Comparative Bioactivities of Chemically Modified Fucoidan and λ-Carrageenan toward Cells Encapsulated in Covalently Cross-Linked Hydrogels.

The sulfated marine polysaccharides, fucoidan and λ-carrageenan, are known to possess anti-inflammatory, immunomodulatory, and cellular protective properties. Although they hold considerable promise for tissue engineering constructs, their covalent cross-linking in hydrogels and comparative bioactivities to cells are absent from the literature. Thus, fucoidan and λ-carrageenan were modified with methacrylate groups and were covalently cross-linked with the synthetic polymer poly(vinyl alcohol)-methacrylate (PVA-MA) to form 20 wt % biosynthetic hydrogels. Identical degrees of methacrylation were confirmed by 1H NMR, and covalent conjugation was determined by using a colorimetric 1,9-dimethyl-methylene blue (DMMB) assay. Pancreatic beta cells were encapsulated in the hydrogels, followed by culturing in the 3D environment for a prolonged period of 32 days and evaluation of the cellular functionality by live/dead, adenosine 5'-triphosphate (ATP) level, and insulin secretion. The results confirmed that fucoidan and λ-carrageenan exhibited ∼12% methacrylate substitution, which generated hydrogels with stable conjugation of the polysaccharides with PVA-MA. The cells encapsulated in the PVA-fucoidan hydrogels demonstrated consistently high ATP levels over the culture period. Furthermore, only cells in the PVA-fucoidan hydrogels retained glucose responsiveness, demonstrating comparatively higher insulin secretion in response to glucose. In contrast, cells in the PVA-λ-carrageenan and the PVA control hydrogels lost all glucose responsiveness. The present work confirms the superior effects of chemically modified fucoidan over λ-carrageenan on pancreatic beta cell survival and function in covalently cross-linked hydrogels, thereby illustrating the importance of differential polysaccharide structural features on their biological effects.

In vitro cell compatibility study of rose bengal-chitosan adhesives.

BACKGROUND AND OBJECTIVES: Photochemical tissue bonding (PTB) using rose bengal (RB) in conjunction with light is an alternative technique to repair tissue without suturing. It was recently demonstrated that laser-irradiated chitosan films, incorporating RB, bonded firmly to calf intestine in vitro. It is thus required to investigate the possible cytotoxic effects of the RB-chitosan adhesive on cells before testing its application to in vivo models. MATERIALS AND METHODS: Adhesive films, based on chitosan and containing ~0.1 wt% RB were fabricated. Their cytotoxicity was assessed by growing human and murine fibroblasts either in media in which adhesive strips had been incubated, or directly on the adhesive. The adhesive was either laser-irradiated or not. Cells were stained after 48 hours with Trypan blue and the number of live and dead cells was recorded for cell viability. RESULTS: Murine and human fibroblasts grew confluent on the adhesives with no apparent morphological changes or any exclusion zone. Cell numbers of murine fibroblasts were not significantly different when cultured in media that was extracted from irradiated (86 ± 7%) and non-irradiated adhesive (89 ± 4%). A similar result was obtained for the human fibroblasts. CONCLUSIONS: These findings support that the RB-chitosan films induced negligible toxicity and growth retardation in murine and human fibroblasts.

3D bioprinting of dual-crosslinked nanocellulose hydrogels for tissue engineering applications.

Hydrogels based on cellulose nanofibrils (CNFs) have been widely used as scaffolds for biomedical applications, however, the poor mechanical properties of CNF hydrogels limit their use as ink for 3D bioprinting in order to generate scaffolds for tissue engineering applications. In this study, a dual crosslinkable hydrogel ink composed of a poly(ethylene glycol) (PEG) star polymer and 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO)-oxidized nanocellulose fibers (CNFs) is presented. As the resulting hydrogel had low structural integrity, at first crosslinking of CNFs was introduced by Ca2+. Strong physical interactions between CNFs and Ca2+ cations allowed easy regulation of the viscosity of the inks for extrusion printing raising the solution viscosity by more than 1.5 times depending on the amount of Ca2+ added. The resulting hydrogel had high structural integrity and was further stabilized in a second step by photo crosslinking of PEG under visible light. In only a few seconds, hydrogels with Young's modulus between ∼10 and 30 kPa were obtained just by altering the CNF and Ca2+ content. 3D printed hydrogels supported fibroblasts with excellent cell viability and proliferation. The dual crosslinkable hydrogel ink herein developed is versatile, easy to prepare, and suitable for 3D printing of bioscaffolds with highly tailored viscoelastic and mechanical properties applicable in a wide range of regenerative medicines.

