Photoswitchable Micelles for the Control of Singlet-Oxygen Generation in Photodynamic Therapies.

Inadvertent photosensitizer-activation and singlet-oxygen generation hampers clinical application of photodynamic therapies of superficial tumors or subcutaneous infections. Therefore, a reversible photoswitchable system was designed in micellar nanocarriers using ZnTPP as a photosensitizer and BDTE as a photoswitch. Singlet-oxygen generation upon irradiation didnot occur in closed-switch micelles with ZnTPP/BDTE feeding ratios >1:10. Deliberate switch closure/opening within 65-80 min was possible through thin layers of porcine tissue in vitro, increasing for thicker layers. Inadvertent opening of the switch by simulated daylight, took several tens of hours. Creating deliberate cell damage and prevention of inadvertent damage in vitro and in mice could be done at lower ZnTPP/BDTE feeding ratios (1:5) in open-switch micelles and at higher irradiation intensities than inferred from chemical clues to generate singlet-oxygen. The reduction of inadvertent photosensitizer activation in micellar nanocarriers, while maintaining the ability to kill tumor cells and infectious bacteria established here, brings photodynamic therapies closer to clinical application.

Changes in lens stiffness due to capsular opacification in accommodative lens refilling.

Accommodation may be restored to presbyopic lenses by refilling the lens capsular bag with a soft polymer. After this accommodative lens refilling prevention of capsular opacification is a requirement, since capsular opacification leads to a decreased clarity of the refilled lens. It has been hypothesized that capsular fibrosis causing the capsular opacification results in increased stiffness of the lens capsular bag, therewith contributing to a decrease in accommodative amplitude of the lens. However, the change in viscoelastic properties of refilled lenses due to capsular fibrosis has never been measured directly. In this study we examined natural lenses from enucleated porcine eyes and refilled lenses directly after refilling and after three months of culturing, when capsular fibrosis had developed, and determined their viscoelastic properties with a low load compression tester. Control refilled lenses were included in which capsular opacification was prevented by treatment with actinomycin D. We related lens stiffening to the degree of capsular opacification, as derived from the microscopic images taken with a confocal laser scanning microscope. Overall, the refilled lenses directly after refilling were softer than refilled lenses after three months of culturing, and refilled lenses treated with actinomycin D were softer compared with untreated refilled lenses. The degree of capsular opacification as assessed by microscopy corresponds to an increase in lens stiffness. This indicates that the viscoelastic properties of the refilled lens are influenced by capsular fibrosis and modulated by treatment of the lens epithelium. In conclusion, this study shows that the development of capsular fibrosis negatively affects the viscoelastic properties of isolated, cultured refilled lenses.

Macrophage response to staphylococcal biofilms on crosslinked poly(ethylene) glycol polymer coatings and common biomaterials in vitro.

Biomaterial-associated-infections (BAI) are serious clinical complications that threaten the longevity of implanted devices and lead to high morbidity and mortality. Poly(ethylene)glycol (PEG) coatings have been studied as a strategy to reduce the incidence of BAI by reducing protein deposition that promotes pathogen adhesion and growth on device surfaces. Despite their effectiveness to reduce protein adsorption and a hundred-fold reduction in bacterial adhesion, PEG-based coatings still facilitate weak bacterial adhesion that can form an initial basis for biofilms. Here, we describe a methodology enabling direct, quantitative and detailed qualitative in situ observation of macrophage morphology, migration and phagocytosis of bacteria. In vitro interaction of macrophages with Staphylococcus epidermidis 3399 adhering to commercial, crosslinked PEG-based coatings (OptiChem®) was compared with fluorinated ethylene propylene, silicone rubber and glass. Adhesion, phagocytosis and migration were studied real-time in a parallel-plate-flow-chamber. Macrophages cultured on OptiChem® coatings showed enhanced migration and phagocytosis of bacteria compared to common biomaterials. Bacterial clearance per macrophage on both inert and reactive OptiChem® coatings were about three times higher than on the common biomaterials studied, corresponding with up to 70% reduction in bacterial numbers on OptiChem®, whereas on the biomaterials less than 40% bacterial reduction was obtained. These findings show that bacterial clearance from cross-linked PEG-based coatings by macrophages is more effective than from common biomaterials, possibly resulting from weak adhesion of bacteria on Optichem®. Moreover, macrophages exhibit higher mobility on Optichem® retaining an improved capability to clear bacteria from larger areas than from other common biomaterials, where they appear more immobilized.

