Impact of Bioactive Peptide Motifs on Molecular Structure, Charging, and Nonfouling Properties of Poly(ethylene oxide) Brushes.

Polymer layers capable of suppressing protein adsorption from biological media while presenting extracellular matrix-derived peptide motifs offer valuable new options for biomimetic surface engineering. Herein, we provide detailed insights into physicochemical changes induced in a nonfouling poly(ethylene oxide) (PEO) brush/polydopamine (PDA) system by incorporation of adhesion ligand (RGD) peptides. Brushes with high surface chain densities (σ ≥ 0.5 chains·nm-2) and pronounced hydrophilicity (water contact angles ≤ 10°) were prepared by end-tethering of heterobifunctional PEOs ( Mn ≈ 20 000 g·mol-1) to PDA-modified surfaces from a reactive melt. Using alkyne distal end group on the PEO chains, azidopentanoic-bearing peptides were coupled through a copper-catalyzed Huisgen azide-alkyne "click" cycloaddition reaction. The surface concentration of RGD was tuned from complete saturation of the PEO surface with peptides (1.7 × 105 fmol·cm-2) to values which may induce distinct differences in cell adhesion (<6.0 × 102 fmol·cm-2). Infrared reflection-absorption and X-ray photoelectron spectroscopies proved the PDA-PEO layers covalent structure and the immobilization of RGD peptides. The complete reconstruction of experimental electrohydrodynamics data utilizing mean-field theory predictions further verified the attained brush structure of the end-tethered PEO chains which provided hydrodynamic screening of the PDA anchor. Increasing the surface concentration of immobilized RGD peptides led to increased interfacial charging. Supported by simulations, this observation was attributed to the ionization of functional groups in the amino acid sequence and to the pH-dependent adsorption of water ions (OH- > H3O+) from the electrolyte. Despite the distinct differences observed in the electrokinetic analysis of the surfaces bearing different amounts of RGD, it was found that the peptide presence on PEO(20 000)-PDA layers does not have a significant effect on the nonfouling properties of the system. Notably, the presented PEO(20 000)-PDA layers bearing RGD peptides in the surface concentration range 5.9 to 1.7 × 105 fmol·cm-2 reduced the protein adsorption from fetal bovine serum to less than 30 ng·cm-2, that is, values comparable to the ones obtained for pristine PEO(20 000)-PDA layers.

Macroporous Biodegradable Cryogels of Synthetic Poly(α-amino acids).

We present an investigation of the preparation of highly porous hydrogels based on biodegradable synthetic poly(α-amino acid) as potential tissue engineering scaffolds. Covalently cross-linked gels with permanent pores were formed under cryogenic conditions by free-radical copolymerization of poly[N(5)-(2-hydroxyethyl)-L-glutamine-stat-N(5)-(2-methacryloyl-oxy-ethyl)-L-glutamine] (PHEG-MA) with 2-hydrohyethyl methacrylate (HEMA) and, optionally, N-propargyl acrylamide (PrAAm) as minor comonomers. The morphology of the cryogels showed interconnected polyhedral or laminar pores. The volume content of communicating water-filled pores was >90%. The storage moduli of the swollen cryogels were in the range of 1-6 kPa, even when the water content was >95%. The enzymatic degradation of a cryogel corresponded to the decrease in its storage modulus during incubation with papain, a model enzyme with specificity analogous to wound-healing enzymes. It was shown that cryogels with incorporated alkyne groups can easily be modified with short synthetic peptides using azide-alkyne cycloaddition "click" chemistry, thus providing porous hydrogel scaffolds with biomimetic features.

Toward structured macroporous hydrogel composites: electron beam-initiated polymerization of layered cryogels.

The ability to tailor mechanical properties and architecture is crucial in creating macroporous hydrogel scaffolds for tissue engineering. In the present work, a technique for the modification of the pore size and stiffness of acrylamide-based cryogels is demonstrated via the regulation of an electron beam irradiation dose. The samples were characterized by equilibrium swelling measurements, light and scanning electron microscopy, mercury porosimetry, Brunauer-Emmett-Teller surface area analysis, and stiffness measurements. Their properties were compared to cryogels prepared by a standard redox-initiated radical polymerization. A (125)I radiolabeled azidopentanoyl-GGGRGDSGGGY-NH2 peptide was bound to the surface to determine the concentration of the adhesive sites available for biomimetic modification. The functionality of the prepared substrates was evaluated by in vitro cultivation of adipose-derived stem cells. Moreover, the feasibility of preparing layered cryogels was demonstrated. This may be the key to the future preparation of complex hydrogel-based scaffolds to mimic the extracellular microenvironment in a wide range of applications.

