Responsive Polyesters with Alkene and Carboxylic Acid Side-Groups for Tissue Engineering Applications.

Main chain polyesters have been extensively used in the biomedical field. Despite their many advantages, including biocompatibility, biodegradability, and others, these materials are rather inert and lack specific functionalities which will endow them with additional biological and responsive properties. In this work, novel pH-responsive main chain polyesters have been prepared by a conventional condensation polymerization of a vinyl functionalized diol with a diacid chloride, followed by a photo-induced thiol-ene click reaction to attach functional carboxylic acid side-groups along the polymer chains. Two different mercaptocarboxylic acids were employed, allowing to vary the alkyl chain length of the polymer pendant groups. Moreover, the degree of modification, and as a result, the carboxylic acid content of the polymers, was easily tuned by varying the irradiation time during the click reaction. Both these parameters, were shown to strongly influence the responsive behavior of the polyesters, which presented adjustable pKα values and water solubilities. Finally, the difunctional polyesters bearing the alkene and carboxylic acid functionalities enabled the preparation of cross-linked polyester films by chemically linking the pendant vinyl bonds on the polymer side groups. The biocompatibility of the cross-linked polymers films was assessed in L929 fibroblast cultures and showed that the cell viability, proliferation, and attachment were greatly promoted on the polyester surface, bearing the shorter alkyl chain length side groups and the higher fraction of carboxylic acid functionalities.

Nonlinear rheometry of entangled polymeric rings and ring-linear blends.

We present a comprehensive experimental rheological dataset for purified entangled ring polystyrenes and their blends with linear chains in nonlinear shear and elongation. In particular, data for shear stress growth coefficient, steady-state shear viscosity, and first and second normal stress differences are obtained and discussed as functions of shear rate as well as molecular parameters (molar mass, blend composition and decreasing molar mass of linear component in blend). Over the extended parameter range investigated, rings do not exhibit clear transient undershoot in shear, in contrast to their linear counterparts and ring-linear blends. For the latter, the size of the undershoot and respective strain appear to increase with shear rate. Universal scaling of strain at overshoot and fractional overshoot (ratio of maximum to steady-state shear stress growth coefficient) indicates subtle differences in the shear-rate dependence between rings and linear polymers or their blends. The shear thinning behaviour of pure rings yields a slope nearly identical to predictions (-4/7) of a recent shear slit model and molecular dynamics simulations. Data for the second normal stress difference are reported for rings and ring-linear blends. While N 2 is negative and its absolute value stays below that of N 1 , as for linear polymers, the ratio -N 2 /N 1 is unambiguously larger for rings compared to linear polymer solutions with the same number of entanglements (almost by factor of two), in agreement with recent non-equilibrium molecular dynamics simulations. Further, -N 2 exhibits slightly weaker shear rate dependence compared to N 1 at high rates, and the respective power-law exponents can be rationalized in view of the slit model (3/7) and simulations (0.6), although further work is needed to unravel the molecular original of the observed behaviour. The comparison of shear and elongational stress growth coefficients for blends reflects the effect of ring-linear threading which leads to significant viscosity enhancement in elongation. Along the same lines, the elongational stress is much larger than the first normal stress in shear, and their ratio is much larger for rings and ring-linear blends compared to linear polymers. This conforms the interlocking scenario of rings and their important role in mechanically reinforcing linear matrices.

Biodegradable Chitosan-graft-Poly(l-lactide) Copolymers For Bone Tissue Engineering.

The design and synthesis of new biomaterials with adjustable physicochemical and biological properties for tissue engineering applications have attracted great interest. In this work, chitosan-graft-poly(l-lactide) (CS-g-PLLA) copolymers were prepared by chemically binding poly(l-lactide) (PLLA) chains along chitosan (CS) via the "grafting to" approach to obtain hybrid biomaterials that present enhanced mechanical stability, due to the presence of PLLA, and high bioactivity, conferred by CS. Two graft copolymers were prepared, CS-g-PLLA(80/20) and CS-g-PLLA(50/50), containing 82 wt % and 55 wt % CS, respectively. Degradation studies of compressed discs of the copolymers showed that the degradation rate increased with the CS content of the copolymer. Nanomechanical studies in the dry state indicated that the copolymer with the higher CS content had larger Young modulus, reduced modulus and hardness values, whereas the moduli and hardness decreased rapidly following immersion of the copolymer discs in alpha-MEM cell culture medium for 24 h. Finally, the bioactivity of the hybrid copolymers was evaluated in the adhesion and growth of MC3T3-E1 pre-osteoblastic cells. In vitro studies showed that MC3T3-E1 cells exhibited strong adhesion on both CS-g-PLLA graft copolymer films from the first day in cell culture, whereas the copolymer with the higher PLLA content, CS-g-PLLA(50/50), supported higher cell growth.

Synthesis, Nanomechanical Characterization and Biocompatibility of a Chitosan-Graft-Poly(ε-caprolactone) Copolymer for Soft Tissue Regeneration.

