Distinct Effects of Heparin and Interleukin-4 Functionalization on Macrophage Polarization and In Situ Arterial Tissue Regeneration Using Resorbable Supramolecular Vascular Grafts in Rats.

Two of the greatest challenges for successful application of small-diameter in situ tissue-engineered vascular grafts are 1) preventing thrombus formation and 2) harnessing the inflammatory response to the graft to guide functional tissue regeneration. This study evaluates the in vivo performance of electrospun resorbable elastomeric vascular grafts, dual-functionalized with anti-thrombogenic heparin (hep) and anti-inflammatory interleukin 4 (IL-4) using a supramolecular approach. The regenerative capacity of IL-4/hep, hep-only, and bare grafts is investigated as interposition graft in the rat abdominal aorta, with follow-up at key timepoints in the healing cascade (1, 3, 7 days, and 3 months). Routine analyses are augmented with Raman microspectroscopy, in order to acquire the local molecular fingerprints of the resorbing scaffold and developing tissue. Thrombosis is found not to be a confounding factor in any of the groups. Hep-only-functionalized grafts resulted in adverse tissue remodeling, with cases of local intimal hyperplasia. This is negated with the addition of IL-4, which promoted M2 macrophage polarization and more mature neotissue formation. This study shows that with bioactive functionalization, the early inflammatory response can be modulated and affect the composition of neotissue. Nevertheless, variability between graft outcomes is observed within each group, warranting further evaluation in light of clinical translation.

In Situ Remodeling Overrules Bioinspired Scaffold Architecture of Supramolecular Elastomeric Tissue-Engineered Heart Valves.

In situ tissue engineering that uses resorbable synthetic heart valve scaffolds is an affordable and practical approach for heart valve replacement; therefore, it is attractive for clinical use. This study showed no consistent collagen organization in the predefined direction of electrospun scaffolds made from a resorbable supramolecular elastomer with random or circumferentially aligned fibers, after 12 months of implantation in sheep. These unexpected findings and the observed intervalvular variability highlight the need for a mechanistic understanding of the long-term in situ remodeling processes in large animal models to improve predictability of outcome toward robust and safe clinical application.

Multi-component supramolecular fibers with elastomeric properties and controlled drug release.

Supramolecular materials based on hydrogen bonding ureido-pyrimidinones (UPy) are highly versatile substrates for tissue engineering, as they provide a platform in which specific functions can be introduced in a modular fashion by means of components with matching supramolecular motifs. In this work, a core-shell fiber mesh is generated by coaxial electrospinning of a robust elastomeric UPy-poly(hexamethylene carbonate) (UPy-PC) core with a hydrophilic shell of poly(ethylene glycol) (UPy-PEG), which is exploited to confer drug release properties to the load-bearing core. The effect of PEG chain length and supramolecular crosslink density on mechanical properties and drug elution profiles is investigated. Hydrated UPy-PC/UPy-PEG meshes containing 30 mol% of UPy-PEG have a Young's modulus matching that of UPy-PC meshes of approximately 0.5 MPa, and elongation at break of 600%. Drug release experiments with low molecular weight drugs encapsulated in the UPy-PEG shell during electrospinning reveal a combined role of drug and matrix hydrophilicity on the elution profile. Our results indicate that a hydrophobic drug is retained in the UPy-PEG shell for several days with a maximum drug release of 56 ± 8% after 14 days, a highly water soluble drug undergoes burst release within one day, and the UPy-modification of a highly water soluble compound increases its retention in the UPy-PEG shell up to multiple weeks. Taken together, our results indicate that the proposed multi-component system is a drug delivery vehicle of excellent versatility for applications requiring strong and durable materials.

Biomechanical Behaviour and Biocompatibility of Ureidopyrimidinone-Polycarbonate Electrospun and Polypropylene Meshes in a Hernia Repair in Rabbits.