Cell adhesion and growth enabled by biomimetic oligopeptide modification of a polydopamine-poly(ethylene oxide) protein repulsive surface.

Protein-repulsive surfaces modified with ligands for cell adhesion receptors have been widely developed for controlling the cell adhesion and growth in tissue engineering. However, the question of matrix production and deposition by cells on these surfaces has rarely been addressed. In this study, protein-repulsive polydopamine-poly(ethylene oxide) (PDA-PEO) surfaces were functionalized with an RGD-containing peptide (RGD), with a collagen-derived peptide binding fibronectin (Col), or by a combination of these peptides (RGD + Col, ratio 1:1) in concentrations of 90 fmol/cm(2) and 700 fmol/cm(2) for each peptide type. When seeded with vascular endothelial CPAE cells, the PDA-PEO surfaces proved to be completely non-adhesive for cells. On surfaces with lower peptide concentrations and from days 1 to 3 after seeding, cell adhesion and growth was restored practically only on the RGD-modified surface. However, from days 3 to 7, cell adhesion and growth was improved on surfaces modified with Col and with RGD + Col. At higher peptide concentrations, the cell adhesion and growth was markedly improved on all peptide-modified surfaces in both culture intervals. However, the collagen-derived peptide did not increase the expression of fibronectin in the cells. The deposition of fibronectin on the material surface was generally very low and similar on all peptide-modified surfaces. Nevertheless, the RGD + Col surfaces exhibited the highest cell adhesion stability under a dynamic load, which correlated with the highest expression of talin and vinculin in the cells on these surfaces. A combination of RGD + Col therefore seems to be the most promising for surface modification of biomaterials, e.g. vascular prostheses.

Nonfouling poly(ethylene oxide) layers end-tethered to polydopamine.

Nonfouling surfaces capable of reducing protein adsorption are highly desirable in a wide range of applications. Coating of surfaces with poly(ethylene oxide) (PEO), a water-soluble, nontoxic, and nonimmunogenic polymer, is most frequently used to reduce nonspecific protein adsorption. Here we show how to prepare dense PEO brushes on virtually any substrate by tethering PEO to polydopamine (PDA)-modified surfaces. The chain lengths of hetero-bifunctional PEOs were varied in the range of 45-500 oxyethylene units (M(n) = 2000-20,000). End-tethering of PEO chains was performed through amine and thiol headgroups from reactive polymer melts to minimize excluded volume effects. Surface plasmon resonance (SPR) was applied to investigate the adsorption of model protein solutions and complex biologic medium (human blood plasma) to the densely packed PEO brushes. The level of protein adsorption of human serum albumin and fibrinogen solutions was below the detection limit of the SPR measurements for all PEO chains end-tethered to PDA, thus exceeding the protein resistance of PEO layers tethered directly on gold. It was found that the surface resistance to adsorption of lysozyme and human blood plasma increased with increasing length and brush character of the PEO chains end-tethered to PDA with a similar or better resistance in comparison to PEO layers on gold. Furthermore, the chain density, thickness, swelling, and conformation of PEO layers were determined using spectroscopic ellipsometry (SE), dynamic water contact angle (DCA) measurements, infrared reflection-absorption spectroscopy (IRRAS), and vibrational sum-frequency-generation (VSFG) spectroscopy, the latter in air and water.

Poly(ethylene oxide) layers grafted to dopamine-melanin anchoring layer: stability and resistance to protein adsorption.

