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Upon removal of the unbound antibody, cells were incubated with the Alexa Fluor 488-labeled secondary antibody (1:100 dilution) and TRITC-conjugated phalloidin (1:200 dilution) in the dark for another hour at room temperature

Upon removal of the unbound antibody, cells were incubated with the Alexa Fluor 488-labeled secondary antibody (1:100 dilution) and TRITC-conjugated phalloidin (1:200 dilution) in the dark for another hour at room temperature. 28 days of culture on HA-gHGP, Type I collagen production and mineral deposition were detected in the absence of osteogenic supplements, suggesting induction of osteogenic differentiation. In contrast, cells on the control gels expressed markers for adipogenesis. Overall, the HA-gHGP composite matrix has great promise for directing the osteogenic differentiation of MSCs by providing an adaptable environment through the spatial presentation of cell adhesive modules. relies on the strategic combination DMH-1 of synthetic scaffolds, viable cells and physiologically relevant biological cues and biophysical stimulations [3-5]. While primary cells isolated from the patients represent an optimal cell source, the inaccessibility of many cell types and their relatively short replicative life span post significant challenges for using these cells DMH-1 in tissue engineering [6]. On the other hand, multipotent human mesenchymal stem cells (MSCs) can be obtained from a variety of adult and fetal issues; they can be expanded for more than 50 cell doublings without signs of senescence and be differentiated into osteoblasts, chondrocytes, adipocytes and nerve cells under defined culture DMH-1 conditions [7-9]. MSCs are naturally sensitive to their environment, responding to chemical, physical and mechanical features of their matrices or substrates, as well as the spatial/temporal presentation of biochemical cues [10, 11]. Cellular behaviors, such as adhesion, proliferation, differentiation and migration, can be influenced by custom-designed, synthetic scaffolds that essentially recapitulate the native stem cell niche [12]. Hydrogels are widely employed as artificial matrices for tissue engineering due to their biocompatibility, high porosity and tissue-like elasticity [13-15]. The current investigation aims at developing hyaluronic acid (HA)-based hydrogels that are hierarchically structured, mechanically robust and chemically defined, suitable for use as conducive substrates for the controlled differentiation of bone-marrow-derived human MSCs. HA is a particularly attractive starting material for the fabrication of synthetic matrices due to its inherent bioactivity, biocompatibility, and biodegradability. Found primarily in the ECM of connective tissues (including bone marrow), HA functions in tissue support, lubrication, and modulation of tissue viscoelasticity [16]. More importantly, HA interacts with its cell surface receptors CD44 and RHAMM to activate various signaling pathways that direct a wide spectrum of cell functions [17, 18]. Our group has created a novel HA hydrogel system, referred to as the doubly crosslinked networks (DXNs), comprised of densely crosslinked HA hydrogel particles (HGPs) physically embedded in or covalently connected to a loosely crosslinked secondary network that is also HA-based [19-22]. While the HGPs exhibit inherent mesh size in the order BRAF of 5-10 [21] or 10-20 nm [23] depending on the particular chemistry employed for particle synthesis, the surrounding secondary matrix contains pores of hundreds of nanometers. The mechanical properties and the enzymatic stability of the HA DXNs can be separately tuned by altering the particle size as well as intra- and inter- particle crosslinking [20-22]. Although primary bovine chondrocytes were able to adapt to the three dimensional (3D) microenvironment and synthesize cartilage-specific glycosaminoglycan, the lack of cell-adhesive motifs in these HA DXNs limits their utility in long term culture of anchorage-dependent cells. Because MSC differentiation and subsequent neotissue formation is directly influenced by cell adhesion to their underlying biomaterials [24], imparting cell-adhesive properties to HA DXNs will significantly expand their applicability in regenerative medicine. The ultimate goal of this study was to develop a hybrid HA matrix that can be employed to mediate cell adhesion and to direct the fate of MSCs by simple manipulation of the hydrogel structure and composition. To this end, HA microparticles containing residual aldehyde groups [20, 23] were utilized for gelatin immobilization. Gelatin-conjugated HA HGPs (gHGPs) were successfully synthesized by a reductive amination reaction between the lysine amines on gelatin and the aldedhyde groups on HA HGPs. Subsequently, gHGPs were physically embedded in a photocrosslinked secondary matrix derived from.