Influence of amino acids, buffers, and ph on the γ-irradiation-induced degradation of alginates.

Alginate-based biomaterials and medical devices are commonly subjected to γ-irradiation as a means of sterilization, either in the dry state or the gel (hydrated) state. In this process the alginate chains degrade randomly in a dose-dependent manner, altering alginates' material properties. The addition of free radical scavenging amino acids such as histidine and phenylalanine protects the alginate significantly against degradation, as shown by monitoring changes in the molecular weight distributions using SEC-MALLS and determining the pseudo first order rate constants of degradation. Tris buffer (0.5 M), but not acetate, citrate, or phosphate buffers had a similar effect on the degradation rate. Changes in pH itself had only marginal effects on the rate of alginate degradation and on the protective effect of amino acids. Contrary to previous reports, the chemical composition (M/G profile) of the alginates, including homopolymeric mannuronan, was unaltered following irradiation up to 10 kGy.

Nylon mesh-based 3D scaffolds for the adherent culture of neural stem/progenitor cells.

We developed novel scaffolds for the adherent culture of neural stem/progenitor cells on the woven mesh. Nylon mesh (NM) is an inert material for cell adhesion. We prepared polyacrylic acid-grafted nylon mesh (PAA-NM) by graft polymerization method using gamma-irradiation. Matrigel was covalently immobilized to the carboxyl groups in PAA-NM by chemical conjugation using 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to prepare the Matrigel-immobilized PAA-grafted nylon mesh (M-PAA-NM). Cell adhesion property of mouse neural stem/progenitor cells (NSPCs) between the NM, PAA-NM, and M-PAA-NM was different from each other. The neurosphere-like clusters of NSPCs were weakly bound to NM and PAA-NM without spreading. The NSPCs were firmly adhered to, spread, and covered the surface of M-PAA-NM. We evaluated the state of differentiation by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immnocytochemistry. A neuronal marker ß III tubulin, a glial marker glial fibrillary acidic protein (GFAP) and a mature glial marker S100ß were expressed at a low level in the cultured cells while immature NSPCs marker Nestin and Sox2 were slightly lower without significant statistical difference. We concluded that the M-PAA-NM is a good substrate for adherent culture of NSPCs without triggering their cell differentiation, and also provides the maintenance of their growth with fewer passages in comparison with the conventional suspension culture of NSPCs in neurospheres.

Dense carbon-nanotube coating scaffolds stimulate osteogenic differentiation of mesenchymal stem cells.

Carbon nanotubes (CNTs) have desirable mechanical properties for use as biomaterials in orthopedic and dental area such as bone- and tooth- substitutes. Here, we demonstrate that a glass surface densely coated with single-walled carbon nanotubes (SWNTs) stimulate the osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs). MSCs incubated on SWNT- and multi-walled carbon nanotube (MWNT)-coated glass showed high activities of alkaline phosphatase that are markers for early stage osteogenic differentiation. Expression of Bmp2, Runx2, and Alpl of MSCs showed high level in the early stage for MSC incubation on SWNT- and MWNT-coated surfaces, but only the cells on the SWNT-coated glass showed high expression levels of Bglap (Osteocalcin). The cells on the SWNT-coated glass also contained the most calcium, and their calcium deposits had long needle-shaped crystals. SWNT coating at high density could be part of a new scaffold for bone regeneration.

Transparent biocompatible wool keratin film prepared by mechanical compression of porous keratin hydrogel.

We could prepare a transparent wool keratin film by mechanical compression of the keratin hydrogel, which was prepared by our method previously reported. Optical transmittance of the keratin film was approximately 70% at 400 nm and 80% at 550 nm. The keratin film had higher mechanical strength than the keratin hydrogel estimated from the tensile test. Young's modulus of the keratin film and that of keratin hydrogel were 0.582 ±â€¯0.294 MPa and 0.041 ±â€¯0.008 MPa, respectively. We evaluated degradability of keratin film by tryptic digestion in vitro and that also by implantation test in vivo. The keratin film showed slower degradation rate in the presence of trypsin in vitro, and also that as a subcutaneous implant in mouse in vivo. Biocompatibility is also a key factor for application of keratin as biomaterials. Within several days after subcutaneous implantation of the sample in mouse, an apparent symptom of acute inflammation of tissues, such as swelling of the reddish skin, was not observed. Keratin film remained in the original morphology of sheet-like structure while keratin hydrogel was degraded with many cracks and gaps after implantation for several weeks. We concluded from those results that keratin film was mostly biocompatible without provoking inflammation nor encapsulation, mechanically stronger than the keratin hydrogel, and was more resistant to degradation than the keratin hydrogel.

Clusters of neural stem/progenitor cells cultured on a soft poly(vinyl alcohol) hydrogel crosslinked by gamma irradiation.

Neural stem/progenitor cells (NSPCs) in the central nervous system (CNS) have the capacity to self-renew by proliferation and are multipotent, giving rise to neurons, astrocytes, and oligodendrocytes. NSPCs can be amplified in neurosphere suspension cultures for cell transplantation therapy to treat CNS diseases as well as for in vitro pharmacological/toxicological assays; however, these suspension cultures have certain limitations, including the inconvenience of changing the culture medium as well as difficulty of live imaging. In the present study, we prepared a gamma-crosslinked poly(vinyl alcohol) (PVA) hydrogel and assessed its suitability as a substrate for adherent NSPC cultures. Differentiation was determined by evaluating the expression of the markers nestin (progenitors), ßIII tubulin (neurons), and glial fibrillary acidic protein and S100ß (glia) by immunocytochemistry and quantitative reverse transcriptase PCR. The levels of the marker genes were similar between the two types of culture; although some variability was observed, there were no fold differences in expression. NSPCs adhered to the PVA gel as clusters and grew without differentiating into neurons and glia. The proliferation rate of cells grown on the soft PVA gel [3.75-7.5% (w/v) PVA] was approximately 70% of that of neurospheres in suspension. We conclude that gamma-crosslinked PVA hydrogels can function as a novel scaffold for maintaining adherent NSPCs in an undifferentiated state.

Porous hydrogel of wool keratin prepared by a novel method: an extraction with guanidine/2-mercaptoethanol solution followed by a dialysis.

In this study, we show a novel simple method to prepare a sponge-like porous keratin hydrogel through the extraction of wool keratin in a solution containing guanidine hydrochloride and 2-mercaptoethanol followed by dialysis for both aggregation of keratin and recrosslink. The gel had a highly porous structure and a fast-swelling property in rehydration after freeze-drying. It had also high mechanical strength both in the tensile test and the measurement of dynamic viscoelasticity. Three types of animal cells, PC12 cells, HOS cells and murine embryonic fibroblasts, well attached and grew on the surface of the porous hydrogel.