Although mesh use has significantly improved the outcomes of hernia and pelvic organ prolapse repair, long-term recurrence rates remain unacceptably high. We aim to determine the in vivo degradation and functional outcome of reconstructed abdominal wall defects, using slowly degradable electrospun ureidopyrimidinone moieties incorporated into a polycarbonate backbone (UPy-PC) implant compared to an ultra-lightweight polypropylene (PP) textile mesh with high pore stability. Twenty four New-Zealand rabbits were implanted with UPy-PC or PP to either reinforce a primary fascial defect repair or to cover (referred to as gap bridging) a full-thickness abdominal wall defect. Explants were harvested at 30, 90 and 180 days. The primary outcome measure was uniaxial tensiometry. Secondary outcomes were the recurrence of herniation, morphometry for musculofascial tissue characteristics, inflammatory response and neovascularization. PP explants compromised physiological abdominal wall compliance from 90 days onwards and UPy-PC from 180 days. UPy-PC meshes induced a more vigorous inflammatory response than PP at all time points. We observed progressively more signs of muscle atrophy and intramuscular fatty infiltration in the entire explant area for both mesh types. UPy-PC implants are replaced by a connective tissue stiff enough to prevent abdominal wall herniation in two-thirds of the gap-bridged full-thickness abdominal wall defects. However, in one-third there was sub-clinical herniation. The novel electrospun material did slightly better than the textile PP yet outcomes were still suboptimal. Further research should investigate what drives muscular atrophy, and whether novel polymers would eventually generate a physiological neotissue and can prevent failure and/or avoid collateral damage.

Experimental reconstruction of an abdominal wall defect with electrospun polycaprolactone-ureidopyrimidinone mesh conserves compliance yet may have insufficient strength.

PURPOSE: Electrospun meshes mimic the extracellular matrix, which may improve their integration. We aimed to compare polycaprolactone (PCL) modified with ureidopyrimidinone (UPy) electrospun meshes with ultra-lightweight polypropylene (PP; Restorelle) reference textile meshes for in vivo compliance. We chose UPy-PCL because we have shown it does not compromise biomechanical properties of native tissue, and because it potentially can be bioactivated. METHODS: We performed ex vivo biomechanical cyclic loading in wet conditions and in vivo overlay of full-thickness abdominal wall defects in rats and rabbits. Animals were sacrificed at 7, 42 and 54 days (rats; n = 6/group) and 30 and 90 days (rabbits; n = 3/group). Outcomes were herniation, mesh degradation and mesh dimensions, explant compliance and histology. High failure rates prompted us to provide additional material strength by increasing fiber diameter and mesh thickness, which was further tested in rabbits as a biomechanically more challenging model. RESULTS: Compliance was tested in animals without herniation. In both species, UPy-PCL-explants were as compliant as native tissue. In rats, PP-explants were stiffer. Contraction was similar in UPy-PCL and PP-explants. However, UPy-PCL-meshes macroscopically degraded from 30 days onwards, coinciding with herniation in up to half of animals. Increased fiber and mesh thickness did not improve outcome. Degradation of UPy-PCL is associated with an abundance of foreign body giant cells until UPy-PCL disappears. CONCLUSION: Abdominal wall reconstruction with electrospun UPy-PCL meshes failed in 50%. Degradation coincided with a transient vigorous foreign body reaction. Non-failing UPy-PCL-explants were as compliant as native tissue. Despite that, the high failure rate forces us to explore electrospun meshes based on other polymers.

Early in-situ cellularization of a supramolecular vascular graft is modified by synthetic stromal cell-derived factor-1α derived peptides.

In an in-situ approach towards tissue engineered cardiovascular replacement grafts, cell-free scaffolds are implanted that engage in endogenous tissue formation. Bioactive molecules can be incorporated into such grafts to facilitate cellular recruitment. Stromal cell derived factor 1α (SDF1α) is a powerful chemoattractant of lymphocytes, monocytes and progenitor cells and plays an important role in cellular signaling and tissue repair. Short SDF1α-peptides derived from its receptor-activating domain are capable of activating the SDF1α-specific receptor CXCR4. Here, we show that SDF1α-derived peptides can be chemically modified with a supramolecular four-fold hydrogen bonding ureido-pyrimidinone (UPy) moiety, that allows for the convenient incorporation of the UPy-SDF1α-derived peptides into a UPy-modified polymer scaffold. We hypothesized that a UPy-modified material bioactivated with these UPy-SDF1α-derived peptides can retain and stimulate circulating cells in an anti-inflammatory, pro-tissue formation signaling environment. First, the early recruitment of human peripheral blood mononuclear cells to the scaffolds was analyzed in vitro in a custom-made mesofluidic device applying physiological pulsatile fluid flow. Preferential adhesion of lymphocytes with reduced expression of inflammatory factors TNFα, MCP1 and lymphocyte activation marker CD25 was found in the bioactivated scaffolds, indicating a reduction in inflammatory signaling. As a proof of concept, in-vivo implantation of the bioactivated scaffolds as rat abdominal aorta interposition grafts showed increased cellularity by CD68+ cells after 7 days. These results indicate that a completely synthetic, cell-free biomaterial can attract and stimulate specific leukocyte populations through supramolecular incorporation of short bioactive SDF1α derived peptides.

