Inhibition of γ-irradiation induced adhesion molecules and NO production by alginate in human endothelial cells

Inflammation is a frequent radiation-induced reaction following therapeutic irradiation. Treatment of human umbilical endothelial cells (HUVEC) with gamma-irradiation (gammaIR) induces the expression of adhesion proteins such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Since the upregulation of these proteins on endothelial cell surface has been known to be associated with inflammation, interfering with the expression of adhesion molecules is an important therapeutic target. In the present study, we demonstrate that high mannuronic acid-containing alginate (HMA) inhibits gammaIR induced expression of ICAM-1, VCAM-1, and E-selectin on HUVEC in a dose dependent manner. HMA also inhibited gammaIR induced production of Nitric oxide (NO). These data suggest that HMA has therapeutic potential for the treatment of various inflammatory disorder associated with an increase of endothelial leukocyte adhesion molecules.     

海藻酸钠两性霉素B对杜氏利什曼原虫前鞭毛体作用体外实验研究

目的 治疗犬利什曼病,消灭保虫宿主,达到预防黑热病的目的。方法 观察海藻酸钠两性霉素B在不同浓度对杜氏利什曼原虫（L.d)江苏株及L.d四川人株前鞭毛体的体外作用。采用显微镜下观察试验。结果 实验组3个不同浓度（10μg/ml,5μg/ml、12μg/ml)的药物作用48h后,培养其颜色未变（红）,虫体活动变慢,虫数减少,抑制率升高（L.d江苏株的抑制率由70%升至93%,L.d四川人株由82%升至96%）,染色后见虫体变形,核、动基体不完整,胞质内出现多个空泡,鞭毛脱落,对照组的培养其颜色变黄,虫体运动活跃,大部分呈长梭形,并排成菊花状,染色后虫体呈长梭形,核、动基体,胞膜及鞭毛完整,清晰,结论 海藻酸钠两性霉素B对杜氏利什曼原虫有较强的体外抑制作用。     

Preparation of Various Solid-Lipid Beads for Drug Delivery of Enrofloxacin

Solid-lipid beads were prepared to retard the release rate of enrofloxacin and to mask its bitter taste using carrageenan or sodium alginate as a shell material and either cacao butter or Witepsol W-35 as a solid lipid core. Sodium alginate was a better shell material than carrageenan and the highest loading efficiency was obtained using 2% sodium alginate. The alginate beads had a spherical morphology and a sturdy shell structure. The enrofloxacin release rate at room temperature was greatly reduced. Solid-lipid beads have the potential to mask the bitter taste of enrofloxacin and extend its release rate.     

Initial characterization of the metabolism of intervertebral disc cells encapsulated in microspheres.

Adult, canine intervertebral disc cells were isolated with a sequential digestion of pronase and bacterial collagenase. The nonchondrodystrophoid nucleus pulposus exhibits two populations of cells: large notochordal cells and smaller chondrocyte-like cells. The cells from the transition zone and anulus fibrosus are uniform in size, ranging from 17 to 21 microns. The isolated cells were encapsulated in alginate beads and cultured in Ham's F-12 medium containing 5% heat-inactivated fetal bovine serum. Alginate bead formation requires calcium ions and can be reversed with a suitable chelator, thus releasing viable cells. We observed that 58% of the newly synthesized proteoglycans formed large-molecular-weight aggregates with hyaluronic acid. The proteoglycans contained low amounts of keratan sulfate (KS) (less than 5% of the total glycosaminoglycans synthesized). The chondroitin sulfates (CS) consisted of 51-67% as 6-O-sulfate and 29-39% as 4-O-sulfate, with the remainder (4-10%) present as 4,6-sulfate for all three zones of the disc. The majority of cells synthesized significant amounts of matrix as evidenced by Alcian Blue staining. By immunohistochemical analysis, the matrix contained chondroitin 6-sulfate as demonstrated by monoclonal antibodies to the unsaturated disaccharides remaining on the proteoglycan core after chondroitinase ABC digestion. Keratan sulfate was also present in the majority of the matrices around cells. These results emphasize the similarity of the newly synthesized proteoglycans secreted by cells grown in alginate beads to those synthesized by the neonate disc. These experiments also demonstrate the usefulness of this method as a microculture technique for disc cells.     