In this study, we propose substrate-independent modification for creating a protein-repellent surface based on dopamine-melanin anchoring layer used for subsequent binding of poly(ethylene oxide) (PEO) from melt. We verified that the dopamine-melanin layer can be formed on literally any substrate and could serve as the anchoring layer for subsequent grafting of PEO chains. Grafting of PEO from melt in a temperature range 70-110 °C produces densely packed PEO layers showing exceptionally low protein adsorption when exposed to the whole blood serum or plasma. The PEO layers prepared from melt at 110 °C retained the protein repellent properties for as long as 10 days after their exposure to physiological-like conditions. The PEO-dopamine-melanin modification represents a simple and universal surface modification method for the preparation of protein repellent surfaces that could serve as a nonfouling background in various applications, such as optical biosensors and tissue engineering.

Enzymatic degradation of the hydrogels based on synthetic poly(α-amino acid)s.

Biodegradable hydrogels are studied as potential scaffolds for soft tissue regeneration. In this work biodegradable hydrogels were prepared from synthetic poly(α-amino acid)s, poly(AA)s. The covalently crosslinked gels were formed by radical copolymerization of methacryloylated poly(AA)s, e.g. poly[N (5)-(2-hydroxy-ethyl)-L-glutamine-ran-L-alanine-ran-N (6)-methacryloyl-L-lysine], as a multifunctional macro-monomer with a low-molecular-weight methacrylic monofunctional monomer, e.g. 2-hydroxyethyl methacrylate (HEMA). Methacryloylated copolypeptides were synthesized by polymerization of N-carboxyanhydrides of respective amino acids and subsequent side-chain modification. Due to their polypeptide backbone, synthetic poly(AA)s are cleavable in biological environment by enzyme-catalyzed hydrolysis. The feasibility of enzymatic degradation of poly(AA)s alone and the hydrogels made from them was studied using elastase, a matrix proteinase involved in tissue healing processes, as a model enzyme. Specificity of elastase for cleavage of polypeptide chains behind the L-alanine residues was reflected in faster degradation of L-alanine-containing copolymers as well as of hydrogels composed of them.

Modification of polylactide surfaces with lactide-ethylene oxide functional block copolymers: accessibility of functional groups.

Feasibility of using amphiphilic block copolymers composed of polylactide (PLA) and poly(ethylene oxide) (PEO) blocks for biomimetic surface modification of polylactide-based biomaterials for tissue engineering was investigated. PEO-b-PLA copolymers were deposited on the PLA surface from a solution in PEO-selective solvent. Copolymers with a neutral omega-methoxy end group of the PEO block (mPEO-b-PLA) were used to provide hydrophilic surface of PLLA, which exhibited suppressed nonspecific protein adsorption. Their analogues, containing biotin group at the end of PEO block (bPEO-b-PLA), were used as a model of functional copolymers, carrying a biomimetic group, for example, a cell-adhesion fibronectine-derived peptide sequence. The surface topography of functional groups on the modified surface and their accessibility for interaction with a protein receptor was investigated, taking advantage of specific biotin-avidin interaction, on surfaces modified with a combination of mPEO-b-PLA and bPEO-b-PLA copolymers. The accessibility of model biotin groups for interaction with their protein counterpart was proven through visualization of avidin or avidin-labeled nanospheres with atomic force microscopy.

Adsorption of poly(ethylene oxide)-block-polylactide copolymers on polylactide as studied by ATR-FTIR spectroscopy.

In this study, the adsorption of amphiphilic poly(ethylene oxide)-block-polylactide (mPEO-PLA) copolymers from a selective solvent onto a polylactide surface was studied as a method of polylactide surface modification and its effect on nonspecific protein adsorption was evaluated. A series of well defined mPEO-PLA copolymers was prepared to investigate the effect of copolymer composition on the resulting PEO chain density and on the surface resistance to protein adsorption. The copolymers contained PEO blocks with molecular weights ranging between 5600 and 23,800 and with 16-47 wt% of PLA. The adsorption of both the copolymers and bovine serum albumin was quantified by attenuated total reflection FTIR spectroscopy (ATR-FTIR). In addition to the adsorbed copolymer amount, its actual composition was determined. The PEO chain density on the surface was found to decrease with the molecular weight of the PEO block and to increase with the molecular weight of the PLA block. The adsorbed copolymers displayed the ability to reduce protein adsorption. The maximum reduction within the tested series (by 80%) was achieved with the copolymer containing PEO of MW 5600 and a PLA block of the same MW.

Poly(amino acid)-based fibrous scaffolds modified with surface-pendant peptides for cartilage tissue engineering.

