Electrochemically Mediated Atom Transfer Radical Polymerization Driven by Alternating Current.

Alternating current (AC) and pulsed electrolysis are gaining traction in electro(organic) synthesis due to their advantageous characteristics. We employed AC electrolysis in electrochemically mediated Atom Transfer Radical Polymerization (eATRP) to facilitate the regeneration of the activator CuI complex on Cu0 electrodes. Additionally, Cu0 served as a slow supplemental activator and reducing agent (SARA ATRP), enabling the activation of alkyl halides and the regeneration of the CuI activator through a comproportionation reaction. We harnessed the distinct properties of Cu0 dual regeneration, both chemical and electrochemical, by employing sinusoidal, triangular, and square-wave AC electrolysis alongside some of the most active ATRP catalysts available. Compared to linear waveform (DC electrolysis) or SARA ATRP (without electrolysis), pulsed and AC electrolysis facilitated slightly faster and more controlled polymerizations of acrylates. The same AC electrolysis conditions could successfully polymerize eleven different monomers across different mediums, from water to bulk. Moreover, it proved effective across a spectrum of catalyst activity, from low-activity Cu/2,2-bipyridine to highly active Cu complexes with substituted tripodal amine ligands. Chain extension experiments confirmed the high chain-end fidelity of the produced polymers, yielding functional and high molecular-weight block copolymers. SEM analysis indicated the robustness of the Cu0 electrodes, sustaining at least 15 consecutive polymerizations.

Recycling Polyethylene/Polyamide Multilayer Films with Poly(isoprene-g-Maleic Anhydride) Compatibilizer.

Polymers generally form incompatible mixtures that make the process of recycling difficult, especially the mechanical recycling of mixed plastic waste. One of the most commonly used films in the packaging industry is multilayer films, mainly composed of polyethylene (PE) and polyamide (PA). Recycling these materials with such different molecular structures requires the use of compatibilizers to minimize phase separation and obtain more useful recycled materials. In this work, commercial polyisoprene-graft-maleic anhydride (PI-g-MA) was tested as a compatibilizer for a blend of PE and PA derived from the mechanical recycling of PE/PA multilayer films. Different amounts of PI-g-MA were tested, and the films made with 1.5% PI-g-MA showed the best results in terms of mechanical properties and dart impact. The films were also characterized thermally via thermogravimetric analysis (TG) and differential scanning calorimetry (DSC), using Fourier-transform infrared spectroscopy (FTIR), and morphologically using a scanning electron microscope (SEM). Other parameters, such as tearing and perforation, were analyzed.

Fast-Gelling Polyethylene Glycol/Polyethyleneimine Hydrogels Degradable by Visible-Light.

The treatment of burn wounds remains a clinical challenge due to the need for repeated dressings changes. Therefore, the development of a dressing system that can be atraumatically removed from the wound bed can be considered a breakthrough and improve treatment times. In this work, the development of an injectable, fast-gelling hydrogel is proposed that can change its mechanical properties when exposed to visible light. The hydrogels are prepared by a "click" amino-yne reaction between poly(ethylene glycol) (PEG) functionalized with propiolic acid and the amino groups of poly(ethyleneimine) (PEI). The hydrogels exhibit a fast gelation time, which can be adjusted by changing the weight percentage and molecular weight of the precursors. They also exhibit good swelling ability and adhesion to living tissues. More importantly, their mechanical properties changed upon irradiation with green light. This loss of properties is achieved by a 1 O2 -mediated mechanism, as confirmed by the degradation of the ß-aminoacrylate linker. Moreover, the in vitro cell compatibility results of the hydrogels and their degradation products show good cytocompatibility. Therefore, it is believed that these hydrogels can be considered as materials with great potential for an innovative strategy for the treatment of burn wounds.

Replacing Harmful Flame Retardants with Biodegradable Starch-Based Materials in Polyethylene Formulations.

