Search

An enzyme aggregate of alginate lyase (EC 4.2.2.3) from flavobactierium was prepared using ammonium sulfate. The resultant aggregates upon cross-linking with glutaraldehyde produced insoluble and catalytically active cross-linked enzyme aggregate (CLEA) enzyme. The catalytic activity and stability of the cross-linked enzyme aggregate of alginate lyase (CLEA-AL) was studied in the presence of various pH, temperatures and organic solvents. Reusability, storage stability and surface morphology of the CLEA-AL were also studied. The native enzyme and CLEA-AL exhibited maximum enzyme activity at pH of 6.3 and at a temperature of 40°C. The CLEA-AL has good stability in nonpolar organic solvents and is thermally stable up to 50°C over a period of 8h. By encapsulating CLEA-AL into alginate hydrogel, we demonstrate that alginate hydrogels can be enzymatically degraded in a controlled fashion. The results also showed that degradation of alginate hydrogel with CLEA-AL incorporated beads is slower than native enzyme and therefore, CLEA-AL can be used for controlled degradation and release of various biologics from the degrading gel.

Composite coatings of electrostatically assembled layer-by-layer anionic and cationic polymers combined with an Mg(OH)2 surface treatment serve to provide a protective coating on AZ31 magnesium alloy substrates. These ceramic conversion coating and layer-by-layer polymeric coating combinations reduced the initial and long-term corrosion progression of the AZ31 alloy. X-ray diffraction and Fourier transform infrared spectroscopy confirmed the successful application of coatings. Potentiostatic polarization tests indicate improved initial corrosion resistance. Hydrogen evolution measurements over a 2 week period and magnesium ion levels over a 1 week period indicate longer range corrosion protection and retention of the Mg(OH)2 passivation layer in comparison to the uncoated substrates. Live/dead staining and DNA quantification were used as measures of biocompatibility and proliferation while actin staining and scanning electron microscopy were used to observe the cellular morphology and integration with the coated substrates. The coatings simultaneously provided improved biocompatibility, cellular adhesion and proliferation in comparison to the uncoated alloy surface utilizing both murine pre-osteoblast MC3T3 cells and human mesenchymal stem cells. The implementation of such coatings on magnesium alloy implants could serve to improve the corrosion resistance and cellular integration of these implants with the native tissue while delivering vital drugs or biological elements to the site of implantation.

The development of polyelectrolyte multilayered coatings on magnesium alloy substrates that can be used for controlled delivery of growth factors and required biomolecules from the surface of these degradable implants could have a significant impact in the field of bone tissue regeneration. The current work reports on the fabrication of multilayered coatings of alginate and poly-L-lysine on alkaline- and fluoride-pretreated AZ31 substrates using a layer-by-layer (LbL) technique under physiological conditions. Furthermore, these coatings were surface functionalized by chemical cross-linking and fibronectin immobilization, and the resultant changes in surface properties have been shown to influence the cellular activity of these multilayered films. The physicochemical characteristics of these coated substrates have been investigated using attenuated total reflectance Fourier transform infrared spectroscopy, atomic force microscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy. Cytocompatibility studies using MC3T3-E1 osteoblasts show that the fluoride-pretreated, cross-linked and fibronectin-immobilized LbL-coated substrates are more bioactive and less cytotoxic than the hydroxide-pretreated, cross-linked and fibronectin-immobilized LbL-coated samples. The in vitro degradation results show that the multilayered coatings of these natural polysaccharide- and synthetic polyamino acid-based polyelectrolytes do not alter the degradation kinetics of the substrates; however, the pretreatment conditions have a significant impact on the overall coating degradation behavior. These preliminary results collectively show the potential use of LbL coatings on magnesium-based degradable scaffolds to improve their surface bioactivity.