Enhancement of Biomimetic Enzymatic Mineralization of Gellan Gum Polysaccharide Hydrogels by Plant-Derived Gallotannins.

Mineralization of hydrogel biomaterials with calcium phosphate (CaP) is considered advantageous for bone regeneration. Mineralization can be both induced by the enzyme alkaline phosphatase (ALP) and promoted by calcium-binding biomolecules, such as plant-derived polyphenols. In this study, ALP-loaded gellan gum (GG) hydrogels were enriched with gallotannins, a subclass of polyphenols. Five preparations were compared, namely three tannic acids of differing molecular weight (MW), pentagalloyl glucose (PGG), and a gallotannin-rich extract from mango kernel (Mangifera indica L.). Certain gallotannin preparations promoted mineralization to a greater degree than others. The various gallotannin preparations bound differently to ALP and influenced the size of aggregates of ALP, which may be related to ability to promote mineralization. Human osteoblast-like Saos-2 cells grew in eluate from mineralized hydrogels. Gallotannin incorporation impeded cell growth on hydrogels and did not impart antibacterial activity. In conclusion, gallotannin incorporation aided mineralization but reduced cytocompatibility.

CO2-Driven Nebulization of pH-Sensitive Supramolecular Polymers for Intraperitoneal Hydrogel Formation and the Treatment of Peritoneal Metastasis.

Because peritoneal metastasis (PM) from ovarian cancer is characterized by non-specific symptoms, it is often diagnosed at advanced stages. Pressurized intraperitoneal aerosol chemotherapy (PIPAC) can be considered a promising drug delivery method for unresectable PM. Currently, the efficacy of intraperitoneal (IP) drug delivery is limited by the off-label use of IV chemotherapeutic solutions, which are rapidly cleared from the IP cavity. Hence, this research aimed to improve PM treatment by evaluating a nanoparticle-loaded, pH-switchable supramolecular polymer hydrogel as a controlled release drug delivery system that can be IP nebulized. Moreover, a multidirectional nozzle was developed to allow nebulization of viscous materials such as hydrogels and to reach an even IP gel deposition. We demonstrated that acidification of the nebulized hydrogelator solution by carbon dioxide, used to inflate the IP cavity during laparoscopic surgery, stimulated the in situ gelation, which prolonged the IP hydrogel retention. In vitro experiments indicated that paclitaxel nanocrystals were gradually released from the hydrogel depot formed, which sustained the cytotoxicity of the formulation for 10 days. Finally, after aerosolization of this material in a xenograft model of PM, tumor progression could successfully be delayed, while the overall survival time was significantly increased compared to non-treated animals.

Pectin-bioactive glass self-gelling, injectable composites with high antibacterial activity.

The present work focuses on the development of novel injectable, self-gelling composite hydrogels based on two types of low esterified amidated pectins from citrus peels and apple pomace. Sol-gel-derived, calcium-rich bioactive glass (BG) fillers in a particle form are applied as delivery vehicles for the release of Ca2+ ions to induce internal gelation of pectins. Composites were prepared by a relatively simple mixing technique, using 20% w/v BG particles of two different sizes (2.5 and <45 µm). Smaller particles accelerated pectin gelation slightly faster than bigger ones, which appears to result from the higher rate of Ca2+ ion release. µCT showed inhomogeneous distribution of the BG particles within the hydrogels. All composite hydrogels exhibited strong antibacterial activity against methicilin-resistant Staphylococcus aureus. The mineralization process of pectin-BG composite hydrogels occurred upon incubation in simulated body fluid for 28 days. In vitro studies demonstrated cytocompatibility of composite hydrogels with MC3T3-E1 osteoblastic cells.

Novel injectable, self-gelling hydrogel-microparticle composites for bone regeneration consisting of gellan gum and calcium and magnesium carbonate microparticles.

