A multi-layer scaffold that incorporates bioglass, phosphatidylserine and steroidal saponins loaded collagen microparticles was prepared in this study. Combinatorial processing techniques involving porogen leaching protocol and freeze-drying were used. Drug distribution and microstructure could be controlled mainly by the concentrations of drug-loaded collagen microparticles and porogen as well as freezing temperature for different layers of the scaffolds. Morphological observation, measurement of swelling properties, examination of drug release kinetics, and analysis of cell bioactivity showed that the resultant scaffolds had highly interconnected pores, gradient drug distribution and graded porous micro-architecture, similar swelling ratio for different layers, better drug release kinetics in view of lower initial release and slower average release rate compared to the scaffolds with homogeneous structure, as well as approving support of cell ingrowth. This study suggests that the optimized scaffolds have promising potential for applications in bone tissue engineering.

AbstractScaffolds featuring spatiotemporal control of drug release is highly desirable for bone tissue regeneration. The objective of this study was to construct a scaffold with gradient porosity and drug distribution and evaluate the effect of scaffold structure on drug release kinetics and cell bioactivity. Nano-hydroxyapatite/collagen/phosphatidylserine scaffolds embedded with steroidal saponin loaded collagen microparticles were prepared using a porogen leaching protocol. Morphological characterization showed that the scaffolds consisted of dense layer and loose layer, and pores were interconnective. The microparticles were entrapped at the center of the scaffolds follow a gradient distribution. Release kinetics correlated with the structure. The loose layer showed greater drug release amount as compared to the dense layer. Such differences in release kinetics have distinct effects on cell bioactivity. Cell proliferated much more in loose layer than that in the dense layer. Such spatial and temporal control over drug deposition and delivery within the scaffolds could provide opportunities for tissue regeneration associated with optimum drug doses at wound site, and lessen undesirable drug release and side-effects at uninjured site.

Steroidal saponins are the major pharmacological constituents of ophiopogon japonicus which have been used to treat bone and joint diseases, nerve damage, and hematischesis for hundreds of years in China. The objective of this study was to investigate the therapeutic potential of collagen microspheres loaded with steroidal saponins as an inducer factor for skeletal defects using an in vitro model. The collagen - steroidal saponins composite microspheres were prepared using a water-in-oil-in-water emulsion/solvent evaporation technique. SEM micrographs showed that spherical particles with diameters of tens micrometers were successfully produced. Release kinetics was characterized by an initial burst release reducing to a linear release over the first 8 days. MTT assay showed that the cells attached on composite microspheres were more in comparison with pure collagen microspheres and stimulated significantly higher level of alkaline phosphatase than the counterpart. Moreover, composite microspheres possess osteoinductive activities as demonstrated by the ability to induce cells to commit to crystals in contact coculture. The present finding suggests that sustained delivery of steroidal saponins may be very effective in improving the osteogenic potential of collagen microspheres and the composite microspheres are useful for tissue-engineering applications.