Fucoidan- and carrageenan-based biosynthetic poly(vinyl alcohol) hydrogels for controlled permeation.

Since the permeation of the inflammatory cytokines into hydrogel scaffolds has been shown to cause dysfunction of encapsulated cells, appropriate design strategies to circumvent this are essential. In the present work, it was hypothesized that highly crosslinked PVA-fucoidan and PVA-carrageenan hydrogels can control permeation of the trefoil-shaped inflammatory cytokine IL-1ß while allowing the permeation of the globular protein albumin. PVA, fucoidan, and carrageenans were functionalized with methacrylate groups and the functionalized polymers were co-crosslinked by UV photopolymerization. The resultant hydrogels were characterized physicochemically and the release of fucoidan and carrageenans was quantified by developing a colorimetric assay, which was validated by XPS analysis. The permeability characteristics of the hydrogels were evaluated using bovine serum albumin (BSA), IgG, and IL-1ß. The results demonstrated an increase in hydrogel swelling through the incorporation of the polysaccharides with minimal overall mass loss. The release studies showed hydrogel stability, where the formulations exhibited ~43% retention of fucoidan and ~60-80% retention of carrageenans consistently up to 7 days. The permeation data revealed very low permeation of IgG and IL-1ß through the hydrogels, with <1% permeation after 24 h, while allowing >6% permeation of BSA. These data indicate that such hydrogels can be used as the basis for cytokine-protective implantable devices for clinical applications.

Photochemical tissue bonding with chitosan adhesive films.

BACKGROUND: Photochemical tissue bonding (PTB) is a promising sutureless technique for tissue repair. PTB is often achieved by applying a solution of rose bengal (RB) between two tissue edges, which are irradiated by a green laser to crosslink collagen fibers with minimal heat production. In this study, RB has been incorporated in chitosan films to create a novel tissue adhesive that is laser-activated. METHODS: Adhesive films, based on chitosan and containing ~0.1 wt% RB were manufactured and bonded to calf intestine by a solid state laser (λ = 532 nm, Fluence~110 J/cm2, spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, tested the bonding strength. K-type thermocouples recorded the temperature (T) at the adhesive-tissue interface during laser irradiation. Human fibroblasts were also seeded on the adhesive and cultured for 48 hours to assess cell growth. RESULTS: The RB-chitosan adhesive bonded firmly to the intestine with adhesion strength of 15 ± 2 kPa, (n = 31). The adhesion strength dropped to 0.5 ± 0.1 (n = 8) kPa when the laser was not applied to the adhesive. The average temperature of the adhesive increased from 26°C to 32°C during laser exposure. Fibroblasts grew confluent on the adhesive without morphological changes. CONCLUSION: A new biocompatible chitosan adhesive has been developed that bonds photochemically to tissue with minimal temperature increase.

Porous chitosan adhesives with L-DOPA for enhanced photochemical tissue bonding.

L-3,4-dihydroxyphenylalanine (L-DOPA) is a naturally occurring catechol that is known to increase the adhesive strength of various materials used for tissue repair. With the aim of fortifying a porous and erodible chitosan-based adhesive film, L-DOPA was incorporated in its fabrication for stronger photochemical tissue bonding (PTB), a repair technique that uses light and a photosensitiser to promote tissue adhesion. The results showed that L-DOPA did indeed increase the tissue bonding strength of the films when photoactivated by a green LED, with a maximum strength recorded of approximately 30 kPa, 1.4 times higher than in its absence. The addition of L-DOPA also did not appreciably change the swelling, mechanical and erodible properties of the film. This study showed that strong, porous and erodible adhesive films for PTB made from biocompatible materials can be obtained through a simple inclusion of a natural additive such as L-DOPA, which was simply mixed with chitosan without any chemical modifications. In vitro studies using human fibroblasts showed no negative effect on cell proliferation indicating that these films are biocompatible. The films are convenient for various surgical applications as they can provide strong tissue support and a microporous environment for cellular infusion without the use of sutures. STATEMENT OF SIGNIFICANCE: Tissue adhesives are not as strong as sutures on wounds under stress. Our group has previously demonstrated that strong sutureless tissue repair can be realised with chitosan-based adhesive films that photochemically bond to tissue when irradiated with green light. The advantage of this technique is that films are easier to handle than glues and sutures, and their crosslinking reactions can be controlled with light. However, these films are not optimal for high-tension tissue regenerative applications because of their non-porous structure, which cannot facilitate cell and nutrient exchange at the wound site. The present study resolves this issue, as we obtained a strong and porous photoactivated chitosan-based adhesive film, by simply using freeze drying and adding L-DOPA.