Long-term prevention of capsular opacification after lens-refilling surgery in a rabbit model.

PURPOSE: To reduce capsular opacification by a peri-surgical treatment of the lens capsule with drugs in an in vivo rabbit model. Lens-refilling surgery is a potential therapeutic intervention to treat patients with a cataract lens. The lens material is replaced with an injectable (bio)polymer that retains the natural mechanical and optical lens properties, therewith allowing accommodation. The occurrence of capsular opacification mediated by lens epithelial cells negatively affects accommodation and vision and should be avoided in this lens restoration approach. METHODS: An in vivo rabbit animal model was used with lens replacement with a silicone-based gel-like polymer and concurrent treatment of the lens epithelium with drugs. A case-study approach was applied as both drug combinations and implantation times were varied. The following drugs were investigated for their potential to prevent capsular opacification long-term: actinomycin D, methotrexate, paclitaxel and Tween-20. All were administered in a hyaluronic acid vehicle. The rabbits were clinically followed for periods up to 4 years postimplantation. Eyes, corneas and lenses were analysed post-mortem using MRI and confocal microscopy. RESULTS: Treatment combinations containing actinomycin D generally led to the least appearance of capsular fibrosis. The use of Tween-20 or paclitaxel without actinomycin D resulted in much earlier and pronounced fibrotic responses. The aspect of capsular opacification was highly variable in individual animals. Application of the drugs in a hyaluronic acid vehicle appeared to be a safe method that spared the corneal endothelium. CONCLUSION: The feasibility of long-term prevention of fibrosis over a period of more than 4 years has been demonstrated in lens refilling in the rabbit model.

Nanocarriers with conjugated antimicrobials to eradicate pathogenic biofilms evaluated in murine in vivo and human ex vivo infection models.

Conventional antimicrobials are becoming increasingly ineffective for treating bacterial infection due to the emergence of multi-drug resistant (MDR) pathogens. In addition, the biofilm-mode-of-growth of infecting bacteria impedes antimicrobial penetration in biofilms. Here, we report on poly(ethylene)glycol-poly(ß-amino esters) (PEG-PAE) micelles with conjugated antimicrobials, that can uniquely penetrate biofilms, target themselves to bacterial cell surfaces once inside the low-pH environment of a biofilm and release conjugated antimicrobials through degradation of their ester-linkage with PAE by bacterial lipases. In vitro, PEG-PAE micelles with conjugated Triclosan (PEG-PAE-Triclosan) yielded no inadvertent leakage of their antimicrobial cargo and better killing of MDR Staphylococcus aureus, Escherichia coli and oral streptococcal biofilms than Triclosan in solution. In mice, PEG-PAE-Triclosan micelles with conjugated Triclosan yielded better eradication efficacy towards a MDR S. aureus-infection compared with Triclosan in solution and Triclosan-loaded micelles at equal Triclosan-equivalent concentrations. Ex vivo exposure of multi-species oral biofilms collected from orthodontic patients to PEG-PAE-Triclosan micelles, demonstrated effective bacterial killing at 30-40 fold lower Triclosan-equivalent concentrations than achieved by Triclosan in solution. Importantly, Streptococcus mutans, the main causative organism of dental caries, was preferentially killed by PEG-PAE-Triclosan micelles. Thus PEG-PAE-Triclosan micelles present a promising addendum to the decreasing armamentarium available to combat infection in diverse sites of the body. STATEMENT OF SIGNIFICANCE: pH-adaptive polymeric micelles with conjugated antimicrobials can efficiently eradicate infectious biofilms from diverse body sites in mice and men. An antimicrobial was conjugated through an ester-linkage to a poly(ethylene glycol) (PEG)/poly(ß-amino ester) block copolymer to create micellar nanocarriers. Stable micelle structures were formed by the hydrophobic poly(ß-amino ester) inner core and a hydrophilic PEG outer shell. Thus formed PEG-PAE-Triclosan micelles do not lose their antimicrobial cargo underway to an infection site through the blood circulation, but penetrate and accumulate in biofilms to release antimicrobials once inside a biofilm through degradation of its ester-linkage by bacterial lipases, to kill biofilm-embedded bacteria at lower antimicrobial concentrations than when applied in solution. PEG-PAE-Triclosan micelles effectively eradicate biofilms of multi-drug-resistant pathogens and oral bacteria, most notably highly cariogenic Streptococcus mutans, in mice and men respectively, and possess excellent clinical translation possibilities.

