Preparation of TGF-beta1/affinity-bound alginate macroporous scaffolds

Spatio-temporal presentation of growth factors is one of the key attributes of the cell's microenvironment. The design of macroporous alginate scaffolds, wherein TGF-b1 or BMP-4 is electrostatically bound to affinity binding sites of alginate sulfate, mimicking their presentation by the extracellular matrix (ECM), was previously shown to enable sustained presentation and release of each factor, thus increasing their biological activity. Specifically, TGF-b1/affinity-bound scaffolds induced the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded within these scaffolds. The prolonged activity of the affinity-bound TGF-b1 enabled efficient induction of signaling pathways leading to chondrogenesis, up to the appearance of committed chondrocytes. Similarly, BMP-4 affinity-bound to the macroporous alginate scaffold enabled efficient induction of osteogenic differentiation in hMSC constructs. Subsequent construction of a multicompartment inductive system, spatially-presenting TGF-b1 and BMP-4 in two distinct layers, enabled complete differentiation of hMSC to chondrocytes and osteoblasts, depending on the type of factor in use in the respective layer. This paper describes in detail the preparation method of the TGF-b1 or BMP4/ affinity-bound alginate scaffolds, and the set of analyses performed to characterize the resultant scaffolds, including release profile study, released factor bioactivity, and functionality of the scaffolds as hMSC-inductive scaffolds.