A One Step Procedure toward Conductive Suspensions of Liposome-Polyaniline Complexes.

Interaction of conjugated polymers with liposomes is an attractive approach that benefits from both systems' characteristics such as electroactivity and enhanced interaction with cells. Conjugated polymer-liposome complexes have been investigated for bioimaging, drug delivery, and photothermal therapy. Their fabrication has largely been achieved by multistep procedures that require first the synthesis and processing of the conjugated polymer. Here, a new one step fabrication approach is reported based on in situ polymerization of a conjugated monomer precursor around liposomes. Polyaniline (PANI) doped with phytic acid is synthesized via oxidative polymerization in the presence of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) vesicles to produce a conductive aqueous suspension of Liposome-PANI complexes. PANI interacts with liposomes without disrupting the bilayer as shown using differential scanning calorimetry and fluorescence quenching studies of the hydrophobic Nile red probe. The electronic conductivity of the Liposome-PANI complexes, which stems from the doped PANI accessible on the liposome surface, is confirmed using conductive atomic force microscopy and electrochemical impedance spectroscopy. Further, short-term in vitro cell studies show that the complexes colocalize with the cell membrane without reducing cell proliferation. This study presents a novel fabrication route to conductive suspensions of conjugated polymer-liposome complexes suitable for potential applications at the biointerface.

Network structure and macromolecular drug release from poly(vinyl alcohol) hydrogels fabricated via two crosslinking strategies.

Injectable hydrogels have potential biomedical applications ranging from tissue fillers to drug delivery vehicles. This study focussed on evaluating the structure of poly(vinyl alcohol) (PVA) hydrogels of variable solid content and high molecular weight model drug release from the networks formed via either conventional photo-polymerization compared with chemical initiation of polymerization using an oxidation-reduction (redox) reaction. Swelling behaviour was characterised in water to assess the structural properties. Model drugs, FITC-Dextran, 20 kDa (FD20) and 4 kDa (FD4) were loaded in the hydrogels prior to curing and drug release studies conducted. Redox-cured hydrogels were more swollen than UV-cured systems, lost approximately 20% of their polymer mass compared to only 5% from UV-cured hydrogels and subsequently exhibited networks of larger mesh sizes. Also, networks of variable solid contents showed different structural properties with systems of higher polymer concentration exhibiting a smaller mesh size. The difference in structural properties of the networks affected release of FD20, being faster in redox-cured than UV-cured hydrogels, and slower from systems of higher solid content. Release of FD4 was faster than FD20 from networks of same solid content. This study suggested that PVA hydrogels can be cured by redox-initiation to function as a controlled delivery system for macromolecular drugs.

Conjugated Polymers in Bioelectronics: Addressing the Interface Challenge.

Conjugated polymers are the material of choice for organic bioelectronic interfaces as they combine mechanical flexibility with electric and ionic conductivity. Their attractive properties are largely demonstrated in vitro, while the in vivo applications are limited to the coating of inorganic electrodes, where they are used to improve the intimate electronic contact between the device and the tissue. However, there has not been a commensurate rise in the in vivo applications of entirely organic implantable electronic devices based on conjugated polymers. To date, there is no comprehensive understanding of how these devices will interface with real biological systems. With the push toward increasingly thinner and more flexible next generation medical implants, this limitation remains a major detractor in the translation of conjugated polymers toward biological applications. This research news article examines the few reported in vivo studies and attempts to establish why there is such a dearth in the literature.

Porous Chitosan Films Support Stem Cells and Facilitate Sutureless Tissue Repair.

Photochemical tissue bonding with chitosan-based adhesive films is an experimental surgical technique that avoids the risk of thermal tissue injuries and the use of sutures to maintain strong tissue connection. This technique is advantageous over other tissue repair methods as it is minimally invasive and does not require mixing of multiple components before or during application. To expand the capability of the film to beyond just a tissue bonding device and promote tissue regeneration, in this study, we designed bioadhesive films that could also support stem cells. The films were modified with oligomeric chitosan to tune their erodibility and made porous through freeze-drying for better tissue integration. Of note, porous adhesive films (pore diameter ∼110 µm), with 10% of the chitosan being oligomeric, could retain similar tissue bonding strengths (13-15 kPa) to that of the nonporous chitosan-based adhesives used in previous studies when photoactivated. When tested in vitro, these films exhibited a mass loss of ∼20% after 7 days, swelling ratios of ∼270-300%, a percentage elongation of ∼90%, and both a tensile strength and Young's modulus of ∼1 MPa. The physical properties of the films were suitable for maintaining the viability and multipotency of bone-marrow-derived human mesenchymal stem cells over the duration of culture. Thus, these biocompatible, photoactivated porous, and erodible adhesive films show promise for applications in controlled cell delivery and regenerative medicine.