In vivo experiments with tracheostoma tissue connector prototypes.

In cancer patients who have undergone total surgical removal of the larynx, ideally voice rehabilitation should be performed using a shunt valve (placed in a fistula of the tracheo-esophageal wall) and a tracheostoma valve (TSV) to enable hands-free tracheo-esophageal speech. A tracheostoma is created by suturing the trachea into the lower anterior part of the neck, and a TSV is a device that can be placed at the stoma. Unfortunately, many patients are unable to use a TSV, mainly due to fixation difficulties. To improve the fixation of the TSV, tracheostoma tissue connector (TS-TC) prototypes have been designed. Prototype 1 consisted of a titanium ring, inner diameter 30 mm, with a circular polypropylene mesh glued to it with silicone adhesive. Four holes had been drilled into the ring for the insertion of sub- and percutaneous screws. Prototype 2 consisted of a silicone rubber ring, inner diameter 30 mm, combined with polypropylene mesh and four titanium inserts that functioned as a base plate for the insertion of sub- and percutaneous screws. In adult female goats a tracheostoma was created and the prototypes were implanted. After 6 weeks of subcutaneous implantation, percutaneous screws were inserted. After twelve weeks, the experiment was terminated and the implants with the surrounding tissues were processed and examined histologically. The clinical appearance during weeks 7-12 varied from very poor to relatively good. Histologically, the implants showed a uniform inflammatory response. We found that all the tissue surrounding the screws showed signs of epithelial down growth. It was concluded that the two-stage implantation procedure of our prototype TS-TCs in this animal model was unsuccessful. Additional research efforts are necessary to improve tissue immobilization and to devise reliable fixation systems for TSVs.

Screening Platform for Cell Contact Guidance Based on Inorganic Biomaterial Micro/nanotopographical Gradients.

High-throughput screening (HTS) methods based on topography gradients or arrays have been extensively used to investigate cell-material interactions. However, it is a huge technological challenge to cost efficiently prepare topographical gradients of inorganic biomaterials due to their inherent material properties. Here, we developed a novel strategy translating PDMS-based wrinkled topography gradients with amplitudes from 49 to 2561 nm and wavelengths between 464 and 7121 nm to inorganic biomaterials (SiO2, Ti/TiO2, Cr/CrO3, and Al2O3) which are frequently used clinical materials. Optimal substratum conditions promoted human bone-marrow derived mesenchymal stem cell alignment, elongation, cytoskeleton arrangement, filopodia development as well as cell adhesion in vitro, which depended both on topography and interface material. This study displays a positive correlation between cell alignment and the orientation of cytoskeleton, filopodia, and focal adhesions. This platform vastly minimizes the experimental efforts both for inorganic material interface engineering and cell biological assessments in a facile and effective approach. The practical application of the HTS technology is expected to aid in the acceleration of developments of inorganic clinical biomaterials.

Self-assembled nanofiber coatings for controlling cell responses.

