Vascular cell viability on polyvinyl alcohol hydrogels modified with water-soluble and -insoluble chitosan.

Polyvinyl alcohol (PVA) hydrogels blended with chitosan or other biological macromolecules have shown promise for cell culture and tissue engineering. This study investigates the attachment and growth of bovine aortic endothelial (BAEC) and smooth muscle cells (BASMC) on the PVA hydrogels modified with water soluble and water insoluble chitosan. Cell adhesion on the surface of the membranes was examined by phase contrast microscopy while cell morphologies were studied using immunocytochemistry staining with EC and SMC specific biomarkers (F-actin and alpha actin respectively). Cells cultured on 6% PVA, 0.4% chitosan (water soluble and insoluble) hydrogel membranes displayed excellent adhesion and spreading characteristics, in addition to negligible cell structural morphological changes in comparison to a polystyrene control. Similar vascular cell adhesion features were apparent on PVA membranes blended with water-soluble and -insoluble chitosan. Fluorescent activated cell sorter (FACS) analysis was used to determine BAEC and BASMC proliferation and cell viability. Apoptotic levels in BAEC after 7 days were 12.8% +/- 2.5% on the PVA- chitosan WS-1 membrane and 10.1% +/- 1.5% on the control well (n = 3) while comparable results were also noted for BASMC. Equivalent proliferative activity was apparent for BAEC on the control and PVA-chitosan membrane after 7 days, while BASMC showed increased proliferative activity on the membranes. These results indicate that the PVA-chitosan blended hydrogel membranes show promise for cell culture and tissue engineering applications.

Purification and characterization of a Z-pro-prolinal-insensitive Z-Gly-Pro-7-amino-4-methyl coumarin-hydrolyzing peptidase from bovine serum--a new proline-specific peptidase.

The study of a new proline-specific peptidase from bovine serum is presented. The enzyme readily cleaves the prolyl oligopeptidase (PO) substrate Z-Gly-Pro-MCA, liberating the fluorophore MCA, thus allowing quantification of enzyme activity. Unlike PO, however, this peptidase is completely insensitive to the PO-specific inhibitor Z-Pro-prolinal and has been designated Z-Pro-prolinal-insensitive Z-Gly-Pro-MCA-hydrolyzing peptidase (ZIP). The two peptidases were successfully separated from each other by phenyl Sepharose hydrophobic interaction chromatography and the subsequent purification focused on the isolation of ZIP from bovine serum. In addition to phenyl Sepharose, calcium phosphate cellulose and DEAE anion-exchange chromatography were employed in the purification, with an overall enzyme yield of 33% and a purification factor of 4023. SDS-PAGE and size-exclusion chromatography indicated a dimeric structure with a relative molecular mass of 174 kDa. The enzyme was stable over the pH range 2.5-10.0. Optimal activity was detected in the pH range 7.4-8.0. Isoelectric focusing revealed a pI of 5.68. Inhibition by AEBSF suggests the peptidase may be a serine protease and ZIP possibly contains a cysteine residue near the active site. alpha(2)M failed to inhibit activity, suggesting oligopeptidase specificity. HPLC analysis revealed a broad substrate specificity for proline-containing peptides. Kinetic analysis indicated that ZIP had a high affinity for Z-Gly-Pro-MCA with a K(m) of 54 microM deduced. Bovine serum ZIP exhibits biophysical characteristics both similar to and different from those of PO isolated from a number of sources and may serve an important physiological function in the degradation of bioactive oligopeptides.