The suitability of hydrogel biomaterials for bone regeneration can be improved by incorporation of an inorganic phase in particle form, thus maintaining hydrogel injectability. In this study, carbonate microparticles containing different amounts of calcium (Ca) and magnesium (Mg) were added to solutions of the anionic polysaccharide gellan gum (GG) to crosslink GG by release of Ca2+ and Mg2+ from microparticles and thereby induce formation of hydrogel-microparticle composites. It was hypothesized that increasing Mg content of microparticles would promote GG hydrogel formation. The effect of Mg incorporation on cytocompatibility and cell growth was also studied. Microparticles were formed by mixing Ca2+ and Mg2+ and [Formula: see text] ions in varying concentrations. Microparticles were characterized physiochemically and subsequently mixed with GG solution to form hydrogel-microparticle composites. The elemental Ca:Mg ratio in the mineral formed was similar to the Ca:Mg ratio of the ions added. In the absence of Mg, vaterite was formed. At low Mg content, magnesian calcite was formed. Increasing the Mg content further caused formation of amorphous mineral. Microparticles of vaterite and magnesium calcite did not induce GG hydrogel formation, but addition of Mg-richer amorphous microparticles induced gelation within 20 min. Microparticles were dispersed homogeneously in hydrogels. MG-63 osteoblast-like cells were cultured in eluate from hydrogel-microparticle composites and on the composites themselves. All composites were cytocompatible. Cell growth was highest on composites containing particles with an equimolar Ca:Mg ratio. In summary, carbonate microparticles containing a sufficient amount of Mg induced GG hydrogel formation, resulting in injectable, cytocompatible hydrogel-microparticle composites.

Enrichment of enzymatically mineralized gellan gum hydrogels with phlorotannin-rich Ecklonia cava extract Seanol(®) to endow antibacterial properties and promote mineralization.

Hydrogels offer several advantages as biomaterials for bone regeneration, including ease of incorporation of soluble substances such as mineralization-promoting enzymes and antibacterial agents. Mineralization with calcium phosphate (CaP) increases bioactivity, while antibacterial activity reduces the risk of infection. Here, gellan gum (GG) hydrogels were enriched with alkaline phosphatase (ALP) and/or Seanol(®), a seaweed extract rich in phlorotannins (brown algae-derived polyphenols), to induce mineralization with CaP and increase antibacterial activity, respectively. The sample groups were unmineralized hydrogels, denoted as GG, GG/ALP, GG/Seanol and GG/Seanol/ALP, and hydrogels incubated in mineralization medium (0.1 M calcium glycerophosphate), denoted as GG/ALP_min, GG/Seanol_min and GG/Seanol/ALP_min. Seanol(®) enhanced mineralization with CaP and also increased compressive modulus. Seanol(®) and ALP interacted in a non-covalent manner. Release of Seanol(®) occurred in a burst phase and was impeded by ALP-mediated mineralization. Groups GG/Seanol and GG/ALP/Seanol exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. GG/Seanol/ALP_min, but not GG/Seanol_min, retained some antibacterial activity. Eluates taken from groups GG/ALP_min, GG/Seanol_min and GG/ALP/Seanol_min displayed comparable cytotoxicity towards MG-63 osteoblast-like cells. These results suggest that enrichment of hydrogel biomaterials with phlorotannin-rich extracts is a promising strategy to increase mineralizability and antibacterial activity.

Polylactide nanofibers with hydroxyapatite as growth substrates for osteoblast-like cells.

Various types of nanofibers are increasingly used in tissue engineering, mainly for their ability to mimic the architecture of tissue at the nanoscale. We evaluated the adhesion, growth, viability, and differentiation of human osteoblast-like MG 63 cells on polylactide (PLA) nanofibers prepared by needle-less electrospinning and loaded with 5 or 15 wt % of hydroxyapatite (HA) nanoparticles. On day 7 after seeding, the cell number was the highest on samples with 15 wt % of HA. This result was confirmed by the XTT test, especially after dynamic cultivation, when the number of metabolically active cells on these samples was even higher than on control polystyrene. Staining with a live/dead kit showed that the viability of cells on all nanofibrous scaffolds was very high and comparable to that on control polystyrene dishes. An enzyme-linked immunosorbent assay revealed that the concentration of osteocalcin was also higher in cells on samples with 15 wt % of HA. There was no immune activation of cells (measured by production of TNF-alpha), associated with the incorporation of HA. Moreover, the addition of HA suppressed the creep behavior of the scaffolds in their dry state. Thus, nanofibrous PLA scaffolds have potential for bone tissue engineering, particularly those with 15 wt % of HA.

Injectable self-gelling composites for bone tissue engineering based on gellan gum hydrogel enriched with different bioglasses.