Tissue regeneration necessitates the development of appropriate scaffolds that facilitate cell growth and tissue development by providing a suitable substrate for cell attachment, proliferation, and differentiation. The optimized scaffolds should be biocompatible, biodegradable, and exhibit proper mechanical behavior. In the present study, the nanomechanical behavior of a chitosan-graft-poly(ε-caprolactone) copolymer, in hydrated and dry state, was investigated and compared to those of the individual homopolymers, chitosan (CS) and poly(ε-caprolactone) (PCL). Hardness and elastic modulus values were calculated, and the time-dependent behavior of the samples was studied. Submersion of PCL and the graft copolymer in α-MEM suggested the deterioration of the measured mechanical properties as a result of the samples' degradation. However, even after three days of degradation, the graft copolymer presented sufficient mechanical strength and elastic properties, which resemble those reported for soft tissues. The in vitro biological evaluation of the material clearly demonstrated that the CS-g-PCL copolymer supports the growth of Wharton's jelly mesenchymal stem cells and tissue formation with a simultaneous material degradation. Both the mechanical and biological data render the CS-g-PCL copolymer appropriate as a scaffold in a cell-laden construct for soft tissue engineering.

Multiphoton 3D Printing of Biopolymer-Based Hydrogels.

Multiphoton lithography, based on multiphoton polymerization, is a powerful technique for the fabrication of complex three-dimensional (3D) structures. Herein, we report on the photostructuring of novel biopolymer-based hybrid hydrogels, comprising gelatin methacrylamide and a water-soluble chitosan derivative, via multiphoton polymerization. The nontoxic, Food and Drug Administration-approved, biocompatible photosensitizer eosin Y was exploited as the sole photoinitiator, without the coinitiators and/or comonomer that are commonly used, allowing for further expansion of the available wavelengths up to 800 nm. Importantly, the obtained hybrid material exhibits excellent biocompatibility, evidenced by the increased proliferation of dental pulp stem cells, compared with the individual components and the polystyrene control, after 7 days in culture. Additionally, the 3D hybrid scaffolds promote the matrix mineralization, following their functionalization with bone morphogenetic protein 2. These tailor-made synthetic, biocompatible materials pave the way for further opportunities in 3D scaffold fabrication, including in situ and in vivo biofabrication.

Osteogenic Potential of Pre-Osteoblastic Cells on a Chitosan-graft-Polycaprolactone Copolymer.

A chitosan-graft-polycaprolactone (CS-g-PCL) copolymer synthesized via a multi-step process was evaluated as a potential biomaterial for the adhesion and growth of MC3T3-E1 pre-osteoblastic cells. A strong adhesion of the MC3T3-E1 cells with a characteristic spindle-shaped morphology was observed from the first days of cell culture onto the copolymer surfaces. The viability and proliferation of the cells on the CS-g-PCL surfaces, after 3 and 7 days in culture, were significantly higher compared to the cells cultured on the tissue culture treated polystyrene (TCPS) control. The osteogenic potential of the pre-osteoblastic cells cultured on CS-g-PCL surfaces was evaluated by determining various osteogenic differentiation markers and was compared to the TCPS control surface. Specifically, alkaline phosphatase activity levels show significantly higher values at both time points compared to TCPS, while secreted collagen into the extracellular matrix was found to be higher on day 7. Calcium biomineralization deposited into the matrix is significantly higher for the CS-g-PCL copolymer after 14 days in culture, while the levels of intracellular osteopontin were significantly higher on the CS-g-PCL surfaces compared to TCPS. The enhanced osteogenic response of the MC3T3-E1 pre-osteoblasts cultured on CS-g-PCL reveals that the copolymer underpins the cell functions towards bone tissue formation and is thus an attractive candidate for use in bone tissue engineering.

Biodegradation of weathered polystyrene films in seawater microcosms.

A microcosm experiment was conducted at two phases in order to investigate the ability of indigenous consortia alone or bioaugmented to degrade weathered polystyrene (PS) films under simulated marine conditions. Viable populations were developed on PS surfaces in a time dependent way towards convergent biofilm communities, enriched with hydrocarbon and xenobiotics degradation genes. Members of Alphaproteobacteria and Gammaproteobacteria were highly enriched in the acclimated plastic associated assemblages while the abundance of plastic associated genera was significantly increased in the acclimated indigenous communities. Both tailored consortia efficiently reduced the weight of PS films. Concerning the molecular weight distribution, a decrease in the number-average molecular weight of films subjected to microbial treatment was observed. Moreover, alteration in the intensity of functional groups was noticed with Fourier transform infrared spectrophotometry (FTIR) along with signs of bio-erosion on the PS surface. The results suggest that acclimated marine populations are capable of degrading weathered PS pieces.

Immunomodulatory Potential of Chitosan-graft-poly(ε-caprolactone) Copolymers toward the Polarization of Bone-Marrow-Derived Macrophages.

