Thermoreversible Protein Hydrogel as Cell Scaffold

Abstract

A thermoreversible fibrillar hydrogel has been formed from an aqueous lysozyme solution in the presence of dithiothreitol (DTT). Its physical properties and potential as a tissue engineering scaffold have been explored. Hydrogels were prepared by dissolving 3 mM protein in a 20 mM DTT/water mixture, heating to 85 degrees C and cooling at room temperature. No gel was observed for the equivalent sample without DTT. The elastic nature of the gel formed was confirmed by rheology, and the storage modulus of our gel was found to be of the same order of magnitude as for other cross-linked biopolymers. Micro differential scanning calorimetry (microDSC) experiments confirmed that the hydrogel was thermally reversible and that gelation and melting occurs through a solid-liquid-like first-order transition. Infrared spectroscopy of the hydrogel and transmission electron microscopy studies of very dilute samples revealed the presence of beta-sheet-rich fibrils that were approximately 4-6 nm in diameter and 1 mum in length. These fibrils are thought to self-assemble along their long axes to form larger fibers that become physically entangled to form the three-dimensional network observed in both cryo-scanning electron microscopy (cryo-SEM) and small-angle neutron scattering (SANS) studies. The hydrogel was subsequently cultured with 3T3 fibroblasts and cells spread extensively after 7 days and stretched actin filaments formed that were roughly parallel to each other, indicating the development of organized actin filaments in the form of stress fibers in cells.

Redox modulated hydrogelation of a self-assembling short peptide amphiphile

“…No secondary structural variation was observed after adding H 2 O 2 into the system, showing that the promotion of disulfide bond formation between the nanofibers did not cause detectable changes to the β-sheet structure (data not shown). Furthermore, complementary FTIR characterizations in D 2 O also indicated the predominance of β-sheet conformation ( Figure 6(b)), where the two peaks centering at 1621 and 1675 cm -1 are indicative of intermolecular anti-parallel β-sheet structure [36][37][38][39].…”

In vitro culture and characterization of enteric neural precursor cells from human gut biopsy specimens using polymer scaffold

“…Though ENS progenitors from the ganglionic gut of children diagnosed with and without Hirschsprung's disease have been isolated, characterized and reported earlier (12), a study similar to ours using a TGP which has proven to yield an increased number of neural progenitors (13), makes this unique. Apart from this, among the various types of thermo-reversible hydrogels reported for cell culture in the literature, the TGP used in this study is novel because it is a purely synthetic hydrogel and does not contain any biological components like proteins such as Hen Egg White Lysozyme (14) or poly-saccharides such as Chitosan (15) which are used in hydrogels to improve properties like cell-adhesion (14). Further, the TGP has been proven to maintain the three-dimensional morphology (16) of different kinds of stem cells, pre-cursor cells and adult cells without alteration of their gene expression (17) for longer periods of time in contrast to other neurosphere like bodies (NLBs) were observed in culture between the 8th day and 12th day ( Figure 1) including the aganglionic sample from Patient I with Hirschsprung's disease.…”

“…Metzger et al generated NLBs from postnatal human gut mucosal tissue and after transplantation; the cells from NLBs colonized aganglionic chick and human hindgut to generate ganglia-like structures, enteric neurons and glia (1). In this study, we have examined the feasibility of culturing NLBs obtained from routine thermo-reversible hydrogels reported (14). Previous studies have suggested that from the 10th day onwards, NLBs can be observed in culture and NLBs have been grown in culture up to 28 days (2,10).…”

Instrumentation and metrology for single RNA counting in biological complexes or nanoparticles by a single-molecule dual-view system

“…Scanning tunneling microscopy (STM) (Mikkelsen et al 2004;Kibsgaard et al 2006), transmission electron microscopy (TEM) (Kastner et al 1991;Pante et al 1997;Fu et al 2004), scanning electron microscopy (SEM) (Elias et al 2005;Yan et al 2006), atomic force microscopy (AFM) (Gould et al 1988;Marti et al 1988;Chen et al 2000), and optical tweezers (Block et al 1990;Finer et al 1994;Svoboda and Block 1994;Johnson et al 2007) have been used to image nanostructured materials, including nanomotors, with resolution in the nanometer scale. However, direct imaging or counting of biomolecules remains challenging.…”