Hydrogels of biocompatible calcium-crosslinkable polysaccharide gellan gum (GG) were enriched with bioglass particles to enhance (i) mineralization with calcium phosphate (CaP); (ii) antibacterial properties and (iii) growth of bone-forming cells for future bone regeneration applications. Three bioglasses were compared, namely one calcium-rich and one calcium-poor preparation both produced by a sol-gel technique (hereafter referred to as A2 and S2, respectively) and one preparation of composition close to that of the commonly used 45S5 type (hereafter referred to as NBG). Incubation in SBF for 7 d, 14 d and 21 d caused apatite formation in bioglass-containing but not in bioglass-free samples, as confirmed by FTIR, XRD, SEM, ICP-OES, and measurements of dry mass, i.e. mass attributable to polymer and mineral and not water. Mechanical testing revealed an increase in compressive modulus in samples containing S2 and NBG but not A2. Antibacterial testing using biofilm-forming meticillin-resistant staphylococcus aureus (MRSA) showed markedly higher antibacterial activity of samples containing A2 and S2 than samples containing NBG and bioglass-free samples. Cell biological characterization using rat mesenchymal stem cells (rMSCs) revealed a stimulatory effect of NBG on rMSC differentiation. The addition of bioglass thus promotes GG mineralizability and, depending on bioglass type, antibacterial properties and rMSC differentiation.

Magnesium-enhanced enzymatically mineralized platelet-rich fibrin for bone regeneration applications.

Membranes of the autologous blood-derived biomaterial platelet-rich fibrin (PRF) were mineralized enzymatically with calcium phosphate (CaP) by the incorporation of alkaline phosphatase (ALP) followed by incubation for 3 days in solutions of either 0.1 M calcium glycerophosphate (CaGP) or a combination of CaGP and magnesium glycerophosphate (CaGP:MgGP; both 0.05 M), resulting in the formation of two different PRF-mineral composites. Fourier transform infrared spectroscopy, transmission electron microscopy and selected area electron diffraction examinations showed that the CaP formed was amorphous. Inductively coupled plasma optical emission spectroscopy analysis revealed similar amounts of Ca and P in both composite types, while a smaller amount of Mg (Ca:Mg molar ratio = 10) was detected in the composites formed in the CaGP:MgGP solution, which was supported by the results of energy-dispersive x-ray spectroscopy-based elemental mapping. Scanning electron microscopy (SEM) imaging showed that the mineral deposits in PRF incubated in the CaGP:MgGP solution were markedly smaller. The mass percentage attributable to the mineral phase was similar in both composite types. MTT and WST tests with SAOS-2 cells revealed that incubation in the CaGP:MgGP solution had no negative effect on cytocompatibility and cell proliferation compared to the CaGP solution. Cells on all samples displayed a well-spread morphology as revealed by SEM imaging. In conclusion, the incorporation of Mg reduces mineral deposit dimensions and promotes cell proliferation.

The use of liposomes to differentiate between the effects of nickel accumulation and altered food quality in Daphnia magna exposed to dietary nickel.

A potential drawback of traditional dietary metal toxicity studies is that it is difficult to distinguish between the direct toxicity of the metal and indirect effects caused by altered concentrations of essential nutrients in the metal-contaminated diet. In previous studies it has become clear that this can hamper the study of the real impact of dietary metal exposure and also complicates the analysis of the mechanisms of dietary metal toxicity in filter-feeding freshwater invertebrates like Daphnia magna. This problem has been partly circumvented by the production of liposomes, since these vectors are invulnerable to metal-induced food quality shifts and as such can be applied to study the mechanisms of dietary metal toxicity without the confounding effect of nutritional quality shifts. The aim of current study was to evaluate if there is relevance for dietary Ni toxicity under natural exposures, i.e., when D. magna is exposed to dietary Ni via living algae, and secondly, to quantify how nutritional quality shifts contribute to the toxic effects that are observed when algae are used as contaminated food vectors. For this aim, liposomes were prepared by the hydration of phosphatidylcholine in media containing 0 (control), 10, 50, 100 and 500 mg Ni/L. The liposome particles were then mixed with uncontaminated green algae in a 1/10 ratio (on a dry wt basis) to make up diets with constant nutrient quality and varying Ni contents (i.e., 1.2 µg Ni/g dry wt in the control and 18.7, 140.3, 165.0 and 501.6 µg Ni/g dry wt in the Ni-contaminated diet, respectively). A second food type was prepared on the basis of a 1/10 mixture (on a dry weight basis) of control liposomes and Ni-contaminated algae, representing a diet that differed in Ni content (i.e., 1.2, 26.8, 84.7, 262.3 and 742.7 µg Ni/g dry wt) and concentrations of essential nutrients (in terms of P and omega 3 poly-unsaturated fatty acids like eicosapentaenoic acid and α-linolenic acid). Both diets were then simultaneously fed to D. magna during a 21-day chronic bioassay, using reproduction, growth, survival, ingestion rate and Ni bioaccumulation as endpoints. Ni delivered by liposomes caused a significant inhibition of reproduction and growth when the metal accumulated to minimum levels of 11.9 and 20.0 µg Ni/g dry wt after 7 and 14 days, respectively. Using algae as Ni vector, similar effects of dietary Ni exposure occurred when algae had been pre-exposed to concentrations of at least 133 µg/L of bioavailable Ni (i.e., Ni2+), which is similar to the reproductive EC50 of waterborne Ni exposure for D. magna (115 µg Ni2+/L). While this may have some consequences for predicting chronic Ni toxicity in this range of Ni concentrations with the biotic ligand model--which could be further improved by including the dietary toxicity pathway in this model, the occurrence of such high concentrations in the field is very rare. Hence, there seems to be very little environmental relevance for dietary Ni toxicity to D. magna. Finally, besides the direct effects of Ni there was no evidence that nutritional quality shifts could have affected daphnids' growth, but it is very likely that the impairment of reproduction at toxic exposure levels of Ni was also partly the result of reduced fatty acid levels.

Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase.

Alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, is incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These are collagen type I, a mussel-protein-inspired adhesive consisting of PEG substituted with catechol groups, cPEG, and the PEG/fumaric acid copolymer OPF. After incubation in Ca-GP solution, FTIR, EDS, SEM, XRD, SAED, ICP-OES, and von Kossa staining confirm CaP formation. The amount of mineral formed decreases in the order cPEG > collagen > OPF. The mineral:polymer ratio decreases in the order collagen > cPEG > OPF. Mineralization increases Young's modulus, most profoundly for cPEG. Such enzymatically mineralized hydrogel/CaP composites may find application as bone regeneration materials.

Liposomes as an alternative delivery system for investigating dietary metal toxicity to Daphnia magna.

Dietary metal toxicity studies with invertebrates such as Daphnia magna are often performed using metal-contaminated algae as a food source. A drawback of this approach is that it is difficult to distinguish between the direct toxicity of the metal and indirect effects caused by a reduced essential nutrient content in the metal-contaminated diet, due to prior exposure of the algae to the metal. This hampers the study of the mechanisms of dietary metal toxicity in filter-feeding freshwater invertebrates. The aim of the present study was to develop a technique for producing metal-contaminated liposomes as an alternative delivery system of dietary metals. These liposomes are not vulnerable to metal-induced shifts in nutrient quality. Liposomes were prepared by the hydration of phosphatidylcholine in media containing either 0 (control) or 50mg Ni/L. The liposomes had average diameters of 19.31 (control) and 10.48 µm (Ni-laden), i.e., a size appropriate for ingestion by D. magna. The liposome particles were then mixed with uncontaminated green algae in a 1/10 ratio (on a dry wt. basis) to make up two diets that differed in Ni content (i.e., 2.0 µg Ni/g dry wt. in the control and 144.2 µg Ni/g dry wt. in the Ni-contaminated diet, respectively). This diet was then fed to D. magna during a 21-day chronic bioassay. The experiment showed that the Ni content and the size distribution of the liposomes were stable for at least 7 days. Also the use of phosphatidylcholine as a liposome component did not affect the reproduction of the daphnids. Exposure to increased level of dietary Ni resulted in 100% mortality after 14 days of exposure and in an increased whole-body Ni concentration in D. magna of 14.9 and 20.4 µg Ni/g dry wt. after 7 and 14 days of exposure, respectively. The Ni-exposed daphnids also exhibited a reduced size (i.e., 30% smaller than the control) after 7 days and a completely halted growth between day 7 and day 14. In terms of reproduction, the size of the first brood (number of juveniles) of the Ni-exposed daphnids was significantly reduced (by 85%) compared to the control. None of the Ni-exposed individuals were able to produce a second brood before dying. The algal ingestion rate - after correction for the indirect effect of a reduced size - was increased (by 68%) by dietary Ni after 6 days of exposure compared to the control, but was severely reduced (by 80% compared to the control) after 13 days. These data suggest that an inhibition of the ingestion process may have contributed to the observed effects of dietary Ni on growth and reproduction beyond 6 days of exposure, although the involvement of other mechanisms cannot be excluded. The mechanism(s) which led to the reduced growth during the first week of exposure remain unclear, although inhibition of the ingestion process can likely be excluded here as an explanation. Overall, this paper demonstrates, using this new method of delivering dietary Ni via liposome carriers and thus excluding potential diet quality shifts, that dietary Ni can indeed induce toxic effects in D. magna. This method may therefore be a promising tool to help further elucidate the mechanisms of dietary metal toxicity to filter-feeding invertebrates.