Porous silk fibroin 3-D scaffolds for delivery of bone morphogenetic protein-2 in vitro and in vivo.

Bone morphogenetic protein-2 (BMP-2) plays a key role in osteogenesis. Biomaterials used for the sustained delivery of BMP-2 in vivo have shown therapeutic benefits. In the present study, BMP-2 was loaded in porous silk fibroin scaffolds derived from silkworm cocoons (2.4 +/- 0.14 microg per scaffold). The release profile of BMP-2 under dynamic culture conditions (spinner flasks) showed that after 1 week in culture 25% of the initial BMP-2 was retained adsorbed to the scaffold; up to 4 weeks no additional BMP-2 was released. BMP-2 induced human bone marrow stromal cells (hMSCs) to undergo osteogenic differentiation when the seeded scaffolds were cultured in medium supplemented with osteogenic stimulants for 4 weeks, based on elevated alkaline phosphatase activity, calcium deposition, and transcript levels for bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa-1. Micro-computed tomography revealed densely deposited mineral at the center of the scaffolds. In contrast, hMSCs cultured in control scaffolds (no BMP-2) exhibited limited osteogenesis. When implanted in critical sized cranial defects in mice, scaffolds loaded with BMP-2 and seeded with hMSCs resulted in significant bone ingrowth. These results were qualitatively similar to scaffolds loaded with BMP-2 but no hMSCs or with BMP-2 and hMSCs but not pregrown into bone-like tissue. Bone-related outcomes were improved when compared with the scaffold controls implanted without BMP-2. These studies illustrate the potential use of slow degrading silk fibroin 3-D scaffolds loaded with BMP-2, in combination with hMSCs, in osteogenesis studies in vitro and in vivo, and provide a new range of material properties for these applications.

Bone morphogenetic protein-2 decorated silk fibroin films induce osteogenic differentiation of human bone marrow stromal cells.

Bone morphogenetic protein (BMP)-2 has a critical role in bone formation and regeneration. Therefore, the ability to immobilize this molecule in certain matrices may be crucial in bone tissue engineering. Using carbodiimide chemistry, BMP-2 was directly immobilized on silk fibroin films. Whereas human bone marrow stromal cells 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 of collagen type I, bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa1. 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. The results illustrate that BMP-2 covalently coupled on silk biomaterial matrices retains biological function in vitro based on the induction of osteogenic markers in seeded bone marrow stromal cells.