Fabrication and characteristics of chitosan sponge as a tissue engineering scaffold.

Cells, growth factors, and scaffolds are the three main factors required to create a tissue-engineered construct. After the appearance of bovine spongiform encephalopathy (BSE), considerable attention has therefore been focused on nonbovine materials. In this study, we examined the properties of a chitosan porous scaffold. A porous chitosan sponge was prepared by the controlled freezing and lyophilization of different concentrations of chitosan solutions. The materials were examined by scanning electron microscopy, and the porosity, tensile strength, and basic fibroblast growth factor (bFGF) release profiles from chitosan sponge were examined in vitro. The morphology of the chitosan scaffolds presented a typical microporous structure, with the pore size ranging from 50 to 200 µm. The porosity of chitosan scaffolds with different concentrations was approximately 75-85%. A decreasing tendency for porosity was observed as the concentration of the chitosan increased. The relationship between the tensile properties and chitosan concentration indicated that the ultimate tensile strength for the sponge increased with a higher concentration. The in vitro bFGF release study showed that the higher the concentration of chitosan solution became, the longer the releasing time of the bFGF from the chitosan sponge was.

Biological safety of fish (tilapia) collagen.

Marine collagen derived from fish scales, skin, and bone has been widely investigated for application as a scaffold and carrier due to its bioactive properties, including excellent biocompatibility, low antigenicity, and high biodegradability and cell growth potential. Fish type I collagen is an effective material as a biodegradable scaffold or spacer replicating the natural extracellular matrix, which serves to spatially organize cells, providing them with environmental signals and directing site-specific cellular regulation. This study was conducted to confirm the safety of fish (tilapia) atelocollagen for use in clinical application. We performed in vitro and in vivo biological studies of medical materials to investigate the safety of fish collagen. The extract of fish collagen gel was examined to clarify its sterility. All present sterility tests concerning bacteria and viruses (including endotoxin) yielded negative results, and all evaluations of cell toxicity, sensitization, chromosomal aberrations, intracutaneous reactions, acute systemic toxicity, pyrogenic reactions, and hemolysis were negative according to the criteria of the ISO and the Ministry of Health, Labour and Welfare of Japan. The present study demonstrated that atelocollagen prepared from tilapia is a promising biomaterial for use as a scaffold in regenerative medicine.

Effects of hypoxia on pluripotency in murine iPS cells.

Retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) or three factors, excluding c-Myc, has been shown to initiate a reprogramming process that results in the transformation of murine fibroblasts to induced pluripotent stem (iPS) cells, and there has been a rapid increase in the number of iPS cell-based preclinical trials. In this study, the effects of these transcription factors were evaluated regarding the growth and differentiation of murine iPS cells under hypoxia. Based on the results of RT-PCR and alizarin red S staining, there were no statistical differences in the growth and differentiation of iPS cells or the induction of iPS cells to osteoblasts under hypoxia between the transcription factor groups. Furthermore, the function of hypoxia inducible factors (HIFs) in murine iPS cells under hypoxia was investigated in relation to the morphology and expression of transcription factors using RT-PCR and Western blotting. The HIF-2α knockdown group exhibited a decrease in the colony size of the iPS cells. The HIF-2α or -3α knockdown group demonstrated a statistically significant decrease in the transcription factor expression compared to that observed in the control group. These results demonstrate that HIF-2α among HIFs is the most influential candidate for the maintenance of the pluripotency of murine iPS cells.

Early gene and protein expression associated with osteoblast differentiation in response to fish collagen peptides powder.

This study was designed to investigate the biological effects of fish collagen peptide (FCP) on human osteoblasts. Human osteoblasts were treated with 0.1% FCP, which was the optimal concentration confirmed by the increase in alkaline phosphatase activity. After one, three, five and seven days of culture, the number of FCP-treated cells increased significantly compared with untreated cells. In a real-time PCR analysis, the expression of osteocalcin, osteopontin, BMP-2 and integrin ß3 mRNAs in FCP-treated cells showed increases compared with untreated cells after three days of culture. After seven days of culture, the expression levels of osteopontin and integrin ß3 were still higher in the FCP-treated cells than in untreated cells. The production of osteocalcin, osteopontin and integrin ß3 proteins in FCP-treated cells also showed increases after seven days of culture. Furthermore, FCP accelerated matrix mineralization in the cultures. The present study indicates the potential utility of FCP as a biomaterial.

Impact of zinc fingers and homeoboxes 3 on the regulation of mesenchymal stem cell osteogenic differentiation.

We propose zinc fingers and homeoboxes 3 (ZHX3) as new osteogenic markers for mesenchymal stem cells (MSC). ZHX3 mRNA expression was upregulated within 1-6 h after incubation of MSCs in the osteogenic induction medium, and reached maximum levels after 24 h of incubation. Two to 4 days later, ZHX3 mRNA levels had decreased sharply. Maximal mRNA levels were 3- to 5-fold higher than those in the undifferentiated state. In contrast, Runt-related transcription factor2 (RUNX2) mRNA expression was downregulated at 2-4 h after incubation, and levels were only enhanced 1.4-fold after 12 and 24 h of incubation. Further, Osterix mRNA levels increased only 1.6-fold after 4 and 24 h of incubation. Thus, ZHX3 expression may be a better marker of MSC osteogenic differentiation than RUNX2 or Osterix expression at the initial stage of differentiation. Knockdown of ZHX3 using 2 distinct small interfering RNA (siRNA) oligonucleotides had little effect on cell morphology or on MSC proliferation, regardless of the differentiation state of the cells. However, ZHX3 siRNAs suppressed Osterix, but not RUNX2 mRNA expression, within 1 h of osteogenic differentiation, and this suppression was sustained for at least 24 h. The 2 ZHX3 siRNAs also suppressed alkaline phosphatase induction and matrix mineralization (assessed using alizarin red staining), and, further, suppressed the calcium content of the cultures at a later stage of differentiation (days 6-21). The effects of ZHX3 siRNAs on the osteogenic differentiation were comparable to those of RUNX2 and Osterix siRNAs. These findings suggest that ZHX3 is involved in the switch from the undifferentiated state of MSC to an osteogenic program, and that ZHX3 may be useful as an early osteogenic differentiation marker.