Oil encapsulation in core–shell alginate capsules by inverse gelation. I: dripping methodology

The production of capsules by inverse gelation consists of adding dropwise oil containing calcium dispersion into an alginate bath. A dripping technique to produce capsules from oil-in-water (O/W) emulsions was proposed by Abang. However, little is known about the oil encapsulation using water-in-oil (W/O) emulsions. This work aims to develop a new method of W/O emulsions encapsulation by inverse gelation. The success of the W/O emulsion encapsulation is due to three factors: 1) use of an emulsion with moderate stability (50?min); 2) production of an emulsion with at least 90?g/L of CaCl₂ and 3) addition of ethanol (20% v/v) into the alginate bath. Both wet and dry capsules were obtained with a spherical shape with diameters of 7 and 3.6?mm, respectively. All volume of oil was encapsulated and the oil loading in the wet and dry capsules was of 23 and 68% v/v, respectively.     

Chondrogenesis of hMSC in affinity-bound TGF-beta scaffolds

Herein we describe a bio-inspired, affinity binding alginate-sulfate scaffold, designed for the presentation and sustained release of transforming growth factor beta 1 (TGF-β1), and examine its effects on the chondrogenesis of human mesenchymal stem cells (hMSCs). When attached to matrix via affinity interactions with alginate sulfate, TGF-β1 loading was significantly greater and its initial release from the scaffold was attenuated compared to its burst release (>90%) from scaffolds lacking alginate-sulfate. The sustained TGF-β1 release was further supported by the prolonged activation (14 d) of Smad-dependent (Smad2) and Smad-independent (ERK1/2) signaling pathways in the seeded hMSCs. Such presentation of TGF-β1 led to hMSC chondrogenic differentiation; differentiated chondrocytes with deposited collagen type II were seen within three weeks of in vitro hMSC seeding. By contrast, in scaffolds lacking alginate-sulfate, the effect of TGF-β1 was short-term and hMSCs could not reach a similar differentiation degree. When hMSC constructs were subcutaneously implanted in nude mice, chondrocytes with deposited type II collagen and aggrecan typical of the articular cartilage were found in the TGF-β1 affinity-bound constructs. Our results highlight the fundamental importance of appropriate factor presentation to its biological activity, namely - inducing efficient stem cell differentiation.     

Single and dual crosslinked oxidized methacrylated alginate/PEG hydrogels for bioadhesive applications

A degradable, cytocompatible bioadhesive can facilitate surgical procedures and minimize patient pain and post-surgical complications. In this study a bioadhesive hydrogel system based on oxidized methacrylated alginate/8-arm poly(ethylene glycol) amine (OMA/PEG) has been developed, and the bioadhesive characteristics of the crosslinked OMA/PEG hydrogels evaluated. Here we demonstrate that the swelling behavior, degradation profiles, and storage moduli of crosslinked OMA/PEG hydrogels are tunable by varying the degree of alginate oxidation. The crosslinked OMA/PEG hydrogels exhibit cytocompatibility when cultured with human bone marrow-derived mesenchymal stem cells. In addition, the adhesion strength of these hydrogels, controllable by varying the alginate oxidation level and measured using a porcine skin model, is superior to commercially available fibrin glue. This OMA/PEG hydrogel system with controllable biodegradation and mechanical properties and adhesion strength may be a promising bioadhesive for clinical use in biomedical applications, such as drug delivery, wound closure and healing, biomedical device implantation, and tissue engineering.     

Young porcine endocrine pancreatic islets cultured in fibrin and alginate gels show improved resistance towards human monocytes

Aim

To investigate the protective function of alginate and fibrin gels used to embed porcine endocrine pancreatic islets towards human monocytes.

