethyl-2-oxazoline) (PEtOx) with a coumarin derivative. The thermoresponsive behavior of the POx in aqueous solutions was investigated, showing a clear difference in the cloud point temperature (T cp ) between the polymers synthesized via different strategies. The hydrogel formation was achieved by irradiation with UV light by the following two methods: on the one hand with the pure coumarin-containing POx and on the other hand mixed with poly(ethylene glycol) diacrylate (PEGDA). The obtained hydrogels were compared regarding their swelling degree and their gel content, whereas the swelling degree in water of prepared hydrogels can be tuned by using POx by varying the coumarin content. Supplementary Information: The online version contains supplementary material available at 10.1007/s00706-022-03013-8.

Synthesis of a Liquid Lignin-Based Methacrylate Resin and Its Application in 3D Printing without Any Reactive Diluents.

3D printing of bio-based and renewable polymers such as lignin has gained research attention during the last few decades. We report on the synthesis and characterization of a liquid lignin-based photopolymer and its application in additive manufacturing (AM). Wheat straw soda lignin is liquified in an oxyalkylation reaction with propylene oxide under alkaline conditions and modified with methacryloyl chloride to obtain a lignin-based methacrylate resin. Ninety percent of the functional hydroxyl groups are grafted during the synthesis. The photopolymerization efficiency was evaluated by real-time-NIR-photorheology experiments with two different photoinitiators, leading to double bond conversions (DBC) of ≥80%. 3D-printing experiments of the methacrylated lignin were performed with the hot lithography technology. For the first time, a light-curable lignin derivative with a lignin content of over 30% was successfully 3D printed via vat photopolymerization without any reactive diluents, which is a significant improvement over current state-of-the-art solutions. This outstanding result is a motivating proof of concept and a promising starting point for the in-depth evaluation of bio-based precursors as an alternative to nonrenewable derivatives for 3D printing.

Guiding cell migration in 3D with high-resolution photografting.

Multi-photon lithography (MPL) has proven to be a suitable tool to precisely control the microenvironment of cells in terms of the biochemical and biophysical properties of the hydrogel matrix. In this work, we present a novel method, based on multi-photon photografting of 4,4'-diazido-2,2'-stilbenedisulfonic acid (DSSA), and its capabilities to induce cell alignment, directional cell migration and endothelial sprouting in a gelatin-based hydrogel matrix. DSSA-photografting allows for the fabrication of complex patterns at a high-resolution and is a biocompatible, universally applicable and straightforward process that is comparably fast. We have demonstrated the preferential orientation of human adipose-derived stem cells (hASCs) in response to a photografted pattern. Co-culture spheroids of hASCs and human umbilical vein endothelial cells (HUVECs) have been utilized to study the directional migration of hASCs into the modified regions. Subsequently, we have highlighted the dependence of endothelial sprouting on the presence of hASCs and demonstrated the potential of photografting to control the direction of the sprouts. MPL-induced DSSA-photografting has been established as a promising method to selectively alter the microenvironment of cells.

A disulfide-based linker for thiol-norbornene conjugation: formation and cleavage of hydrogels by the use of light.

Photolabile groups are the key components of photo-responsive polymers, dynamically tunable materials with multiple applications in materials and life sciences. They usually consist of a chromophore and a labile bond and are inherently light sensitive. An exception are disulfides, simple reversible linkages, which become photocleavable upon addition of a photoinitiator. Despite their practical features, disulfides are rarely utilized due to their impractical formation. Here, we report a disulfide-based linker series bearing norbornene terminals for facile crosslinking of thiol-functionalized macromers via light-triggered thiol-ene conjugation (TEC). Besides finding a highly reactive lead compound, we also identify an unexpected TEC-retardation, strongly dependent on the molecular linker structure and affecting hydrogel stability. Finally, we present a useful method for localized disulfide cleavage by two-photon irradiation permitting micropatterning of disulfide-crosslinked networks.

High-Throughput and Combinatorial Approaches for the Development of Multifunctional Polymers.