In this study, fibrous scaffolds based on poly(γ-benzyl-l-glutamate) (PBLG) were investigated in terms of the chondrogenic differentiation potential of human tooth germ stem cells (HTGSCs). Through the solution-assisted bonding of the fibres, fully connected scaffolds with pore sizes in the range 20-400 µm were prepared. Biomimetic modification of the PBLG scaffolds was achieved by a two-step reaction procedure: first, aminolysis of the PBLG fibres' surface layers was performed, which resulted in an increase in the hydrophilicity of the fibrous scaffolds after the introduction of N5 -hydroxyethyl-l-glutamine units; and second, modification with the short peptide sequence azidopentanoyl-GGGRGDSGGGY-NH2 , using the 'click' reaction on the previously modified scaffold with 2-propynyl side-chains, was performed. Radio-assay of the 125 I-labelled peptide was used to evaluate the RGD density in the fibrous scaffolds (which varied in the range 10-3 -10 pm/cm2 ). All the PBLG scaffolds, especially with density 90 ± 20 fm/cm2 and 200 ± 100 fm/cm2 RGD, were found to be potentially suitable for growth and chondrogenic differentiation of HTGSCs. Copyright © 2015 John Wiley & Sons, Ltd.

Polymer scaffolds with no skin-effect for tissue engineering applications fabricated by thermally induced phase separation.

Thermally induced phase separation (TIPS) based methods are widely used for the fabrication of porous scaffolds for tissue engineering and related applications. However, formation of a less-/non-porous layer at the scaffold's outer surface at the air-liquid interface, often known as the skin-effect, restricts the cell infiltration inside the scaffold and therefore limits its efficacy. To this end, we demonstrate a TIPS-based process involving the exposure of the just quenched poly(lactide-co-caprolactone):dioxane phases to the pure dioxane for a short time while still being under the quenching strength, herein after termed as the second quenching (2Q). Scanning electron microscopy, mercury intrusion porosimetry and contact angle analysis revealed a direct correlation between the time of 2Q and the gradual disappearance of the skin, followed by the widening of the outer pores and the formation of the fibrous filaments over the surface, with no effect on the internal pore architecture and the overall porosity of scaffolds. The experiments at various quenching temperatures and polymer concentrations revealed the versatility of 2Q in removing the skin. In addition, the in vitro cell culture studies with the human primary fibroblasts showed that the scaffolds prepared by the TIPS based 2Q process, with the optimal exposure time, resulted in a higher cell seeding and viability in contrast to the scaffolds prepared by the regular TIPS. Thus, TIPS including the 2Q step is a facile, versatile and innovative approach to fabricate the polymer scaffolds with a skin-free and fully open porous surface morphology for achieving a better cell response in tissue engineering and related applications.

A frame-supported ultrathin electrospun polymer membrane for transplantation of retinal pigment epithelial cells.

We report on the design and fabrication of a frame-supported nanofibrous membrane for the transplantation of retinal pigment epithelial (RPE) cells, which is a promising therapeutic option for the treatment of degenerative retinal disorders. The membranous cell carrier prepared from 640 nm-thick poly(DL-lactide) fibres uniquely combines high porosity, large pore size and low thickness, to maximize the nutrient supply to the transplanted cells in the subretinal space and thus to enhance the therapeutic effect of the transplantation. The carrier was prepared by electrospinning, which made it easy to embed a 95 µm-thick circular supporting frame 2 mm in diameter. Implantations into enucleated porcine eyes showed that the frame enabled the ultrathin membrane to be handled without irreversible folding, and allowed the membrane to regain its flat shape when inserted into the subretinal space. We further demonstrated that the minimum membrane thickness compatible with the surgical procedure and instrumentation employed here was as low as 4 µm. Primary porcine RPE cells cultivated on the membranes formed a confluent monolayer, expressed RPE-specific differentiation markers and showed transepithelial resistance close to that of the native RPE. Most importantly, the majority of the RPE cells transplanted into the subretinal space remained viable. The ultrathin, highly porous, and surgically convenient cell carrier presented here has the potential to improve the integration and the functionality of transplanted RPE cells.

Cellular Responses Modulated by FGF-2 Adsorbed on Albumin/Heparin Layer-by-Layer Assemblies.

