Reparative Effects of Astaxanthin-Hyaluronan Nanoaggregates against Retrorsine-CCl4-Induced Liver Fibrosis and Necrosis.

Astaxanthin (Asta), a xanthophyll carotenoid, has been reported to be a strong antioxidative agent and has anti-inflammatory, antitumor and free radical-scavenging activities. However, inadequate stability and water solubility results in its low bioavailability. This study incorporated Asta into hydrophilic hyaluronan nanoparticles (HAn) to produce Asta-HAn aggregates (AHAna) using an electrostatic field system and investigated the restorative effects of AHAna on retrorsine-CCl4-induced liver fibrosis in rats in vivo. Transmission electron microscopy (TEM) revealed that the prepared HAn were approximately 15 ± 2.1 nm in diameter and after the incorporation of Asta into HAn, the size increased to 210-500 nm. The incorporation efficiency of Asta was approximately 93% and approximately 54% of Asta was released after incubation for 18 h. Significant reductions in alanine aminotransferase and aspartate aminotransferase levels were observed after the rats were intraperitoneally injected with AHAna. Histopathological findings revealed the greatest reduction in hepatic fibrosis and hepatocyte necrosis in the rats after 2 weeks of intraperitoneal injection with AHAna, which is consistent with the data acquired from serum biochemical analysis. The restorative effects on liver damage displayed by AHAna in vivo demonstrated that Asta aggregated through HAn incorporation exerts therapeutic effects on liver fibrosis and necrosis.

Anticancer Effects of Sinulariolide-Conjugated Hyaluronan Nanoparticles on Lung Adenocarcinoma Cells.

Lung cancer is one of the most clinically challenging malignant diseases worldwide. Sinulariolide (SNL), extracted from the farmed coral species Sinularia flexibilis, has been used for suppressing malignant cells. For developing anticancer therapeutic agents, we aimed to find an alternative for non-small cell lung cancer treatment by using SNL as the target drug. We investigated the SNL bioactivity on A549 lung cancer cells by conjugating SNL with hyaluronan nanoparticles to form HA/SNL aggregates by using a high-voltage electrostatic field system. SNL was toxic on A549 cells with an IC50 of 75 µg/mL. The anticancer effects of HA/SNL aggregates were assessed through cell viability assay, apoptosis assays, cell cycle analyses, and western blotting. The size of HA/SNL aggregates was approximately 33-77 nm in diameter with a thin continuous layer after aggregating numerous HA nanoparticles. Flow cytometric analysis revealed that the HA/SNL aggregate-induced apoptosis was more effective at a lower SNL dose of 25 µg/mL than pure SNL. Western blotting indicated that caspases-3, -8, and -9 and Bcl-xL and Bax played crucial roles in the apoptotic signal transduction pathway. In summary, HA/SNL aggregates exerted stronger anticancer effects on A549 cells than did pure SNL via mitochondria-related pathways.

Pepino polyphenolic extract improved oxidative, inflammatory and glycative stress in the sciatic nerves of diabetic mice.

The effect of pepino polyphenolic extract (PPE) on diabetic neuropathy was examined. Using HPLC/ESI-MS-MS analysis, PPE was demonstrated to contain coumaroyl and caffeoyl derivatives among polyphenols. PPE at 0.5 or 1% was supplied to diabetic mice for 12 weeks. The PPE intake at two doses significantly improved glycaemic control. These treatments reserved the glutathione (GSH) level, and decreased the thiobarbituric acid reactive substances (TBARS) level, reactive oxygen species (ROS), interleukin (IL)-6, tumour necrosis factor (TNF)-alpha, fructose, and glycation intermediates and precursors of advanced glycation end products (AGEs), such as methylglyoxal (MG) and N-(carboxymethyl)lysine (CML), in the sciatic nerves of diabetic mice. In a histological study of sciatic nerves, PPE had the effects in improving the nerves of diabetic mice, showing disorganization of the fascicle with numerous small myelinated fibers. The PPE intake at two doses retained the activity, and the protein and mRNA levels of glutathione peroxidase (GPX), and decreased protein expressions of aldose reductase (AR) and the receptor for the advanced glycation end product (RAGE) in sciatic nerves. These findings support that pepino polyphenolic extract could attenuate oxidative, inflammatory and glycative stress in diabetic peripheral nerves.