In tissue engineering, the use of biomaterials as templates or scaffolds to guide tissue development in vivo provokes the inevitable action of the immune system of the host. This induced immune response often determines the success of the scaffold, including angiogenesis and regeneration or failure causing inflammation and fibrosis. Therefore, it is crucial to predict or even better to promote the proper immune response following implantation. The aim of the present study was to evaluate the immunomodulatory potential of chitosan-graft-poly(ε-caprolactone) copolymers (CS-g-PCL) by analyzing the differentiation of primary bone marrow derived macrophages (BMDM) cultured in vitro on copolymer thin films. In order to evaluate the role of the chitosan content of the copolymer on macrophage polarization, two different copolymers containing 50 and 78% w/w chitosan were studied. Our data from cytokines secretion detection by ELISA show that the CS-g-PCL copolymer significantly decreases the secretion of the inducible levels of pro-inflammatory cytokines IL-12/23 by 31% ± 6, and thus possesses anti-inflammatory ability. Moreover, this anti-inflammatory action is correlated with the increased chitosan content of the copolymer. In addition, the CS-g-PCL copolymer significantly enhances the production of Arg1, the hallmark of M2 polarized macrophages, as shown by semiquantitative RT-PCR analysis, and this enhancement is 4-fold higher for the copolymer with the lower chitosan content. Although further in vivo experimentation is required to predict the outcome of the in situ engraftment of the copolymer, our results so far suggest that the CS-g-PCL copolymers possess anti-inflammatory activity and favor the transition of M1 to M2 macrophages, which are essential prerequisites for proper tissue remodeling.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) immobilized on laser-fabricated 3D scaffolds enhance osteogenesis.

The regeneration of bone via a tissue engineering approach involves components from the macroscopic to the nanoscopic level, including appropriate 3D scaffolds, cells and growth factors. In this study, hexagonal scaffolds of different diagonals were fabricated by Direct Laser Writing using a photopolymerizable hybrid material. The proliferation of bone marrow (BM) mesenchymal stem cells (MSCs) cultured on structures with various diagonals, 50, 100, 150 and 200µm increased significantly after 10days in culture, however without significant differences among them. Next, recombinant human bone morphogenetic protein 2 (rhBMP-2) was immobilized onto the hybrid material both via covalent binding and physical adsorption. Both immobilization types exhibited similar high releaseate bioactivity profiles and a sustained delivery of rhBMP-2. The collagen and calcium levels produced in the extracellular matrix (ECM) were significantly elevated for the samples functionalized with BMP-2 compared to those in the osteogenic medium. Furthermore, significant upregulation of gene expression in both types of BMP-2 immobilized scaffolds was observed for alkaline phosphatase (ALPL) and osteocalcin (BGLAP) at days 7, 14, and 21, for RUNX2 at day 21, and for osteonectin (SPARC) at days 7 and 14. The results suggest that the release of bioactive rhBMP-2 from the hybrid scaffolds enhance the control over the osteogenic differentiation during cell culture.

Wharton's jelly Mesenchymal Stem Cell response on chitosan-graft-poly (ε-caprolactone) copolymer for myocardium tissue engineering.

Cell therapy and tissue engineering attract increasing attention as a potential approach for cardiac repair. Although a plethora of interesting concepts in the emerging field of cardiac stem cell-based tissue engineering are reported, there are still challenges that this field needs to overcome to achieve therapeutic translation into the clinical praxis. Engineering biomaterial scaffolds that facilitate stem cell engraftment, survival and homing are crucial for successful cellular cardiomyoplasty after myocardial infarction (MI). In this study we investigate for the first time the cellular response of Wharton's jelly (WJ) Mesenchymal Stem Cells (MSCs) on a copolymeric material comprising chitosan (CS) and poly(ε-caprolactone) (PCL). First we synthesize a copolymer consisting of poly(ε-caprolactone) grafted on a chemically modified chitosan-backbone (CS-g-PCL). Furthermore, we investigate the morphology, viability and proliferation of WJMSCs on material coatings and examine the cellular response from different donors. Our results show strong cell adhesion on the CS-g- PCL material surface from the first hours in culture, and a proliferation increase after 3 and 7 days. These findings support the potential use of our proposed cell-material combination in myocardium tissue engineering.

Microporous polystyrene particles for selective carbon dioxide capture.

This study presents the synthesis of microporous polystyrene particles and the potential use of these materials in CO(2) capture for biogas purification. Highly cross-linked polystyrene particles are synthesized by the emulsion copolymerization of styrene (St) and divinylbenzene (DVB) in water. The cross-link density of the polymer is varied by altering the St/DVB molar ratio. The size and the morphology of the particles are characterized by scanning and transmission electron microscopy. Following supercritical point drying with carbon dioxide or lyophilization from benzene, the polystyrene nanoparticles exhibit a significant surface area and permanent microporosity. The dried particles comprising 35 mol % St and 65 mol % DVB possess the largest surface area, ∼205 m(2)/g measured by Brunauer-Emmett-Teller and ∼185 m(2)/g measured by the Dubinin-Radushkevich method, and a total pore volume of 1.10 cm(3)/g. Low pressure measurements suggest that the microporous polystyrene particles exhibit a good separation performance of CO(2) over CH(4), with separation factors in the range of ∼7-13 (268 K, CO(2)/CH(4) = 5/95 gas mixture), which renders them attractive candidates for use in gas separation processes.