High-throughput (HT) development of new multifunctional polymers is accomplished by the combination of different HT tools established in polymer sciences in the last decade. Important advances are robotic/HT synthesis of polymer libraries, the HT characterization of polymers, and the application of spatially resolved polymer library formats, explicitly microarray and gradient libraries. HT polymer synthesis enables the generation of material libraries with combinatorial design motifs. Polymer composition, molecular weight, macromolecular architecture, etc. may be varied in a systematic, fine-graded manner to obtain libraries with high chemical diversity and sufficient compositional resolution as model systems for the screening of these materials for the functions aimed. HT characterization allows a fast assessment of complementary properties, which are employed to decipher quantitative structure-properties relationships. Moreover, these methods facilitate the HT determination of important surface parameters by spatially resolved characterization methods, including time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy. Here current methods for the high-throughput robotic synthesis of multifunctional polymers as well as their characterization are presented and advantages as well as present limitations are discussed.

Gelatin methacryloyl as environment for chondrocytes and cell delivery to superficial cartilage defects.

Cartilage damage typically starts at its surface, either due to wear or trauma. Treatment of these superficial defects is important in preventing degradation and osteoarthritis. Biomaterials currently used for deep cartilage defects lack appropriate properties for this application. Therefore, we investigated photo-crosslinked gelatin methacryloyl (gelMA) as a candidate for treatment of surface defects. It allows for liquid application, filling of surface defects and forming a protective layer after UV-crosslinking, thereby keeping therapeutic cells in place. gelMA and photo-initiator lithium phenyl-2,4,6-trimethyl-benzoylphosphinate (Li-TPO) concentration were optimized for application as a carrier to create a favorable environment for human articular chondrocytes (hAC). Primary hAC were used in passages 3 and 5, encapsulated into two different gelMA concentrations (7.5 wt% (soft) and 10 wt% (stiff)) and cultivated for 3 weeks with TGF-ß3 (0, 1 and 10 ng/mL). Higher TGF-ß3 concentrations induced spherical cell morphology independent of gelMA stiffness, while low TGF-ß3 concentrations only induced rounded morphology in stiff gelMA. Gene expression did not vary across gel stiffnesses. As a functional model gelMA was loaded with two different cell types (hAC and/or human adipose-derived stem cells [ASC/TERT1]) and applied to human osteochondral osteoarthritic plugs. GelMA attached to the cartilage, smoothened the surface and retained cells in place. Resistance against shear forces was tested using a tribometer, simulating normal human gait and revealing maintained cell viability. In conclusion gelMA is a versatile, biocompatible material with good bonding capabilities to cartilage matrix, allowing sealing and smoothening of superficial cartilage defects while simultaneously delivering therapeutic cells for tissue regeneration.

Increasing the Microfabrication Performance of Synthetic Hydrogel Precursors through Molecular Design.

Implementation of hydrogel precursors in two-photon polymerization (2PP) technology provides promising opportunities in the tissue engineering field thanks to their soft characteristics and similarity to extracellular matrix. Most of the hydrogels, however, are prone to post-fabrication deformations, leading to a mismatch between the computer-aided design and the printed structure. In the present work, we have developed novel synthetic hydrogel precursors to overcome the limitations associated with 2PP processing of conventional hydrogel precursors such as post-processing deformations and a narrow processing window. The precursors are based on a poly(ethylene glycol) backbone containing urethane linkers and are, on average, functionalized with six acrylate terminal groups (three on each terminal group). As a benchmark material, we exploited a precursor with an identical backbone and urethane linkers, albeit functionalized with two acrylate groups, that were reported as state-of-the-art. An in-depth characterization of the hexafunctional precursors revealed a reduced swelling ratio (<0.7) and higher stiffness (>36 MPa Young's modulus) compared to their difunctional analogs. The superior physical properties of the newly developed hydrogels lead to 2PP-based fabrication of stable microstructures with excellent shape fidelity at laser scanning speeds up to at least 90 mm s-1, in contrast with the distorted structures of conventional difunctional precursors. The hydrogel films and microscaffolds revealed a good cell interactivity after functionalization of their surface with a gelatin methacrylamide-based coating. The proposed synthesis strategy provides a one-pot and scalable synthesis of hydrogel building blocks that can overcome the current limitations associated with 2PP fabrication of hydrogel microstructures.