In a typical cell culture system, growth factors immobilized on the cell culture surfaces can serve as a reservoir of bio-signaling molecules, without the need to supplement them additionally into the culture medium. In this paper, we report on the fabrication of albumin/heparin (Alb/Hep) assemblies for controlled binding of basic fibroblast growth factor (FGF-2). The surfaces were constructed by layer-by-layer adsorption of polyelectrolytes albumin and heparin and were subsequently stabilized by covalent crosslinking with glutaraldehyde. An analysis of the surface morphology by atomic force microscopy showed that two Alb/Hep bilayers are required to cover the surface of substrate. The formation of the Alb/Hep assemblies was monitored by the surface plasmon resonance (SPR), the infrared multiinternal reflection spectroscopy (FTIR MIRS) and UV/VIS spectroscopy. The adsorption of FGF-2 on the cross-linked Alb/Hep was followed by SPR. The results revealed that FGF-2 binds to the Alb/Hep assembly in a dose and time-dependent manner up to the surface concentration of 120 ng/cm(2). The bioactivity of the adsorbed FGF-2 was assessed in experiments in vitro, using calf pulmonary arterial endothelial cells (CPAE). CPAE cells could attach and proliferate on Alb/Hep surfaces. The adsorbed FGF-2 was bioactive and stimulated both the proliferation and the differentiation of CPAE cells. The improvement was more pronounced at a lower FGF-2 surface concentration (30 ng/cm(2)) than on surfaces with a higher concentration of FGF-2 (120 ng/cm(2)).

Self-assembled anchor layers/polysaccharide coatings on titanium surfaces: a study of functionalization and stability.

Composite materials based on a titanium support and a thin, alginate hydrogel could be used in bone tissue engineering as a scaffold material that provides biologically active molecules. The main objective of this contribution is to characterize the activation and the functionalization of titanium surfaces by the covalent immobilization of anchoring layers of self-assembled bisphosphonate neridronate monolayers and polymer films of 3-aminopropyltriethoxysilane and biomimetic poly(dopamine). These were further used to bind a bio-functional alginate coating. The success of the titanium surface activation, anchoring layer formation and alginate immobilization, as well as the stability upon immersion under physiological-like conditions, are demonstrated by different surface sensitive techniques such as spectroscopic ellipsometry, infrared reflection-absorption spectroscopy and X-ray photoelectron spectroscopy. The changes in morphology and the established continuity of the layers are examined by scanning electron microscopy, surface profilometry and atomic force microscopy. The changes in hydrophilicity after each modification step are further examined by contact angle goniometry.

Biochemical evidence for transcytotic absorption of polyaspartamide from the rat lung: effects of temperature and metabolic inhibitors.

Airway-to-perfusate polyhydroxyethylaspartamide (PHEA) absorption was studied in the isolated perfused rat lung at a reduced temperature and by the use of metabolic inhibitors, to kinetically clarify the mechanisms and cellular pathways of its active absorption. Fluorophore-labeled PHEA (F-PHEA; 7.4 kDa) was administered into the airways, and its absorption followed with time at 25 degrees C and in the presence of 2,4-dinitrophenol (DNP), ouabain (OUA), monensin (MON), and nocodazole (NOC). Across-dose absorption profiles were analyzed using a kinetic model incorporating active (V(max,P) and K(m,P)) and passive (k(a,P)) absorption from the pulmonary lung region alongside the competing, pulmonary-to-bronchial mucociliary escalator (k(E)). The model was validated at 25 degrees C and a lack of perturbation on the k(a,P) and k(E) values for passively absorbed solutes confirmed by studying the disposition of sodium fluorescein and 4.4 kDa fluorescein isothiocyanate-labeled dextran. F-PHEA absorption was significantly suppressed at 25 degrees C, compared with 37 degrees C, because of a significant decrease in the value of the maximum rate of active absorption, V(max,P) (4.37 --> 0.67 microg/min; p < 0.05), whereas the carrier-affinity term, K(m,P), was statistically unchanged. F-PHEA's active absorption was also significantly inhibited by DNP (> or =0.5 mM), OUA (> or =50 microM), MON (> or =10 microM), and NOC (> or =1 microM), whereas these inhibitors had no significant effect on the values for k(a,P) and k(E). Thus, F-PHEA's pulmonary active absorption in the rat lung was temperature- and adenosine 5'-triphosphate-derived intracellular energy-dependent (DNP and OUA inhibition) and apparently mediated via transcytosis through cytoplasmic endosomes and microtubules (MON and NOC inhibition).