The addition of toxic flame retardants to commercially available polymers is often required for safety reasons due to the high flammability of these materials. In this work, the preparation and incorporation of efficient biodegradable starch-based flame retardants into a low-density polyethylene (LDPE) matrix was investigated. Thermoplastic starch was first obtained by plasticizing starch with glycerol/water or glycerol/water/choline phytate to obtain TPS-G and TPS-G-CPA, respectively. Various LDPE/TPS blends were prepared by means of melt blending using polyethylene graft maleic anhydride as a compatibilizer and by varying the content of TPS and a halogenated commercial flame retardant. By replacing 38% and 76% of the harmful commercial flame retardant with safe TPS-G-CPA and TPS-G, respectively, blends with promising fire behavior were obtained, while the limiting oxygen index (LOI ≈ 28%) remained the same. The presence of choline phytate improved both the charring ability and fire retardancy of starch and resulted in a 43% reduction in fire growth index compared to the blend with commercial flame retardant only, as confirmed by means of cone calorimetry. Standard UL 94 vertical tests showed that blends containing TPS exhibited dripping behavior (rated V2), while those with commercial flame retardant were rated V0. Overall, this work demonstrates the potential of starch as a natural flame retardant that could reduce the cost and increase the safety of polymer-based materials.

Facile Synthesis of Highly Stretchable, Tough, and Photodegradable Hydrogels.

Recently, highly stretchable and tough hydrogels that are photodegradable on-demand have been reported. Unfortunately, the preparation procedure is complex due to the hydrophobic nature of the photocrosslinkers. Herein, a simple method is reported to prepare photodegradable double-network (DN) hydrogels that exhibit high stretchability, toughness, and biocompatibility. Hydrophilic ortho-nitrobenzyl (ONB) crosslinkers incorporating different poly(ethylene glycol) (PEG) backbones (600, 1000, and 2000 g mol-1 ) are synthesized. These photodegradable DN hydrogels are prepared by the irreversible crosslinking of chains by using such ONB crosslinkers, and the reversible ionic crosslinking between sodium alginate and divalent cations (Ca2+ ). Remarkable mechanical properties are obtained by combining ionic and covalent crosslinking and their synergistic effect, and by reducing the length of the PEG backbone. The rapid on-demand degradation of these hydrogels is also demonstrated by using cytocompatible light wavelength (λ = 365 nm) that degrades the photosensitive ONB units. The authors have successfully used these hydrogels as skin-worn sensors for monitoring human respiration and physical activities. A combination of excellent mechanical properties, facile fabrication, and on-demand degradation holds promise for their application as the next generation of substrates or active sensors eco-friendly for bioelectronics, biosensors, wearable computing, and stretchable electronics.

Targeted downregulation of MYC mediated by a highly efficient lactobionic acid-based glycoplex to enhance chemosensitivity in human hepatocellular carcinoma cells.

The chemosensitization of tumor cells by gene therapy represents a promising strategy for hepatocellular carcinoma (HCC) treatment. In this regard, HCC-specific and highly efficient gene delivery nanocarriers are urgently needed. For this purpose, novel lactobionic acid-based gene delivery nanosystems were developed to downregulate c-MYC expression and sensitize tumor cells to low concentration of sorafenib (SF). A library of tailor-made cationic glycopolymers, based on poly(2-aminoethyl methacrylate hydrochloride) (PAMA) and poly(2-lactobionamidoethyl methacrylate) (PLAMA) were synthesized by a straightforward activators regenerated by electron transfer atom transfer radical polymerization. The nanocarriers prepared with PAMA114-co-PLAMA20 glycopolymer were the most efficient for gene delivery. These glycoplexes specifically bound to the asialoglycoprotein receptor and were internalized through the clathrin-coated pit endocytic pathway. c-MYC expression was significantly downregulated by MYC short-hairpin RNA (MYC shRNA), resulting in efficient inhibition of tumor cells proliferation and a high levels apoptosis in 2D and 3D HCC-tumor models. Moreover, c-MYC silencing increased the sensitivity of HCC cells to SF (IC50 for MYC shRNA + SF 1.9 µM compared to 6.9 µM for control shRNA + SF). Overall, the data obtained demonstrated the great potential of PAMA114-co-PLAMA20/MYC shRNA nanosystems combined with low doses of SF for the treatment of HCC.

Glycopolymers Mediate Suicide Gene Therapy in ASGPR-Expressing Hepatocellular Carcinoma Cells in Tandem with Docetaxel.