Methods

Groups of 200 islet equivalents from young pigs were embedded in either a fibrin or in an alginate gel, and as a control seeded in tissue culture polystyrene (TCPS) well plates. The islet cultures were incubated with 2 × 10⁵ human monocytes for 24 h. In addition, both islets and monocytes were separately cultured in TCPS, fibrin and alginate. Islet morphology, viability and function were investigated as well as the secretion of cytokines TNFα, IL-6, and IL-1β.

Results

When freely-floating in TCPS, non-encapsulated islets were surrounded by monocytes and started to disperse after 24 h. In fibrin, monocytes could be found in close proximity to embedded islets, indicating monocyte migration through the gel. In contrast, after 24 h, few monocytes were found close to islets in alginate. Immunofluorescence staining and manual counting showed that integrin expression was higher in fibrin-embedded islet cultures. A TUNEL assay revealed elevated numbers of apoptotic cells for islets in TCPS wells compared to fibrin and alginate cultures. Insulin secretion was higher with islets embedded in fibrin and alginate when compared to non-encapsulated islets. TNFα, IL-6 and IL-1β were found in high concentrations in the media of co-cultures and monocyte mono-culture in fibrin.

Conclusion

Both alginate and fibrin provide key structural support and offer some protection for the islets towards human monocytes. Fibrin itself triggers the cytokine secretion from monocytes.     

Paracrine exchanges of molecular signals between alginate-encapsulated pericytes and freely suspended endothelial cells within a 3D protein gel

Paracrine signals, essential for the proper survival and functioning of tissues, may be mimicked by delivery of therapeutic proteins within engineered tissue constructs. Conventional delivery methods are of limited duration and are unresponsive to the local environment. We developed a system for sustained and regulated delivery of paracrine signals by encapsulating living cells of one type in alginate beads and co-suspending these cell-loaded particles along with unencapsulated cells of a second type within a 3D protein gel. This system was applied to vascular tissue engineering by placing human placental microvascular pericytes (PCs) in the particulate alginate phase and human umbilical vein endothelial cells (HUVECs) in the protein gel phase. Particle characteristics were optimized to keep the encapsulated PCs viable for at least two weeks. Encapsulated PCs were bioactive in vitro, secreting hepatocyte growth factor, an angiogenic protein, and responding to externally applied HUVEC-derived signals. Encapsulated PCs influenced HUVEC behavior in the surrounding gel by enhancing the formation of vessel-like structures when compared to empty alginate bead controls. In vivo, encapsulated PCs modulated the process of vascular self-assembly by HUVECs in 3D gels following implantation into immunodeficient mice. We conclude that alginate encapsulated cells can provide functional paracrine signals within engineered tissues.     

Fabrication and characterization of monodisperse PLGA–alginate core–shell microspheres with monodisperse size and homogeneous shells for controlled drug release

Monodisperse PLGA–alginate core–shell microspheres with controlled size and homogeneous shells were first fabricated using capillary microfluidic devices for the purpose of controlling drug release kinetics. Sizes of PLGA cores were readily controlled by the geometries of microfluidic devices and the fluid flow rates. PLGA microspheres with sizes ranging from 15 to 50 μm were fabricated to investigate the influence of the core size on the release kinetics. Rifampicin was loaded into both monodisperse PLGA microspheres and PLGA–alginate core–shell microspheres as a model drug for the release kinetics studies. The in vitro release of rifampicin showed that the PLGA core of all sizes exhibited sigmoid release patterns, although smaller PLGA cores had a higher release rate and a shorter lag phase. The shell could modulate the drug release kinetics as a buffer layer and a near-zero-order release pattern was observed when the drug release rate of the PLGA core was high enough. The biocompatibility of PLGA–alginate core–shell microspheres was assessed by MTT assay on L929 mouse fibroblasts cell line and no obvious cytotoxicity was found. This technique provides a convenient method to control the drug release kinetics of the PLGA microsphere by delicately controlling the microstructures. The obtained monodisperse PLGA–alginate core–shell microspheres with monodisperse size and homogeneous shells could be a promising device for controlled drug release.