Nanofibers are thought to enhance cell adhesion, growth, and function. We demonstrate that the choice of building blocks in self-assembling nanofiber systems can be used to control cell behavior. The use of 2 D-coated, self-assembled nanofibers in controlling lens epithelial cells, fibroblasts, and mesenchymal stem cells was investigated, focusing on gene and protein expression related to the fibrotic response. To this end, three nanofibers with different characteristics (morphology, topography, and wettability) were compared with two standard materials frequently used in culturing cells, TCPS, and a collagen type I coating. Cell metabolic activity, cell morphology, and gene and protein expression were analyzed. The most hydrophilic nanofiber with more compact network consisting of small fibers proved to provide a beneficial 2 D environment for cell proliferation and matrix formation while decreasing the fibrotic/stress behavior in all cell lines when compared with TCPS and the collagen type I coating. This nanofiber demonstrates the potential to be used as a biomimetic coating to study the development of fibrosis through epithelial-to-mesenchymal transition. This study also shows that nanofiber structures do not enhance cell function by definition, because the physico-chemical characteristics of the nanofibers influence cell behavior as well and actually can be used to regulate cell behavior toward suboptimal performance. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2252-2265, 2017.

Accommodative lens refilling in rhesus monkeys.

PURPOSE: Accommodation can be restored to presbyopic human eyes by refilling the capsular bag with a soft polymer. This study was conducted to test whether accommodation, measurable as changes in optical refraction, can be restored with a newly developed refilling polymer in a rhesus monkey model. A specific intra- and postoperative treatment protocol was used to minimize postoperative inflammation and to delay capsular opacification. METHODS: Nine adolescent rhesus monkeys underwent refilling of the lens capsular bag with a polymer. In the first four monkeys (group A) the surgical procedure was followed by two weekly subconjunctival injections of corticosteroids. In a second group of five monkeys (group B) a treatment intended to delay the development of capsular opacification was applied during the surgery, and, in the postoperative period, eye drops and two subconjunctival injections of corticosteroids were applied. Accommodation was stimulated with carbachol iontophoresis or pilocarpine and was measured with a Hartinger refractometer at regular times during a follow-up period of 37 weeks in five monkeys. In one monkey, lens thickness changes were measured with A-scan ultrasound. RESULTS: In group A, refraction measurement was possible in one monkey. In the three other animals in group A, postoperative inflammation and capsular opacification prevented refraction measurements. In group B, the maximum accommodative amplitude of the surgically treated eyes was 6.3 D. In three monkeys the accommodative amplitude decreased to almost 0 D after 37 weeks. In the two other monkeys, the accommodative amplitude remained stable at +/-4 D during the follow-up period. In group B, capsular opacification developed in the postoperative period, but refraction measurements could still be performed during the whole follow-up period of 37 weeks. CONCLUSIONS: A certain level of accommodation can be restored after lens refilling in adolescent rhesus monkeys. During the follow-up period refraction measurements were possible in all five monkeys that underwent the treatment designed to prevent inflammation and capsular opacification.

Effects of peptide ratios in nanofibre-based hydrogels for the prevention of capsular opacification.

PURPOSE: To moderate the capsular opacification (CO) response after lens surgery, an experimental study was performed in which nanofibre-based hydrogels (nanogels) with different ratios of attached peptides were applied to provide extracellular matrix-related cues for lens epithelial cells (LECs) in a porcine eye model. METHODS: The lens content was removed, and the capsules were refilled with nanogel. Lenses were divided into two groups, the first group (n = 34) was refilled with nanogels containing different ratios of two laminin-derived peptides (IKVAV + YIGSR), and the latter group (n = 26) was refilled with nanogel combinations of a fibronectin-derived and a type IV collagen-derived peptide (RGDS + DGEA). Two lenses were refilled with culture medium to investigate the effect of the medium on LECs. After refilling, lenses were extracted and cultured for 3 weeks. Lens epithelial cells (LECs) were assessed for morphology and alpha-smooth muscle actin (αSMA) expression using confocal laser scanning microscopy. RESULTS: Differences were seen in cell morphology between lenses refilled with nanogels with IKVAV + YIGSR and RGDS + DGEA peptides. In nanogels with IKVAV + YIGSR peptides, differences in LEC morphology were largest when ratios between the peptides were unequal, whereas LEC responses from the RGDS + DGEA refilled groups showed variation in LEC morphology dependent on the total quantity of mixed-in peptides. The culture medium did not induce proliferation or transformation of LECs. CONCLUSIONS: Ratios and concentrations of cell adhesion-mediating peptides both can direct the LEC response, depending on the adhesion molecules of origin, by influencing LEC proliferation and transformation. Nanogels with incorporated peptides may be tuned towards CO prevention.