Thiol-Ene Cross-linking of Poly(ethylene glycol) within High Internal Phase Emulsions: Degradable Hydrophilic PolyHIPEs for Controlled Drug Release.

Macroporous polymer monoliths prepared from high internal phase emulsions (HIPEs) can be found in various biomedical applications. While typically water-in-oil HIPEs are applied for polyHIPE preparation, they are not suitable for hydrophilic polyHIPE preparation. Herein, direct oil-in-water emulsions based on water-soluble poly(ethylene glycol)diacrylate or poly(ethylene glycol)dimethacrylate were developed. Furthermore, the incorporation of a hydrophilic water-miscible thiol, ethoxylated trimethylolpropane tris(3-mercaptopropionate) (ETTMP) was reported for the first time within thiol-ene polyHIPEs. Due to the transparency of the emulsions, rapid curing via photopolymerization was feasible. The average pore diameters of the resulting polyHIPEs ranged between 1.2 and 3.6 µm, and porosity of up to 90% was achieved. The water uptake of the materials reached up to 1000% by weight. Drug loading and release were demonstrated, employing salicylic acid as a model drug. Porous profile and biodegradability add to the usefulness of the material for biomedical applications.

Polymer Networks for Enrichment of Calcium Ions.

In this study, solvogels containing (2-((2-(ethoxycarbonyl)prop-2-en-1-yl)oxy)-ethyl) phosphonic acid (ECPA) and N,N'-diethyl-1,3-bis-(acrylamido)propane (BNEAA) as the crosslinker are synthesized by UV induced crosslinking photopolymerization in various solvents. The polymerization of the ECPA monomer is monitored by the conversion of double bonds with in situ attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The morphology of the networks is characterized by in situ photorheology, solid state NMR spectroscopy, and scanning electron microscopy (SEM) of the dried gels. It is demonstrated that the storage modulus is not only determined by the crosslinker content in the gel, but also by the solvent used for preparation. The networks turn out to be porous structures with G' being governed by a rigid, phase-separated polymer phase rather than by entropic elasticity. The external and internal pKa values of the poly(ECPA-co-BNEAA) gels were determined by titration with a specially designed method and compared to the calculated values. The polymer-immobilized phosphonic acid groups in the hydrogels induce buffering behavior into the system without using a dissolved buffer. The calcium accumulation in the gels is studied by means of a double diffusion cell filled with calcium ion-containing solutions. The successful accumulation of hydroxyapatite within the gels is shown by a combination of SEM, energy-dispersive X-ray spectroscopy (EDX) and wide-angle X-ray scattering (WAXS).

Differentiation of physical and chemical cross-linking in gelatin methacryloyl hydrogels.

Gelatin methacryloyl (GM) hydrogels have been investigated for almost 20 years, especially for biomedical applications. Recently, strengthening effects of a sequential cross-linking procedure, whereby GM hydrogel precursor solutions are cooled before chemical cross-linking, were reported. It was hypothesized that physical and enhanced chemical cross-linking of the GM hydrogels contribute to the observed strengthening effects. However, a detailed investigation is missing so far. In this contribution, we aimed to reveal the impact of physical and chemical cross-linking on strengthening of sequentially cross-linked GM and gelatin methacryloyl acetyl (GMA) hydrogels. We investigated physical and chemical cross-linking of three different GM(A) derivatives (GM10, GM2A8 and GM2), which provided systematically varied ratios of side-group modifications. GM10 contained the highest methacryloylation degree (DM), reducing its ability to cross-link physically. GM2 had the lowest DM and showed physical cross-linking. The total modification degree, determining the physical cross-linking ability, of GM2A8 was comparable to that of GM10, but the chemical cross-linking ability was comparable to GM2. At first, we measured the double bond conversion (DBC) kinetics during chemical GM(A) cross-linking quantitatively in real-time via near infrared spectroscopy-photorheology and showed that the DBC decreased due to sequential cross-linking. Furthermore, results of circular dichroism spectroscopy and differential scanning calorimetry indicated gelation and conformation changes, which increased storage moduli of all GM(A) hydrogels due to sequential cross-linking. The data suggested that the total cross-link density determines hydrogel stiffness, regardless of the physical or chemical nature of the cross-links.

