Bioinductive protein-based scaffolds for human mesenchymal stem cells differentiation.

摘要

Silks are biocompatible and biodegradable polymers with excellent mechanical properties. The present dissertation explores fundamental aspects of silk related to pluripotent adult human bone marrow stromal cells (hMSCs) in combination with bone morphogenetic protein-2 (BMP-2) for bone regeneration. Silk fibroin, extracted from Bombyx mori silkworm cocoons, was processed into 3-D porous scaffolds decorated with a pentapeptide containing the cell attachment sequence RGD. Scaffolds were fixed on tissue culture plates, seeded with hMSCs and cultured in osteogenic medium supplemented with BMP-2. Micro-computer tomography (muCT) showed the development of long interconnected and organized bone-like trabeculae with cuboid cells. The X-ray diffraction pattern of the deposited bone corresponded to hydroxyapatite composition present in the native bone.; To increase osteoconductivity, BMP-2 was loaded in the porous silk fibroin scaffolds. The release profile of BMP-2 under dynamic culture conditions (spinner flasks) showed an initial burst after 1 week in culture; up to 4 weeks no additional BMP-2 was released. BMP-2 induced hMSCs to undergo osteogenic differentiation when the seeded scaffolds were cultured for 4 weeks, based on elevated alkaline phosphatase activity, calcium deposition and transcript levels for bone-characteristic markers (bone sialoprotein, osteopontin, osteocalcin, BMP-2 and cbfa1). muCT revealed densely deposited mineral at the center of the scaffolds.; Using carbodiimide chemistry, BMP-2 was covalently immobilized on silk fibroin films. Whereas hMSCs cultured on unmodified silk fibroin films in the presence of osteogenic stimulants exhibited little if any osteogenesis, the same cells cultured on BMP-2 decorated films in the presence of osteogenic stimulants differentiated into an osteoblastic lineage as assessed by their significantly elevated alkaline phosphatase activity, calcium deposition and higher transcript levels for bone-characteristic markers. Using cell culture inserts, it was demonstrated that differentiation was induced by the immobilized protein and not by protein released into the culture medium. Comparison with a similar amount of medium supplemented BMP-2, where no additional protein was added with medium changes, showed that delivery of BMP-2 immobilized on the biomaterial surface was more efficient than soluble delivery. This is the first reported biologically active BMP-2 covalently immobilized on protein-based scaffolds used to induce differentiation of hMSCs toward an osteogenic outcome. (Abstract shortened by UMI.)