Non-Covalently Stabilized Alginate Hydrogels as Functional Cell Scaffold Material.

Biopolymers are an attractive class of compounds for being used in biomedical applications as they are widely available from biomass. Their drawback is the lack of mechanical stability and the ability to tune this properly. Covalent chemical cross-linking is an often used approach but it limits usability due to legislation as well as the need of advanced and specialized knowledge by end users such as clinicians. Here, increased and tunable mechanical properties are achieved of alginate-based hydrogels with non-covalent approaches using linear polyethyleneimine (LPEI) as a polyelectrolyte rather than only multivalent metal ions (Ca2+ ). Gel stiffness increases with increasing LPEI content. Gel morphology changes from a thin fibrous mesh for alginate-Ca2+ to thicker fibrous networks when LPEI is introduced. The gels are able to efficiently release encapsulated small molecular dyes and the gels are able to host cells. For the cell encapsulation human skin fibroblasts (HSkF) and human bone marrow-derived mesenchymal stem cells (hBM-MSC) are used. HSkF can be successfully incorporated without diminished viability while the matrix components and gel preparation method are not compatible with hBM-MSC. The newly developed alginate-based system is regarded as a potential candidate for wound dressing materials.

Directional nanotopographic gradients: a high-throughput screening platform for cell contact guidance.

A novel approach was developed using PDMS-substrates with surface-aligned nanotopography gradients, varying unidirectional in amplitude and wavelength, for studying cell behavior with regard to adhesion and alignment. The gradients target more surface feature parameters simultaneously and provide more information with fewer experiments and are therefore vastly superior with respect to individual topography substrates. Cellular adhesion experiments on non-gradient aligned nanowrinkled surfaces displayed a linear relationship of osteoblast cell adhesion with respect to topography aspect ratio. Additionally, an aspect ratio of 0.25 was found to be most efficient for cell alignment. Modification of the surface preparation method allowed us to develop an approach for creating surface nanotopography gradients which innovatively provided a superior data collection with fewer experiments showing that 1) low amplitude with small wavenumber is best for osteoblast cell adhesion 2) indeed higher aspect ratios are favorable for alignment however only with features between 80-180 nm in amplitude and 450-750 nm in wavelength with a clear transition between adhesion and alignment efficiency and 3) disproved a linear relationship of cell adhesion towards aspect ratio as was found for single feature substrate analysis.

Macrophage phagocytic activity toward adhering staphylococci on cationic and patterned hydrogel coatings versus common biomaterials.