Single-Molecule Force Spectroscopy Reveals Adhesion-by-Demand in Statherin at the Protein-Hydroxyapatite Interface.

Achieving strong adhesion in wet environments remains a technological challenge in biomedical applications demanding biocompatibility. Attention for adhesive motifs meeting such demands has largely been focused on marine organisms. However, bioadhesion to inorganic surfaces is also present in the human body, in the hard tissues of teeth and bones, and is mediated through serines (S). The specific amino acid sequence DpSpSEEKC has been previously suggested to be responsible for the strong binding abilities of the protein statherin to hydroxyapatite, where pS denotes phosphorylated serine. Notably, similar sequences are present in the non-collagenous bone protein osteopontin (OPN) and the mussel foot protein 5 (Mefp5). OPN has previously been shown to promote fracture toughness and physiological damage formation. Here, we investigated the adhesion strength of the motif D(pS)(pS)EEKC on substrates of hydroxyapatite, TiO2, and mica using atomic force microscopy (AFM) single-molecule force spectroscopy (SMFS). Specifically, we investigated the dependence of adhesion force on phosphorylation of serines by comparing findings with the unphosphorylated variant DSSEEKC. Our results show that high adhesion forces of over 1 nN on hydroxyapatite and on TiO2 are only present for the phosphorylated variant D(pS)(pS)EEKC. This warrants further exploitation of this motif or similar residues in technological applications. Further, the dependence of adhesion force on phosphorylation suggests that biological systems potentially employ an adhesion-by-demand mechanism via expression of enzymes that up- or down-regulate phosphorylation, to increase or decrease adhesion forces, respectively.

Assessment of a long-term in vitro model to characterize the mechanical behavior and macrophage-mediated degradation of a novel, degradable, electrospun poly-urethane vascular graft.

An assessment tool to evaluate the degradation of biodegradable materials in a more physiological environment is still needed. Macrophages are critical players in host response, remodeling and degradation. In this study, a cell culture model using monocyte-derived primary macrophages was established to study the degradation, macro-/micro-mechanical behavior and inflammatory behavior of a new designed, biodegradable thermoplastic polyurethane (TPU) scaffold, over an extended period of time in vitro. For in vivo study, the scaffolds were implanted subcutaneously in a rat model for up to 36 weeks. TPU scaffolds were fabricated via the electrospinning method. This technique provided a fibrous scaffold with an average fiber diameter of 1.39 ± 0.76 µm and an average pore size of 7.5 ± 1.1 µm. The results showed that TPU scaffolds supported the attachment and migration of macrophages throughout the three-dimensional matrix. Scaffold degradation could be detected in localized areas, emphasizing the role of adherent macrophages in scaffold degradation. Weight loss, molecular weight and biomechanical strength reduction were evident in the presence of the primary macrophage cells. TPU favored the switch from initial pro-inflammatory response of macrophages to an anti-inflammatory response over time both in vitro and in vivo. Expression of MMP-2 and MMP-9 (the key enzymes in tissue remodeling based on ECM modifications) was also evident in vitro and in vivo. This study showed that the primary monocyte-derived cell culture model represents a promising tool to characterize the degradation, mechanical behavior as well as biocompatibility of the scaffolds during an extended period of observation.

Thiol-Gelatin-Norbornene Bioink for Laser-Based High-Definition Bioprinting.