Synthesis and characterization of radiopaque iodine-containing degradable PVA hydrogels.

Poly (vinyl alcohol) (PVA) hydrogels are highly attractive for biomedical applications, especially for controlled release of drugs and proteins. Recently, degradable PVA hydrogels have been described, having the advantage that the material disappears over time from the implantation site. Herein, we report the synthesis of radiopaque degradable PVA, which gives a further advantage that the position of the hydrogel can precisely be determined by X-ray fluoroscopy. Radiopacity has been introduced by replacing 0.5% of the pendent alcohol groups on the PVA with 4-iodobenzoylchloride. This level of substitution rendered the polymer adequately radiopaque. The subsequent modification of 0.8% of the pendent hydroxyl groups with an ester acrylate functional group allowed for cross-linking of the macromers. The radiopaque hydrogels degraded over a time span of 140 days. Rheology data suggested that the macromer solutions were appropriate for injection.

Drug-delivery study and estimation of polymer-solvent interaction parameter for bisacrylate ester-modified Pluronic hydrogels.

In this study, Pluronic F127 hydrogels were characterised as an injectable system for the controlled release of drugs with variable molecular weights (FITC-Dextran at 70 and 40 kDa). In addition, the polymer-solvent interaction parameter (chi) was successfully estimated. Pluronic hydrogels (10-25 wt.%) were redox cured and their swelling behaviour investigated in PBS (pH 7.45) at 37 degrees C. After swelling to equilibrium, the hydrogels were compressed and the rubber-elasticity theory was applied to evaluate chi. Tensile tests proved the hydrogels were elastic and their chi values ranged between 0.50 and 0.53. The full drug load could be delivered over a period of approximately 15 h suggesting that redox cured Pluronic F127 hydrogels can function as injectable systems for controlled and sustained release of macromolecules.

A flexible polyaniline-based bioelectronic patch.

Bioelectronic materials based on conjugated polymers are being developed in the hope to interface with electroresponsive tissues. We have recently demonstrated that a polyaniline chitosan patch can efficiently electro-couple with cardiac tissue modulating its electrophysiology. As a promising bioelectronic material that can be tailored to different types of devices, we investigate here the impact of varying the synthesis conditions and time of the in situ polymerization of aniline (An) on the sheet resistance of the bioelectronic patch. The sheet resistance increases significantly for samples that have either the lowest molar ratio of oxidant to monomer or the highest molar ratio of dopant to monomer, while the polymerization time does not have a significant effect on the electrical properties. Conductive atomic force microscopy reveals that the patch with the lowest sheet resistance has a connected network of the conductive phase. In contrast, patches with higher sheet resistances exhibit conductive areas of lower current signals or isolated conductive islands of high current signals. Having identified the formulation that results in patches with optimal electrical properties, we used it to fabricate patches that were implanted in rats for two weeks. It is shown that the patch retains an electroactive nature, and only mild inflammation is observed with fibrous tissue encapsulating the patch.

The effect of redox polymerisation on degradation and cell responses to poly (vinyl alcohol) hydrogels.

Biocompatible, degradable hydrogel systems that can cure in situ following injection as a liquid are useful as a base for tissue engineering and drug delivery. In this study, poly (vinyl alcohol) (PVA) polymers were modified with degradable crosslinkers and formulated for either ultraviolet (UV) light initiation or chemical initiation using an oxidation/reduction (redox) curing method. A major objective was to compare the properties of degradable PVA hydrogels formed via two routes of curing. The effect of macromer concentration, degree of hydrolysis and functional group density on the degradation profiles was investigated. Also, since the hydrogels have been designed to be injected as a liquid for in situ curing, the effect of modified macromer solutions and degradation products on cell growth was investigated. Total degradation times ranged from approximately 20 days up to 120 days and increased in direct proportion with percent macromer. Initiation method (UV or redox) did not significantly impact on time for total degradation. While aqueous solutions of the modified macromer induced some cell growth inhibition, mainly associated with oxidative solutions, degradation products showed relatively low cell growth inhibition. Degradable PVA hydrogels tailored to produce networks with various degradation profiles can be cured by redox initiation and have potential as injectable polymers for soft-tissue engineering and drug delivery.