Unusual analyte-matrix adduct ions and mechanism of their formation in MALDI TOF MS of benzene-1,3,5-tricarboxamide and urea compounds.

Analyte-matrix adducts are normally absent under typical matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) conditions. Interestingly, though, in the analysis of several types of organic compounds synthesized in our laboratory, analyte-matrix adduct ion peaks were always recorded when common MALDI matrices such as 4-hydroxy-α-cyanocinnamic acid (CHCA) were used. These compounds are mainly those with a benzene-1,3,5-tricarboxamide (BTA) or urea moiety, which are important building blocks to make new functional supramolecular materials. The possible mechanism of the adduct formation was investigated. A shared feature of the compounds studied is that they can form intermolecular hydrogen bonding with matrices like CHCA. The intermolecular hydrogen bonding will make the association between analyte ions and matrix molecules stronger. As a result, the analyte ions and matrix molecules in MALDI clusters will become more difficult to be separated from each other. Furthermore, it was found that analyte ions were mainly adducted with matrix salts, which is probably due to the much lower volatility of the salts compared with that of their corresponding matrix acids. It seems that the analyte-matrix adduct formation for our compounds are caused by the incomplete evaporation of matrix molecules from the MALDI clusters because of the combined effects of enhanced intermolecular interaction between analyte-matrix and of the low volatility of matrix salts. Based on these findings, strategies to suppress the analyte-matrix adduction are briefly discussed. In return, the positive results of using these strategies support the proposed mechanism of the analyte-matrix adduct formation.

Thioamides: versatile bonds to induce directional and cooperative hydrogen bonding in supramolecular polymers.

The amide bond is a versatile functional group and its directional hydrogen-bonding capabilities are widely applied in, for example, supramolecular chemistry. The potential of the thioamide bond, in contrast, is virtually unexplored as a structuring moiety in hydrogen-bonding-based self-assembling systems. We report herein the synthesis and characterisation of a new self-assembling motif comprising thioamides to induce directional hydrogen bonding. N,N',N''-Trialkylbenzene-1,3,5-tris(carbothioamide)s (thioBTAs) with either achiral or chiral side-chains have been readily obtained by treating their amide-based precursors with P2S5. The thioBTAs showed thermotropic liquid crystalline behaviour and a columnar mesophase was assigned. IR spectroscopy revealed that strong, three-fold, intermolecular hydrogen-bonding interactions stabilise the columnar structures. In apolar alkane solutions, thioBTAs self-assemble into one-dimensional, helical supramolecular polymers stabilised by three-fold hydrogen bonding. Concentration- and temperature-dependent self-assembly studies performed by using a combination of UV and CD spectroscopy demonstrated a cooperative supramolecular polymerisation mechanism and a strong amplification of supramolecular chirality. The high dipole moment of the thioamide bond in combination with the anisotropic shape of the resulting cylindrical aggregate gives rise to sufficiently strong depolarised light scattering to enable depolarised dynamic light scattering (DDLS) experiments in dilute alkane solution. The rotational and translational diffusion coefficients, D(trans) and D(rot), were obtained from the DDLS measurements, and the average length, L, and diameter, d, of the thioBTA aggregates were derived (L = 490 nm and d = 3.6 nm). These measured values are in good agreement with the value L(w) = 755 nm obtained from fitting the temperature-dependent CD data by using a recently developed equilibrium model. This experimental verification validates our common practice for determining the length of BTA-based supramolecular polymers from model fits to experimental CD data. The ability of thioamides to induce cooperative supramolecular polymerisation makes them effective and broadly applicable in supramolecular chemistry.

Supramolecular polymerization in water harnessing both hydrophobic effects and hydrogen bond formation.

The formation of supramolecular polymers in water through rational design of a benzene-1,3,5-tricarboxamide (BTA) motif is presented. Intermolecular hydrogen bonding and hydrophobic effects cooperate in the self-assembly into long fibrillar aggregates. Minimal changes in molecular structure significantly affect the internal packing of the aggregates.

Network Formation in an Orthogonally Self-Assembling System.

