Biphasic calcium phosphate scaffolds with controlled pore size distribution prepared by in-situ foaming.

In this study, a reproducible method of fabricating hierarchically 3D porous scaffolds with high porosity and pore interconnectivity is reported. The method is based on in-situ foaming of a dispersion of diisocyanate, polyol, water and hydroxyapatite (HA) to form a hard foamed HA/polyurethane composite which after heat treatment provided a bi-phase calcium phosphate scaffold. This technique, combining the advantages of polymer sponge and direct foaming methods, provides a better control over the macrostructure of the scaffold. A modification of the multi-scaled porous macrostructure of scaffolds produced by changing the ratio of input reactants and by sintering temperature was studied. The pore morphology, size, and distribution were characterized using a scanning electron microscope and mercury porosimetry. The pores were open and interconnected with multi-scale (from several nanometres to millimetres) sizes convenient for using in tissue engineering applications. The bioactivity was confirmed by growing an apatite layer on the surfaces after immersion in simulated body fluid. The material was biocompatible, as shown by using normal human adipose tissue-derived stem cells (ASC). When seeded onto the scaffolds, the ASC adhered and remained healthy while maintaining their typical morphology.

Iminodiacetic acid-modified magnetic poly(2-hydroxyethyl methacrylate)-based microspheres for phosphopeptide enrichment.

Magnetic non-porous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres prepared by the dispersion polymerization and modified with iminodiacetic acid (IDA) were employed for the IMAC separation of phosphopeptides. Fe(3+) and Ga(3+) ions immobilized on IDA-modified magnetic microspheres were used for the enrichment of phosphopeptides from the proteolytic digests of two model proteins differing in their physico-chemical properties and phosphate group content: porcine pepsin A and bovine α-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The phosphopeptides separated from the proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ability of the prepared Fe(3+)- and Ga(3+)-IDA-modified magnetic microspheres to capture phosphopeptides from complex mixtures was shown on an example of bovine milk proteolytic digest.

Study of pepsin phosphorylation using immobilized metal affinity chromatography.

The interactions of pepsin with immobilized trivalent metal ions and the participation of the enzyme phosphate group in this process were investigated using high performance immobilized metal affinity chromatography. Two different sorbents were used: the newly prepared one, consisting of Ga(3+ )chelate of (6-amino-1-hydroxyhexane-1,1-diyl) bis(phosphonic acid) covalently bound to a methacrylate support (BP-Ga(3+)), and the commercial one, containing immobilized Fe(3+ )ions (POROS MC20-Fe(3+)). The comparison of the behavior of porcine pepsin A and its partially dephosphorylated form on both sorbents showed that both forms of pepsin were adsorbed under the same conditions. To eliminate the participation of free carboxyl groups in pepsin adsorption, both enzyme forms were modified by amidation or esterification. Native enzyme and its partially dephosphorylated form both with modified carboxyl groups differed in their interaction with immobilized Ga(3+ )and Fe(3+). Phosphorylated pepsin molecules with esterified carboxyl groups were adsorbed on both sorbents while nonphosphorylated ones with esterified carboxyl groups were not adsorbed.

Affinity chromatography of porcine pepsin A using quinolin-8-ol as ligand.

Stationary phase containing quinolin-8-ol immobilized on macroporous methacrylate support for the affinity chromatography of porcine pepsin A is described. Optimized chromatographic conditions for separation of porcine pepsin A on this stationary phase were found investigating the influence of pH, concentration, ionic strength and chemical composition of the used mobile phases. The stationary phase shows a good reproducibility of chromatographic analyses (relative standard deviation, +/-2%), a high recovery (ca. 93%) and a satisfactory capacity (13 mg pepsin A/1 mL stationary phase) for porcine pepsin A. The obtained findings confirm the applicability of affinity chromatography on the stationary phase with immobilized quinolin-8-ol to the isolation and determination of porcine pepsin A.