Effects of culturing media on hepatocytes differentiation using Volvox sphere as co-culturing vehicle.

Volvox sphere is a unique design to mimic natural volvox consists of a large outer-sphere that contains smaller inner-spheres, which provide three-dimensional (3D) environment to culture cells. The purpose of this study is to co-culture mesenchymal stem cells (MSCs) and AML12 liver cells in Volvox spheres and to evaluate the effects of two media, DMEM and DMEM/F12 on the cultured cells. The results of this study shows that the 3D Volvox sphere can successfully be applied for co-culture of MSCs and AML12 liver cells, and the MSCs are able to differentiate into hepatocyte-like cells expressing hepatocyte-specific markers including albumin (ALB), alpha feto-protein (AFP) and cytokeratin 18 (CK18) mRNA expressions and producing CK18 and ALB proteins. Interestingly, the MSCs expressed higher ALB, AFP and CK18 mRNA expression at the initial 7-day culture by using DMEM, whereas, the MSCs expressed more mRNA expressions from 7-day to 14-day by the usage of DMEM/F12. The result demonstrated that DMEM and DMEM/F12 media could affect MSCs behaviors during a 14-day culture.

The production of volvox spheres and their potential application in multi-drugs encapsulation and release.

Volvox sphere is a bio-mimicking concept of an innovative biomaterial structure of a sphere that contains smaller microspheres which then encapsulate chemicals, drugs and/or cells. The volvox spheres were produced via a high-voltage electrostatic field system, using alginate as the primary material. Encapsulated materials tested in this study include staining dyes, nuclear fast red and trypan blue, and model drugs, bovine serum albumin (BSA) and cytochrome c (CytC). The external morphology of the volvox spheres was observed via electron microscopy whereas the internal structure of the volvox spheres was observed via an optical microscope with the aid of the staining dyes, since alginate is colorless and transparent. The diameter of the microspheres was about 200 to 300 µm, whereas the diameter of the volvox spheres was about 1500 µm. Volvox spheres were durable, retaining about 95% of their mass after 4 weeks. Factors affecting entrapment efficiency, such as temperature and concentration of the bivalent cross-linker, were compared followed by a 7-day in vitro release study. The encapsulation efficiency of CytC within the microspheres was higher at cold (~4°C) and warm (~50°C) temperatures whereas temperature has no obvious effect on the BSA encapsulation. High crosslinking concentration (25% w/v) of calcium chloride has resulted higher entrapment efficiency for BSA but not for CytC. Furthermore, volvox spheres showed a different release pattern of BSA and CytC when compared to microspheres encapsulating BSA and CytC. Despite the fact that the mechanisms behind remain unclear and further investigation is required, this study demonstrates the potential of the volvox spheres for drug delivery.

Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds.

Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porous collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 µm porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.

Repair of chondral defects with allogenous chondrocyte-seeded hyaluronan/collagen II microspheres in a rabbit model.

Using a recently established method to prepare hyaluronan/collagen II (HA/Col II) microspheres for a novel biomaterial to couple with living cells/tissues, this animal model study evaluated the effects on a 4-week healing process of chondral defects by the implantation of allogenous chondrocyte-seeded HA/Col II microspheres that had been cultured in vitro for 7 days prior to implantation compared with unseeded HA/Col II microspheres or an untreated wound. Four weeks postsurgery, the untreated group's defect was filled with translucent soft tissue. At the same time, the edges and demarcation lines of the healing defects that were implanted with either HA/Col II microspheres or chondrocyte-seeded HA/Col II microspheres were infused yet recognizable. Furthermore, the new tissues were well integrated into the surrounding articular cartilage. Less glycosaminoglycan (GAG) staining was observed in the defects implanted with HA/Col II microspheres, which indicated that most of the repair tissues were derived from fibrocartilage formation. Conversely, more GAG staining appeared in the defect implanted with chondrocyte-seeded HA/Col II microspheres, which demonstrated a higher level of hyaline cartilage regeneration. Due to the short healing period assigned to this study, the repaired cartilage showed limited incorporation into the surrounding host cartilage and some loose connection to the subchondral bone.