Many supramolecular motifs self-assemble into nanorods, forming the basis of the mechanical properties of supramolecular polymers. When integrated as end-caps in a bifunctional telechelic polymer, the motifs can phase segregate into the same or into another nanorod. In the latter case, a functional cross-link is formed by the bridging chain that strengthens the polymer network. This study introduces a supramolecular polymeric system that consists of two different nanorod forming supramolecular motifs. When end-capped to monofunctional polymers, these supramolecular motifs self-assemble in an orthogonal fashion in two separate types of noncross-linked nanorods, resulting in a viscous liquid lacking macroscopic properties. The addition of 15 mol % of an α,ω-telechelic polymer containing both supramolecular motifs, each on one end, transforms this viscous sticky liquid to a solid material with elastomeric properties due to network formation between the two types of nanorods.

Single-chain folding of polymers for catalytic systems in water.

Enzymes are a source of inspiration for chemists attempting to create versatile synthetic catalysts. In order to arrive at a polymeric chain carrying catalytic units separated spatially, it is a prerequisite to fold these polymers in water into well-defined compartmentalized architectures thus creating a catalytic core. Herein, we report the synthesis, physical properties, and catalytic activity of a water-soluble segmented terpolymer in which a helical structure in the apolar core is created around a ruthenium-based catalyst. The supramolecular chirality of this catalytic system is the result of the self-assembly of benzene-1,3,5-tricarboxamide side chains, while the catalyst arises from the sequential ruthenium-catalyzed living radical polymerization of the different monomers followed by ligand exchange. The polymers exhibit a two-state folding process and show transfer hydrogenation in water.

Probing the limits of the majority-rules principle in a dynamic supramolecular polymer.

By systematic variation of the chemical structure of benzene-1,3,5-tricarboxamide (BTA) derivatives, the effect of chemical structure on the amplification of chirality was studied and quantified. In combination with temperature-dependent amplification experiments, the limits of the majority-rules principle were also investigated. For all BTA derivatives a high, constant helix reversal penalty was determined, which is related to the intermolecular hydrogen bonds that are present in all studied derivatives. For asymmetrically substituted BTA derivatives an odd-even effect was found in the degree of chiral amplification when changing the position of the stereogenic center with respect to the amide functionality. It was found that the mismatch penalty could be directly related to the number of stereocenters present in the molecules. Increasing this number from one to three resulted in an increase in this energy penalty while leaving the helix reversal penalty unaffected. For the majority-rules principle this implies that a single stereocenter present in the molecule contains sufficient chiral information at the molecular level to result in a chirally amplified state at the supramolecular level. Further evidence that the mismatch penalty is directly related to the number of stereocenters was obtained from mixed majority-rules experiments where two BTA derivatives with different numbers of stereocenters with opposite stereoconfiguration were studied in a majority-rules experiment. Finally, the ultimate limits of chiral amplification for the majority-rules principle were investigated, revealing that, given a certain helix reversal penalty, there is an optimum to which the mismatch penalty can be reduced while also enhancing the degree of chiral amplification. Temperature-dependent majority-rules experiments could indeed confirm these simulations. These findings show the relevance of both energy penalties when trying to enhance the degree of chiral amplification for the majority-rules principle in a one-dimensional helical supramolecular polymer.

Iterative tandem catalysis of secondary diols and diesters to chiral polyesters.

The well-known dynamic kinetic resolution of secondary alcohols and esters was extended to secondary diols and diesters to afford chiral polyesters. This process is an example of iterative tandem catalysis (ITC), a polymerization method where the concurrent action of two fundamentally different catalysts is required to achieve chain growth. In order to procure chiral polyesters of high enantiomeric excess value (ee) and good molecular weight, the catalysts employed need to be complementary and compatible during the polymerization reaction. We here show that Shvo's catalyst and Novozym 435 fulfil these requirements. The optimal polymerization conditions of 1,1'-(1,3-phenylene) diethanol (1,3-diol) and diisopropyl adipate required 2 mol% Shvo's catalyst and 12 mg Novozym 435 per mmol alcohol group in the presence of 0.5 M 2,4-dimethyl-3-pentanol as the hydrogen donor. With these conditions, chiral polyesters were obtained with peak molecular weights up to 15 kDa, an ee value up to 99% and with 1-3 % ketone end groups. Also with the structural isomer, 1,4-diol, a chiral polyester was obtained, albeit with lower molecular weight (8.3 kDa) and slightly lower ee (94%). Aliphatic secondary diols also resulted in enantio-enriched polymers but at most an ee of 46 % was obtained with molecular weights in the range of 3.3-3.7 kDa. This low ee originates from the intrinsic low enantioselectivity of Novozym 435 for this type of secondary aliphatic diols. The results presented here show that ITC can be applied to procure chiral polyesters with good molecular weight and high ee from optically inactive AA-BB type monomers.