Biomaterial-associated-infection causes failure of biomaterial implants. Many new biomaterials have been evaluated for their ability to inhibit bacterial colonization and stimulate tissue-cell-integration, but neglect the role of immune cells. This paper compares macrophage phagocytosis of adhering Staphylococcus aureus on cationic-coatings and patterned poly(ethylene)glycol-hydrogels versus common biomaterials and stainless steel in order to identify surface conditions that promote clearance of adhering bacteria. Staphylococci were allowed to adhere and grow on the materials in a parallel-plate-flow-chamber, after which murine macrophages were introduced. From the decrease in the number of adhering staphylococci, phagocytosis-rates were calculated, and total macrophage displacements during an experiment determined. Hydrophilic surfaces had the lowest phagocytosis-rates, while common biomaterials had intermediate phagocytosis-rates. Patterning of poly(ethylene)glycol-hydrogel coatings increased phagocytosis-rates to the level of common biomaterials, while on cationic-coatings phagocytosis-rates remained relatively low. Likely, phagocytosis-rates on cationic coatings are hampered relative to common biomaterials through strong electrostatic binding of negatively-charged macrophages and staphylococci. On polymeric biomaterials and glass, phagocytosis-rates increased with macrophage displacement, while both parameters increased with biomaterial surface hydrophobicity. Thus hydrophobicity is a necessary surface condition for effective phagocytosis. Concluding, next-generation biomaterials should account for surface effects on phagocytosis in order to enhance the ability of these materials to resist biomaterial-associated-infection.

Plasma-treated polystyrene surfaces: model surfaces for studying cell-biomaterial interactions.

Biocompatibility of biomaterials relates, amongst others, to the absence of adverse cellular reactions and modulation of cell adhesion and subsequent responses. With respect to tissue-engineering applications, most materials need to evoke cell adhesion and spreading, while potentially displaying differential cell function. Adhesion has frequently been studied in a controlled fashion, using adhesion-supporting and -inhibiting substrata. The aim of this study is to create a panel of related materials with gradually changing surface characteristics in order to sustain similar individual cell adhesion and spreading, yet different cell population behaviour. A series of polystyrene materials was created with increasing oxygen surface incorporation and, concurrently, decreasing water-contact angles. Individual cells adhered and spread on all surfaces whilst showing well-developed focal adhesions and stress fibres. Cell populations demonstrated a decreased growth on surfaces with lower wettability. The biochemical activity of cell populations was not influenced by the surface treatment, but cell proliferation on surfaces increased with increasing oxygen incorporation. Furthermore, surface coverage with assembled fibronectin matrix was higher on the substrata with higher wettability. Finally, the expression of the adhesion-related proteins cadherin-5, focal adhesion kinase and RhoA was increased on surfaces with higher wettability. Further explorations of the cell biological basis of the observed differential behaviour will give more detailed answers on the rules governing cell-material interactions.

Prevention of capsule opacification after accommodating lens refilling: pilot study of strategies evaluated in a monkey model.

PURPOSE: To test 2 strategies to prevent capsule opacification after accommodating lens refilling in a rhesus monkey model. SETTING: Animal laboratory and laboratory of European university medical centers. DESIGN: Experimental study. METHODS: Six rhesus monkeys had refilling of the lens capsular bag. In the first strategy, before it was filled with a silicone polymer, the capsular bag was treated with noncommercial sodium hyaluronate 1.0% containing cytotoxic substances. In the second strategy, the capsular bag was filled with clinically used sodium hyaluronate 1.0% (Healon) after treatment with actinomycin-D. Slitlamp inspection was performed during a follow-up of 40 to 50 weeks. After enucleation, magnetic resonance images were obtained and confocal fluorescence imaging was performed. RESULTS: Using the first strategy, capsule opacification developed in all eyes. Using the second strategy, 1 monkey did not develop capsule opacification after a 9-month follow-up. In a second monkey, the lens capsule remained clear for 3 months, after which the hyaluronate refill material was exchanged with a silicone polymer and capsule opacification developed. Combining these results with those in a previous study, the difference in opacification between silicone and sodium hyaluronate as refilling materials was statistically significant (P<.01). CONCLUSIONS: That no capsular bag fibrosis occurred in the presence of hyaluronate suggests that the properties of hyaluronate are the reason that remaining lens epithelial cells do not develop into fibrotic cells. The choice of a suitable lens-refilling material prevents the development of capsule opacification. FINANCIAL DISCLOSURE: Mr. Terwee was an employee of Abbott Medical Optics B.V. during the study period. No other author has a financial or proprietary interest in any material or method mentioned.