Cationic glycopolymers stand out as gene delivery nanosystems due to their inherent biocompatibility and high binding affinity to the asialoglycoprotein receptor (ASGPR), a target receptor overexpressed in hepatocellular carcinoma (HCC) cells. However, their synthesis procedure remains laborious and complex, with problems of solubilization and the need for protection/deprotection steps. Here, a mini-library of well-defined poly(2-aminoethyl methacrylate hydrochloride-co-poly(2-lactobionamidoethyl methacrylate) (PAMA-co-PLAMA) glycopolymers was synthesized by activators regenerated by electron transfer (ARGET) ATRP to develop an efficient gene delivery nanosystem. The glycoplexes generated had suitable physicochemical properties and showed high ASGPR specificity and high transfection efficiency. Moreover, the HSV-TK/GCV suicide gene therapy strategy, mediated by PAMA144-co-PLAMA19-based nanocarriers, resulted in high antitumor activity in 2D and 3D culture models of HCC, which was significantly enhanced by the combination with small amounts of docetaxel. Overall, our results demonstrated the potential of primary-amine polymethacrylate-containing-glycopolymers as HCC-targeted suicide gene delivery nanosystems and highlight the importance of combined strategies for HCC treatment.

pH-responsive nanoparticles based on POEOMA-b-PDPA block copolymers for RNA encapsulation, protection and cell delivery.

The use of gene-based products, such as DNA or RNA, is increasingly being explored for various innovative therapies. However, the success of these strategies is highly dependent on the effective delivery of these biomolecules to target cells. Therefore, the development of pH-responsive nanoparticles comprises the creation of intelligent delivery systems with high therapeutic efficiency. In this work, the pH-responsiveness of the poly(2-(diisopropylamino)ethyl methacrylate)) (PDPA) block was investigated for the encapsulation and delivery of small RNAs (sRNA) to cancer cells. The pH responsiveness was dependent on the protonation profile of the tertiary amines of PDPA, which directly affected the electrostatic interactions established with RNA. Thus, block copolymers based on poly(oligo(ethylene oxide) methyl ether methacrylate) (POEOMA) and PDPA, POEOMA-b-PDPA, were synthesized by supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP). The structure of the block copolymers was characterized by size exclusion chromatography and 1H NMR spectroscopy. The copolymers allowed effective complexation of model sRNAs and a pre-miRNA with efficiencies of about 89 % and 91 %, respectively. The characterization by dynamic light scattering revealed that these systems had sizes between 76 and 1375 nm. It was also found that the morphology of the polyplexes depended on the pH, since the preparation at a pH lower than the pKa of the copolymers resulted in spherical but polydisperse particles, while higher pH values resulted in nanoparticles with more homogeneous size, but altered morphology. Moreover, due to pH-responsiveness, it was achieved the release of RNA at pH higher than the pKa of the copolymers, while maintaining its integrity. The polyplexes also showed a high potential to protect RNA from RNases. The transfection of a lung cancer model (A549) and fibroblast cell lines showed that these polyplexes did not cause cell toxicity. In addition, the polyplexes enabled the effective transfection of the A549 cell line with pre-miRNA-29b and miRNA-29b, resulting in a decrease of expression levels of the target DNMT3B gene by approximately 51 % and 47 %, respectively. Overall, the POEOMA-b-PDPA copolymers proved to be a promising strategy for developing responsive delivery systems, that can play a critical role in some diseases, such as cancer, where pH varies between the intra and extracellular environments.

Scaling-Up an Aqueous Self-Degassing Electrochemically Mediated ATRP in Dispersion for the Preparation of Cellulose-Polymer Composites and Films.

Electrochemically mediated atom transfer radical polymerization (eATRP) is developed in dispersion conditions to assist the preparation of cellulose-based films. Self-degassing conditions are achieved by the addition of sodium pyruvate (SP) as a ROS scavenger, while an aluminum counter electrode provides a simplified and more cost-effective electrochemical setup. Different polyacrylamides were grown on a model cellulose substrate which was previously esterified with 2-bromoisobutyrate (-BriB), serving as initiator groups. Small-scale polymerizations (15 mL) provided optimized conditions to pursue the scale-up up to 1000 mL (scale-up factor ~67). Cellulose-poly(N-isopropylacrylamide) was then chosen to prepare the tunable, thermoresponsive, solvent-free, and flexible films through a dissolution/regeneration method. The produced films were characterized by Fourier-transform infrared (FTIR), scanning electron microscopy (SEM), dynamic scanning calorimetry (DSC), and thermogravimetric analysis (TGA).