Solute disposition in the rat lung in vivo and in vitro: determining regional absorption kinetics in the presence of mucociliary escalator.

Solute absorption from the airways was compared and modeled in vivo and in vitro isolated perfused rat lung (IPRL), and its regional kinetic descriptors in the presence of competing mucociliary escalator were estimated. 7.4 kDa fluorophore-labeled polyhydroxyethylaspartamide (F-PHEA), FITC-labeled dextran 40 (FD-4) and sodium fluorescein (F-Na) were used as model solutes. They were reproducibly administered into the airways in a range of doses in vivo and in vitro IPRL, and their initial deposition and subsequent absorption profiles compared. Each of the absorption data was fitted across doses to a kinetic model in which rate constants for Michaelis-Menten-type active (V(max,P) and K(m,P)) and/or first-order passive (k(a,P)) absorption and mucociliary escalator (k(E)) were estimated simultaneously. Statistically indistinguishable initial solute distribution was ensured in vivo and in vitro. The absorption profiles for F-PHEA were kinetically identical in vivo and in vitro, and their modeling analysis revealed the presence of competing, solute-independent pulmonary-to-bronchial mucociliary escalator with a half-life of 28.9 min. F-PHEA's active absorption was found to be 77 times faster than its passive absorption, yet this was present only in the pulmonary region. Passive solute absorption was inversely related to solute molecular weight [F-PHEA < FD-4 < F-Na]. Bronchial absorption was shown for F-Na in vivo and its rate indistinguishable from that from the pulmonary region. Thus, a single kinetic model was developed, enabling regional absorption kinetic analysis both in vivo and in vitro, in the presence of competing, solute-independent mucociliary escalator.

Adhesion and growth of rat aortic smooth muscle cells on lactide-based polymers.

Biodegradable materials based on polymers of hydroxy acids are studied for application in artificial vascular substitutes. Polymers with functional surfaces are being developed, carrying specific recognition structures to affect selectively the adhesion and proliferation of endothelial cells (EC) and vascular smooth muscle cells (VSMC). This preliminary study focuses on evaluation of adhesion and growth of VSMC on surfaces of polylactide polymers and those modified by amphiphilic polylactide/poly(ethylene oxide) copolymers. Poly(L-lactic acid), PLLA, and poly(DL-lactic acid), PDLLA, and a block copolymer of lactide with a carboxylated poly(ethylene oxide) segment, PLLA-b-PEO-COOH, were synthesized by controlled polymerization of L and D,L-lactide, respectively, and using delta-hydroxy-Z-carboxymethyl-PEO as a macroinitiator for the copolymer. Films of polymers were deposited on glass coverslips by a spin-coating method. Uncoated glass coverslips and Falcon dishes were used as control substrates. VSMC were obtained from the thoracic aorta of young adult male Wistar rats by explantation method and seeded in Dulbecco-Modified Eagle MEM with 10% foetal bovine serum. The number of adhering cells, their shape, size of cell-material contact area and cell population doubling time were evaluated from day 1 to 7 after seeding. It was found that both PLLA and especially PDLLA relatively well supported adhesion and growth of VSMC. However, on carboxylated surfaces of the PLLA-b-PEO-COOH copolymer, a lower number of initially adhering cells (by 37% than on Falcon dishes, pdelta0.05), smaller cell spreading area (by 45% and 37% than on glass and Falcon dishes, respectively, pdelta0.01) and longer doubling time (by 49% and 31% than on glass and Falcon dishes, pdelta0.001). Thus, surfaces coated by a PLA/PEO-COOH copolymer can be used as minimum background surface to reveal the effect of other more specific adhesion structures.

Biodegradable copolymers carrying cell-adhesion peptide sequences.