Physical properties and erosion behavior of poly(trimethylene carbonate-co-ε-caprolactone) networks.

Form-stable resorbable networks are prepared by gamma irradiating trimethylene carbonate (TMC)- and ε-caprolactone (CL)-based (co)polymer films. To evaluate their suitability for biomedical applications, their physical properties and erosion behavior are investigated. Homopolymer and copolymer networks that are amorphous at room temperature are flexible and rubbery with elastic moduli ranging from 1.8 ± 0.3 to 5.2 ± 0.4 MPa and permanent set values as low as 0.9% strain. The elastic moduli of the semicrystalline networks are higher and range from 61 ± 3 to 484 ± 34 MPa. The erosion behavior of (co)polymer networks is investigated in vitro using macrophage cultures, and in vivo by subcutaneous implantation in rats. In macrophage cultures, as well as upon implantation, a surface erosion process is observed for the amorphous (co)polymer networks, while an abrupt decrease in the rate and a change in the nature of the erosion process are observed with increasing crystallinity. These resorbable and form-stable networks with tuneable properties may find application in a broad range of biomedical applications.

An annulus fibrosus closure device based on a biodegradable shape-memory polymer network.

Injuries to the intervertebral disc caused by degeneration or trauma often lead to tearing of the annulus fibrosus (AF) and extrusion of the nucleus pulposus (NP). This can compress nerves and cause lower back pain. In this study, the characteristics of poly(D,L-lactide-co-trimethylene carbonate) networks with shape-memory properties have been evaluated in order to prepare biodegradable AF closure devices that can be implanted minimally invasively. Four different macromers with (D,L-lactide) to trimethylene carbonate (DLLA:TMC) molar ratios of 80:20, 70:30, 60:40 and 40:60 with terminal methacrylate groups and molecular weights of approximately 30 kg mol(-1) were used to prepare the networks by photo-crosslinking. The mechanical properties of the samples and their shape-memory properties were determined at temperatures of 0 °C and 40 °C by tensile tests- and cyclic, thermo-mechanical measurements. At 40 °C all networks showed rubber-like behavior and were flexible with elastic modulus values of 1.7-2.5 MPa, which is in the range of the modulus values of human annulus fibrosus tissue. The shape-memory characteristics of the networks were excellent with values of the shape-fixity and the shape-recovery ratio higher than 98 and 95%, respectively. The switching temperatures were between 10 and 39 °C. In vitro culture and qualitative immunocytochemistry of human annulus fibrosus cells on shape-memory films with DLLA:TMC molar ratios of 60:40 showed very good ability of the networks to support the adhesion and growth of human AF cells. When the polymer network films were coated by adsorption of fibronectin, cell attachment, cell spreading, and extracellular matrix production was further improved. Annulus fibrosus closure devices were prepared from these AF cell-compatible materials by photo-polymerizing the reactive precursors in a mold. Insertion of the multifunctional implant in the disc of a cadaveric canine spine showed that these shape-memory devices could be implanted through a small slit and to some extent deploy self-sufficiently within the disc cavity.

Phagocytosis of bacteria adhering to a biomaterial surface in a surface thermodynamic perspective.

Bacterial biofilms can increase the pathogenicity of infection and constitute a major problem in modern health-care, especially on biomaterial implants and devices. Biofilms are difficult to eradicate by the host immune system, even with antibiotics, and have been the number one cause of biomaterial implant and device failure for decades. Therefore, it is important to understand how immune cells interact with adhering pathogens. This study firstly aims to develop a simple method to quantify phagocytosis of six different strains of staphylococci adhering on a surface with phase-contrast-microscopy. Phagocytosis of adhering staphylococci to a glass surface by phagocytes was quantified in a parallel plate flow chamber, and expressed as a phagocytosis rate, accounting for the number of adhering staphylococci initially present and for the duration of phagocytosis. Murine macrophages were more effective in clearing staphylococci from a surface than human phagocytes, which require differentiation from their monocyte or promyelocytic state during an experiment. Direct visualization of internalization of a GFP-modified S. aureus strain inside phagocytes confirmed the validity of the method proposed. As a second aim, the differences in phagocytosis rates observed were investigated on a surface thermodynamic basis using measured contact angles of liquids on macroscopic lawns of staphylococci and phagocytes, confirming that phagocytosis of adhering pathogens can be regarded as a surface phenomenon. In addition, surface thermodynamics revealed that phagocytosis of adhering pathogens is determined by an interplay of physical attraction between pathogens and phagocytes and the influence of chemo-attractants. For future studies, these results will help to place in vitro experiments and murine infection models in better perspective with respect to human ones.