Dual electrochemical and chemical control in atom transfer radical polymerization with copper electrodes.

In Atom Transfer Radical Polymerization (ATRP), Cu0 acts as a supplemental activator and reducing agent (SARA ATRP) by activating alkyl halides and (re)generating the CuI activator through a comproportionation reaction, respectively. Cu0 is also an unexplored, exciting metal that can act as a cathode in electrochemically mediated ATRP (eATRP). Contrary to conventional inert electrodes, a Cu cathode can trigger a dual catalyst regeneration, simultaneously driven by electrochemistry and comproportionation, if a free ligand is present in solution. The dual regeneration explored herein allowed for introducing the concept of pulsed galvanostatic electrolysis (PGE) in eATRP. During a PGE, the process alternates between a period of constant current electrolysis and a period with no applied current in which polymerization continues via SARA ATRP. The introduction of no electrolysis periods without compromising the overall polymerization rate and control is very attractive, if large current densities are needed. Moreover, it permits a drastic charge saving, which is of unique value for a future scale-up, as electrochemistry coupled to SARA ATRP saves energy, and shortens the equipment usage.

Photo-degradable, tough and highly stretchable hydrogels.

We present for the first time highly stretchable and tough hydrogels with controlled light-triggered photodegradation. A double-network of alginate/polyacrylamide (PAAm) is formed by using covalently and ionically crosslinked subnetworks. The ionic Ca2+ alginate interpenetrates a PAAm network covalently crosslinked by a bifunctional acrylic crosslinker containing the photodegradable o-nitrobenzyl (ONB) core instead of the commonly used methylene bisacrylamide (MBAA). Remarkably, due to the developed protocol, the change of the crosslinker did not affect the hydrogel's mechanical properties. The incorporation of photosensitive components in hydrogels allows external temporal control of their properties and tuneable degradation. Cell viability and cell proliferation assays revealed that hydrogels and their photodegradation products are not cytotoxic to the NIH3T3 cell line. In one example of application, we used these hydrogels for bio-potential acquisition in wearable electrocardiography. Surprisingly, these hydrogels showed a lower skin-electrode impedance, compared to the common medical grade Ag/AgCl electrodes. This work lays the foundation for the next generation of tough and highly stretchable hydrogels that are environmentally friendly and can find applications in a variety of fields such as health, electronics, and energy, as they combine excellent mechanical properties with controlled degradation.

Application of vinyl polymer-based materials as nucleic acids carriers in cancer therapy.

Nucleic acid-based therapies have changed the paradigm of cancer treatment, where conventional treatment modalities still have several limitations in terms of efficacy and severe side effects. However, these biomolecules have a short half-life in vivo, requiring multiple administrations, resulting in severe suffering, discomfort, and poor patient compliance. In the early days of (nano)biotechnology, these problems caused concern in the medical community, but recently it has been recognized that these challenges can be overcome by developing innovative formulations. This review focuses on the use of vinyl polymer-based materials for the protection and delivery of nucleic acids in cancer. First, an overview of the properties of nucleic acids and their versatility as drugs is provided. Then, key information on the achievements to date, the most effective delivery methods, and the evaluation of functionalization approaches (stimulatory strategies) are critically discussed to highlight the importance of vinyl polymers in the new cancer treatment approaches. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures.

Development of electrospun mats based on hydrophobic hydroxypropyl cellulose derivatives.

In this work, hydroxypropyl cellulose esters (HPCE) with long aliphatic chains were prepared and innovatively used in electrospinning to obtain hydroxypropyl cellulose (HPC)-based mats with enhanced resistance to moist environments. The described approach is very simple and does not require any post-treatment (e.g. cross-linking step) to overcome a major problem concerning the premature loss of properties of cellulose-based materials when in contact with moisture. HPCE-based electrospun mats were characterized in terms of their morphology, swelling ability and in vitro hydrolytic degradation. The mats exhibited a swelling capacity of over 115%, depending on the degree of substitution. The in vitro hydrolytic degradation tests showed the high structural integrity of the mats (< 5% weight loss) over a period of 30 days. The in vitro cytotoxicity tests showed that the mats of HPC esters are cytocompatible and promote the adhesion, proliferation and spreading of NIH3T3 fibroblast cells. These data suggest that the HPCE mats may be interesting materials for wound dressings, as well as for other tissue engineering applications.