A conducting polymer with enhanced electronic stability applied in cardiac models.

Electrically active constructs can have a beneficial effect on electroresponsive tissues, such as the brain, heart, and nervous system. Conducting polymers (CPs) are being considered as components of these constructs because of their intrinsic electroactive and flexible nature. However, their clinical application has been largely hampered by their short operational time due to a decrease in their electronic properties. We show that, by immobilizing the dopant in the conductive scaffold, we can prevent its electric deterioration. We grew polyaniline (PANI) doped with phytic acid on the surface of a chitosan film. The strong chelation between phytic acid and chitosan led to a conductive patch with retained electroactivity, low surface resistivity (35.85 ± 9.40 kilohms per square), and oxidized form after 2 weeks of incubation in physiological medium. Ex vivo experiments revealed that the conductive nature of the patch has an immediate effect on the electrophysiology of the heart. Preliminary in vivo experiments showed that the conductive patch does not induce proarrhythmogenic activities in the heart. Our findings set the foundation for the design of electronically stable CP-based scaffolds. This provides a robust conductive system that could be used at the interface with electroresponsive tissue to better understand the interaction and effect of these materials on the electrophysiology of these tissues.

Lysozyme depolymerization of photo-activated chitosan adhesive films.

Effective tissue bioadhesion of rose bengal-chitosan films can be achieved by photoactivation using a green laser. In this study, lysozyme was incorporated in these films to enhance the rate of depolymerization and assess the laser impact on lysozyme. The lysozyme loaded films exhibited a 21% mass loss after 4 weeks implantation in rats while control films (without lysozyme) had only 7% mass loss. Capillary electrophoresis-mass spectroscopy showed that chitosan degraded into monomers and oligomers of glucosamine and N-acetyl-glucosamine. Irradiation with laser did not affect the depolymerization of adhesive by lysozyme suggesting that the inclusion of lysozyme in the bioadhesive is a viable technique for tailoring the depolymerization.

Tissue repair strength using chitosan adhesives with different physical-chemical characteristics.

A range of chitosan-based biomaterials have recently been used to perform sutureless, laser-activated tissue repair. Laser-activation has the advantage of bonding to tissue through a non-contact, aseptic mechanism. Chitosan adhesive films have also been shown to adhere to sheep intestine strongly without any chemical modification to chitosan. In this study, we continue to investigate chitosan adhesive films and explore the impact on the tissue repair strength and tensile strength characteristics of four types of adhesive film based on chitosan with different molecular weight and degree of deacetylation. Results showed that adhesives based on chitosan with medium molecular weight achieved the highest bonding strength, tensile strength and E-modulus when compared to the other adhesives.

Laser-activated adhesive films for sutureless median nerve anastomosis.

A novel chitosan adhesive film that incorporates the dye 'Rose Bengal' (RB) was used in conjunction with a green laser to repair transected rat median nerves in vivo. Histology and electrophysiological recording assessed the impact of the laser-adhesive technique on nerves. One week post-operatively, the sham-control group (laser-adhesive technique applied on un-transected nerves) conserved the average number and size of myelinated fibres in comparison to its contralateral side and electrophysiological recordings demonstrated no significant difference with un-operated nerves. Twelve weeks after the laser-adhesive anastomoses, nerves were in continuity with regenerated axons that crossed the anastomotic site.

Fabrication and application of rose bengal-chitosan films in laser tissue repair.

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two tissue edges(1,2). These are then irradiated by a laser that is selectively absorbed by the RB. The resulting photochemical reactions supposedly crosslink the collagen fibers in the tissue with minimal heat production(3). In this report, RB has been incorporated in thin chitosan films to fabricate a novel tissue adhesive that is laser-activated. Adhesive films, based on chitosan and containing ~0.1 wt% RB, are fabricated and bonded to calf intestine and rat tibial nerves by a solid state laser (λ=532 nm, Fluence~110 J/cm(2), spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, is used to test the bonding strength. The RB-chitosan adhesive bonds firmly to the intestine with a strength of 15 ± 6 kPa, (n=30). The adhesion strength drops to 2 ± 2 kPa (n=30) when the laser is not applied to the adhesive. The anastomosis of tibial nerves can be also completed without the use of sutures. A novel chitosan adhesive has been fabricated that bonds photochemically to tissue and does not require sutures.