Two-photon polymerization (2PP) is a lithography-based 3D printing method allowing the fabrication of 3D structures with sub-micrometer resolution. This work focuses on the characterization of gelatin-norbornene (Gel-NB) bioinks which enables the embedding of cells via 2PP. The high reactivity of the thiol-ene system allows 2PP processing of cell-containing materials at remarkably high scanning speeds (1000 mm s-1 ) placing this technology in the domain of bioprinting. Atomic force microscopy results demonstrate that the indentation moduli of the produced hydrogel constructs can be adjusted in the 0.2-0.7 kPa range by controlling the 2PP processing parameters. Using this approach gradient 3D constructs are produced and the morphology of the embedded cells is observed in the course of 3 weeks. Furthermore, it is possible to tune the enzymatic degradation of the crosslinked bioink by varying the applied laser power. The 3D printed Gel-NB hydrogel constructs show exceptional biocompatibility, supported cell adhesion, and migration. Furthermore, cells maintain their proliferation capacity demonstrated by Ki-67 immunostaining. Moreover, the results demonstrate that direct embedding of cells provides uniform distribution and high cell loading independently of the pore size of the scaffold. The investigated photosensitive bioink enables high-definition bioprinting of well-defined constructs for long-term cell culture studies.

Hard Block Degradable Polycarbonate Urethanes: Promising Biomaterials for Electrospun Vascular Prostheses.

We report biodegradable thermoplastic polyurethanes for soft tissue engineering applications, where frequently used carboxylic acid ester degradation motifs were substituted with carbonate moieties to achieve superior degradation properties. While the use of carbonates in soft blocks has been reported, their use in hard blocks of thermoplastic polyurethanes is unprecedented. Soft blocks consist of poly(hexamethylene carbonate), and hard blocks combine hexamethylene diisocyanate with the newly synthesized cleavable carbonate chain extender bis(3-hydroxypropylene)carbonate (BHPC), mimicking the motif of poly(trimethylene carbonate) with highly regarded degradation properties. Simultaneously, the mechanical benefits of segmented polyurethanes are exploited. A lower hard block concentration in BHPC-based polymers was more suitable for vascular grafts. Nonacidic degradation products and hard block dependent degradation rates were found. Implantation of BHPC-based electrospun degradable vascular prostheses in a small animal model revealed high patency rates and no signs of aneurysm formations. Specific vascular graft remodeling and only minimal signs of inflammatory reactions were observed.

Long Term Evaluation of Nanofibrous, Bioabsorbable Polycarbonate Urethane Grafts for Small Diameter Vessel Replacement in Rodents.

OBJECTIVE: Biodegradable materials for in situ vascular tissue engineering could meet the increasing clinical demand for sufficient synthetic small diameter vascular substitutes in aortocoronary bypass and peripheral vascular surgery. The aim of this study was to design a new degradable thermoplastic polycarbonate urethane (dPCU) with improved biocompatibility and optimal biomechanical properties. Electrospun conduits made from dPCU were evaluated in short and long term follow up and compared with expanded polytetrafluoroethylene (ePTFE) controls. METHODS: Both conduits were investigated prior to implantation to assess their biocompatibility and inflammatory potential via real time polymerase chain reaction using a macrophage culture. dPCU grafts (n = 28) and ePTFE controls (n = 28) were then implanted into the infrarenal abdominal aorta of Sprague-Dawley rats. After seven days, one, six, and 12 months, grafts were analysed by histology and immunohistochemistry (IHC) and assessed biomechanically. RESULTS: Anti-inflammatory signalling was upregulated in dPCU conduits and increased significantly over time in vitro. dPCU and ePTFE grafts offered excellent long and short term patency rates (92.9% in both groups at 12 months) in the rat model without dilatation or aneurysm formation. In comparison to ePTFE, dPCU grafts showed transmural ingrowth of vascular specific cells resulting in a structured neovessel formation around the graft. The graft material was slowly reduced, while the compliance of the neovessel increased over time. CONCLUSION: The newly designed dPCU grafts have the potential to be safely applied for in situ vascular tissue engineering applications. The degradable substitutes showed good in vivo performance and revealed desirable characteristics such as biomechanical stability, non-thrombogenicity, and minimal inflammatory response after long term implantation.