Development of light-degradable poly(urethane-urea) hydrogel films.

Biocompatible hydrogels are exciting platforms that have stood out in recent years for their outstanding potential for biomedical applications. For these applications, the ability of the material to respond to an external stimulus can be a relevant addition. This responsiveness allows the material to modify its physical properties in such a way that it can deliver molecules that support the healing process or allow easy removal of the films from the tissue. Among the polymers used to produce these systems, polyurethane (PU) and polyurethane-urea (PUU) are some of the most cited examples. In this work, a new hydrogel-sensitive PUU film is proposed. These films are prepared from polyethylene glycol (PEG) and contain a ROS-responsive telechelic ß-aminoacrylate bond. The hydrogel films showed interesting mechanical and thermal properties, good water uptake and low cytotoxicity, which makes them suitable for biomedical applications. More importantly, the hydrogel films exhibited a light-degradable profile through an innovative ROS-mediated cleavage process, as indicated by the loss of mechanical properties.

Process Development for Flexible Films of Industrial Cellulose Pulp Using Superbase Ionic Liquids.

Due to environmental concerns, more attention has been given to the development of bio-based materials for substitution of fossil-based ones. Moreover, paper use is essential in daily routine and several applications of industrial pulp can be developed. In this study, transparent films were produced by industrial cellulose pulp solubilization in tetramethylguanidine based ionic liquids followed by its regeneration. Films were characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), UV/Vis spectroscopy, proton nuclear magnetic resonance (1H-NMR), dynamic scanning calorimetry (DSC), thermal analysis (TG), and X-ray diffraction (XRD). Mechanical tests showed that films have a good elongation property, up to 50%, depending on ionic liquid incorporation. The influence of the conjugated acid and dissolution temperature on mechanical properties were evaluated. These results revealed the potential of this methodology for the preparation of new biobased films.

Light-Activated Antimicrobial Surfaces Using Industrial Varnish Formulations to Mitigate the Incidence of Nosocomial Infections.

Evidence has shown that hospital surfaces are one of the major vehicles of nosocomial infections caused by drug-resistant pathogens. Smart surface coatings presenting multiple antimicrobial activity mechanisms have emerged as an advanced approach to safely prevent this type of infection. In this work, industrial waterborne polyurethane varnish formulations containing for the first time cationic polymeric biocides (SPBs) combined with photosensitizer curcumin were developed to afford contact-active and light-responsive antimicrobial surfaces. SPBs were prepared by atom transfer radical polymerization, which allows control over the polymer features that influence antimicrobial efficiency (e.g., molecular weight), while natural curcumin was employed to impart photodynamic activity to the surface. Antibacterial testing against Gram-negative Escherichia coli revealed that glass surfaces coated with the new formulations displayed photokilling effect under white-light (42 mW/cm2) irradiation within only 15 min of exposure. In addition, it was observed a combined antimicrobial effect between the two biocides (cationic SPB and curcumin), with a higher reduction in the number of viable bacteria observed for the surfaces containing cationic SPB/curcumin mixtures in comparison with the one obtained for surfaces only with polymer or without biocides. The waterborne industrial varnish formulations allowed the formation of homogeneous films without the need for addition of a coalescing agent, which can be potentially applied in diverse surface substrates to reduce bacterial transmission infections in healthcare environments.

Catalytic Halogen Exchange in Supplementary Activator and Reducing Agent Atom Transfer Radical Polymerization for the Synthesis of Block Copolymers.

Synthesis of block copolymers (BCPs) by catalytic halogen exchange (cHE) is reported, using supplemental activator and reducing agent Atom Transfer Radical Polymerization (SARA ATRP). The cHE mechanism is based on the use of a small amount of a copper catalyst in the presence of a suitable excess of halide ions, for the synthesis of block copolymers from macroinitiators with monomers of mismatching reactivity. cHE overcomes the problem of inefficient initiation in block copolymerizations in which the second monomer provides dormant species that are more reactive than the initiator. Model macroinitiators with low dispersity are prepared and extended to afford well-defined block copolymers of various compositions. Combined cHE/SARA ATRP is therefore a simple and potent polymerization tool for the copolymerization of a wide range of monomers allowing the production of tailored block copolymers.