Amphiphilic block copolymers are used to create bioactive surfaces on biodegradable polymer scaffolds for tissue engineering. Cell-selective biomaterials can be prepared using copolymers containing peptide sequences derived from extracellular-matrix proteins (ECM). Here we discuss alternative ways for preparation of amphiphilic block copolymers composed of hydrophobic polylactide (PLA) and hydrophilic poly(ethylene oxide) (PEO) blocks with cell-adhesion peptide sequences. Copolymers PLA-b-PEO were prepared by a living polymerisation of lactide in dioxane with tin(II)2-ethylhexanoate as a catalyst. The following approaches for incorporation of peptides into copolymers were elaborated. (a) First, a side-chain protected Gly-Arg-Gly-Asp-Ser-Gly (GRGDSG) peptide was prepared by solid-phase peptide synthesis (SPPS) and then coupled with delta-hydroxy-Z-amino-PEO in solution. In the second step, the PLA block was grafted to it via a controlled polymerisation of lactide initiated by the hydroxy end-groups of PEO in the side-chain-protected GRGDSG-PEO. Deprotection of the peptide yielded a GRGDSG-b-PEO-b-PLA copolymer, with the peptide attached through its C-end. (b) A protected GRGDSG peptide was built up on a polymer resin and coupled with Z-carboxy-PEO using a solid-phase approach. After cleavage of the delta-hydroxy-PEO-GRGDSG copolymer from the resin, polymerisation of lactide followed by deprotection of the peptide yielded a PLA-b-PEO-b-GRGDSG block copolymer, in which the peptide is linked through its N-terminus.

Direct laser writing of synthetic poly(amino acid) hydrogels and poly(ethylene glycol) diacrylates by two-photon polymerization.

The additive manufacturing technique of direct laser writing by two-photon polymerization (2PP-DLW) enables the fabrication of three-dimensional microstructures with superior accuracy and flexibility. When combined with biomimetic hydrogel materials, 2PP-DLW can be used to recreate the microarchitectures of the extracellular matrix. However, there are currently only a limited number of hydrogels applicable for 2PP-DLW. In order to widen the selection of synthetic biodegradable hydrogels, in this work we studied the 2PP-DLW of methacryloylated and acryloylated poly(α-amino acid)s (poly(AA)s). The performance of these materials was compared to widely used poly(ethylene glycol) diacrylates (PEGdas) in terms of polymerization and damage thresholds, voxel size, line width, post-polymerization swelling and deformation. We found that both methacryloylated and acryloylated poly(AA) hydrogels are suitable to 2PP-DLW with a wider processing window than PEGdas. The poly(AA) with the highest degree of acryloylation showed the greatest potential for 3D microfabrication.

Dip TIPS as a facile and versatile method for fabrication of polymer foams with controlled shape, size and pore architecture for bioengineering applications.

The porous polymer foams act as a template for neotissuegenesis in tissue engineering, and, as a reservoir for cell transplants such as pancreatic islets while simultaneously providing a functional interface with the host body. The fabrication of foams with the controlled shape, size and pore structure is of prime importance in various bioengineering applications. To this end, here we demonstrate a thermally induced phase separation (TIPS) based facile process for the fabrication of polymer foams with a controlled architecture. The setup comprises of a metallic template bar (T), a metallic conducting block (C) and a non-metallic reservoir tube (R), connected in sequence T-C-R. The process hereinafter termed as Dip TIPS, involves the dipping of the T-bar into a polymer solution, followed by filling of the R-tube with a freezing mixture to induce the phase separation of a polymer solution in the immediate vicinity of T-bar; Subsequent free-drying or freeze-extraction steps produced the polymer foams. An easy exchange of the T-bar of a spherical or rectangular shape allowed the fabrication of tubular, open- capsular and flat-sheet shaped foams. A mere change in the quenching time produced the foams with a thickness ranging from hundreds of microns to several millimeters. And, the pore size was conveniently controlled by varying either the polymer concentration or the quenching temperature. Subsequent in vivo studies in brown Norway rats for 4-weeks demonstrated the guided cell infiltration and homogenous cell distribution through the polymer matrix, without any fibrous capsule and necrotic core. In conclusion, the results show the "Dip TIPS" as a facile and adaptable process for the fabrication of anisotropic channeled porous polymer foams of various shapes and sizes for potential applications in tissue engineering, cell transplantation and other related fields.