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Temporally and spatially controlled delivery of growth factors in polymeric scaffolds is crucial for engineering composite tissue structures, such as osteochondral constructs. In the present study, microsphere-mediated growth factor delivery in polymer scaffolds and its impact on osteochondral differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was evaluated. Two growth factors, bone morphogenetic protein 2 (rhBMP-2) and insulin-like growth factor I (rhIGF-I), were incorporated as a single concentration gradient or reverse gradient combining two factors in the scaffolds. To assess the gradient making system and the delivery efficiency of polylactic-co-glycolic acid (PLGA) and silk fibroin microspheres, initially an alginate gel was fabricated into a cylinder shape with microspheres incorporated as gradients. Compared to PLGA microspheres, silk microspheres were more efficient in delivering rhBMP-2, probably due to sustained release of the growth factor, while less efficient in delivering rhIGF-I, likely due to loading efficiency. The growth factor gradients formed were shallow, inducing non-gradient trends in hMSC osteochondral differentiation. Aqueous-derived silk porous scaffolds were used to incorporate silk microspheres using the same gradient process. Both growth factors formed deep and linear concentration gradients in the scaffold, as shown by enzyme-linked immunosorbent assay (ELISA). After seeding with hMSCs and culturing for 5 weeks in a medium containing osteogenic and chondrogenic components, hMSCs exhibited osteogenic and chondrogenic differentiation along the concentration gradients of rhBMP-2 in the single gradient of rhBMP-2 and reverse gradient of rhBMP-2/rhIGF-I, but not the rhIGF-I gradient system, confirming that silk microspheres were more efficient in delivering rhBMP-2 than rhIGF-I for hMSCs osteochondrogenesis. This novel silk microsphere/scaffold system offers a new option for the delivery of multiple growth factors with spatial control in a 3D culture environment for both understanding natural tissue growth process and in vitro engineering complex tissue constructs.

A method was developed to prepare silk fibroin microspheres using lipid vesicles as templates to efficiently load protein drugs in active form for controlled release. The lipid was subsequently removed by methanol or sodium chloride treatments, resulting in silk microspheres consisting of beta-sheet structure and about 2 mum in diameter. NaCl treated microspheres had smoother surfaces compared to the methanol treatments based on SEM analysis, and both types of microspheres had a mixture of multilamellar and unilamellar structures. A model protein drug, horseradish peroxidase, was encapsulated in the microspheres. Freeze-thaw cycles during preparation led to higher loading of the peroxidase due to improved mixing between the silk and drug, while without this process the drug and silk remained in separate layers or domains in microspheres. This partitioning was determined with fluorescein-labeled silk and rhodamine-labeled dextran. Small molecules such as the enzyme substrate 3,3',5,5'-tetramethylbenzidine, Mw=240 Da, and its oxidized product freely diffused through the MeOH- and NaCl-processed silk microspheres so that enzyme loading and activity could be determined. Enzyme activity was retained during processing and in the final microspheres. The enzyme release profile depended on the NaCl-process used in microsphere preparation. The physically cross-linked beta-sheet structure of silk fibroin and the residual lipids in the microspheres played important roles in controlling enzyme release profiles. The silk microspheres have the potential for diverse applications where controlled protein release from biocompatible, mechanically tough, and slowly biodegradable carriers is desirable.

No abstract

Bombyx mori silk fibroin self-assembles on surfaces to form ultrathin nanoscale coatings based on our prior studies using layer-by-layer deposition techniques driven by hydrophobic interactions between silk fibroin protein molecules. In the present study, polylactic-co-glycolic acid (PLGA) and alginate microspheres were used as substrates and coated with silk fibroin. The coatings were visualized by confocal laser scanning microscopy using fluorescein-labeled silk fibroin. On PLGA microspheres the coating was ~1 μm and discontinuous, reflecting the porous surface of these microspheres determined by SEM. In contrast, on alginate microspheres the coating was ~10 μm thick and continuous. The silk fibroin penetrated into the alginate gel matrix. The silk coating on the PLGA microspheres delayed PLGA degradation. The silk coating on the alginate microspheres survived ethylenediamine tetraacetic acid (EDTA) treatment used to remove the Ca +2 -cross-links in the alginate gels to solubilize the alginate. This suggests that alginate microspheres can be used as templates to form silk microcapsules. Horseradish peroxidase (HRP) and tetramethylrhodamineconjugated bovine serum albumin (BSA) as model protein drugs were encapsulated in the PLGA and alginate microspheres with and without the silk fibroin coatings. Drug release was significantly retarded by the silk coatings when compared to uncoated microsphere controls, and was retarded further by methanol-treated silk coating when compared to silk water-based coatings on alginate microspheres. Silk coatings on PLGA and alginate microspheres provide mechanically stable shells as well as a diffusion barrier to the encapsulated protein drugs. This coating technique has potential for biosensor and drug delivery applications due to the aqueous process employed, the ability to control coating thickness and crystalline content, and the biocompatibility of the silk fibroin protein used in the process.