The Impact of the Addition of Compatibilizers on Poly (lactic acid) (PLA) Properties after Extrusion Process.

Poly (lactic acid) (PLA), due to its biodegradability, biocompatibility, and renewability, is one of the most promising biobased polymers for replacing some of the petrol-based materials. Low flexibility of PLA is overcome, by blending it with olefin-based polymers, such as polypropylene (PP). However, the use of compatibilizing agents is required to attain final materials with suitable mechanical properties. Such agents, although essential, can affect PLA structure and, consequently, the mechanical properties of the PLA. To the best of our knowledge, this issue was never studied, and the results can contribute to achieving the best formulations of PLA-based blends according to their final applications. The thermal and mechanical properties of the extruded PLA, with three different commercial compatibilizing agents, were evaluated with the purpose of demonstrating how the compatibilizers can introduce structural differences into the PLA chain during the extrusion process. The combination of crystallinity, molecular weight, and the morphology of the samples after extrusion determines the final mechanical properties of PLA. Despite being a fundamental study, it is our aim to contribute to the sustainability of PLA-based industries. The addition of a 2.5% concentration of C1 compatibilizer seems to have less influence on the final morphology and mechanical properties of the blends.

Development of red-light cleavable PEG-PLA nanoparticles as delivery systems for cancer therapy.

The development of targeted delivery systems can improve the selectivity of cancer drugs. Additionally, a system that promotes the controlled delivery of the drug triggered by an external stimulus in the exact target tissue is highly desirable. Regarding the light stimulus, the NIR window (650-950 nm) is the most suitable due to its higher capacity of penetration in human tissues and less harmful effects on normal cells. In this work, new red-light-responsive nanoparticles for doxorubicin delivery were developed. The nanoparticles were based on cleavable di-block copolymers of poly(ethylene glycol) (PEG) and poly(lactic acid) (PLA) linked by a red-light sensitive segment (1,2-bis(2-hydroxyethylthio)ethylene, BHETE). The PEG-BHETE-PLA copolymers were synthesized under mild conditions and exhibited a narrow polydispersity. The nanoparticles presented a size between 53 and 133 nm, with a doxorubicin loading capacity between 1.2 and 4.4 wt%. Release study of the encapsulated doxorubicin confirms the light-triggered nanoparticle disassembly process. In vitro cytotoxicity tests in MCF7 cell line, for the light-triggered nanoparticles, showed a decrease in cancer cells' viability higher than 25% compared to non-irradiated cells. Due to the promising results obtained with the light-sensitive PEG-BHETE-PLA nanoparticles, these materials have great potential to be used in drug delivery systems for cancer therapy.

A Preliminary Evaluation of the Pro-Chondrogenic Potential of 3D-Bioprinted Poly(ester Urea) Scaffolds.

Degeneration of articular cartilage (AC) is a common healthcare issue that can result in significantly impaired function and mobility for affected patients. The avascular nature of the tissue strongly burdens its regenerative capacity contributing to the development of more serious conditions such as osteoarthritis. Recent advances in bioprinting have prompted the development of alternative tissue engineering therapies for the generation of AC. Particular interest has been dedicated to scaffold-based strategies where 3D substrates are used to guide cellular function and tissue ingrowth. Despite its extensive use in bioprinting, the application of polycaprolactone (PCL) in AC is, however, restricted by properties that inhibit pro-chondrogenic cell phenotypes. This study proposes the use of a new bioprintable poly(ester urea) (PEU) material as an alternative to PCL for the generation of an in vitro model of early chondrogenesis. The polymer was successfully printed into 3D constructs displaying adequate substrate stiffness and increased hydrophilicity compared to PCL. Human chondrocytes cultured on the scaffolds exhibited higher cell viability and improved chondrogenic phenotype with upregulation of genes associated with type II collagen and aggrecan synthesis. Bioprinted PEU scaffolds could, therefore, provide a potential platform for the fabrication of bespoke, pro-chondrogenic tissue engineering constructs.