Biodegradable nanocomposite hydrogel structures with enhanced mechanical properties prepared by photo-crosslinking solutions of poly(trimethylene carbonate)-poly(ethylene glycol)-poly(trimethylene carbonate) macromonomers and nanoclay particles.

Soft hydrogels with elasticity modulus values lower than 100kPa that are tough and biodegradable are of great interest in medicine and in tissue engineering applications. We have developed a series of soft hydrogel structures from different methacrylate-functionalized triblock copolymers of poly(ethylene glycol) (PEG) with poly(trimethylene carbonate) (PTMC) by photo-crosslinking aqueous solutions of the macromonomers in 2.5 and 5wt.% colloidal dispersions of clay nanoparticles (Laponite XLG). The length of the PTMC blocks of the macromonomers and the clay content determined the physicomechanical properties of the obtained hydrogels. While an increase in the PTMC block length in the macromonomers from 0.2 to 5kg/mol resulted in a decrease in the gel content, the addition of 5wt.% Laponite nanoclay to the crosslinking solution lead to very high gel contents of the hydrogels of more than 95%. The effect of PTMC block length on the mechanical properties of the hydrogels was not as pronounced, and soft gels with a compressive modulus of less than 15kPa and toughness values of 25kJm(-3) were obtained. However, the addition of 5wt.% Laponite nanoclay to the formulations considerably increased the compressive modulus and resilience of the hydrogels; swollen nanocomposite networks with compressive modulus and toughness values of up to 67kPa and 200kJm(-3), respectively, could then be obtained. The prepared hydrogels were shown to be enzymatically degradable by cholesterol esterase and by the action of macrophages. With an increase in PTMC block length in the hydrogels, the rates of mass loss increased, while the incorporated Laponite nanoclay suppressed degradation. Nanocomposite hydrogel structures with a designed gyroid pore network architecture were prepared by stereolithography. Furthermore, in the swollen state the porous gyroid structures were mechanically stable and the pore network remained fully open and interconnected.

Resorbable elastomeric networks prepared by photocrosslinking of high-molecular-weight poly(trimethylene carbonate) with photoinitiators and poly(trimethylene carbonate) macromers as crosslinking aids.

Resorbable and elastomeric poly(trimethylene carbonate) (PTMC) networks were efficiently prepared by photoinitiated crosslinking of linear high-molecular-weight PTMC. To crosslink PTMC films, low-molecular-weight PTMC macromers with methacrylate end groups were synthesized and used as crosslinking aids. By exposing PTMC films containing only photoinitiator (Irgacure(®) 2959) or both photoinitiator and PTMC macromers to ultraviolet light, PTMC networks with high gel contents (87-95%) could be obtained. The crosslink density could be readily varied by adjusting the irradiation time or the amount of crosslinking aid used. The formed networks were flexible, with low elastic modulus values ranging from 7.1 to 7.5MPa, and also showed excellent resistance to creep in cyclic tests. In vitro experiments showed that the photocrosslinked PTMC networks could be eroded by macrophages, and upon incubation in aqueous cholesterol esterase enzyme- or potassium dioxide solutions. The rate of surface erosion of photocrosslinked PTMC networks was significantly lower than that observed for films prepared from linear PTMC. These resorbable PTMC elastomeric networks are compatible with cells and may find application in tissue engineering and controlled release.