Silk ionomers for encapsulation and differentiation of human MSCs

Mesenchymal stem cells from human bone marrow (hMSCs), due to their intrinsic self-renewing ability and multilineage potential, can differentiate to various cell types of connective tissues including bone, cartilage, fat, muscle, tendon and stroma, thus representing one of the most significant cell sources for regenerative medicine [1–2]. To fully exploit the potential of stem cells in tissue engineering and regeneration, well-controlled techniques for in vitro cell expansion, maintenance and specific differentiation are needed. The most common methods to induce and direct stem cell differentiation in vitro involve the use of soluble chemical and biochemical factors. In contrast, numerous studies indicate that besides growth factors such as cytokines, chemokines and serum proteins, many other factors have a profound influence on cell differentiation [3]. In particular, hMSCs fate in vitro is dependent on physical and chemical dues from the environment as well as from the complex interactions between cells [4–5]. Cell function can be controlled using tailored biomaterials that mimic the native stem cell niche [6]. The engineering of functional tissues in vitro relies on the accurate combination of biomaterial, cells, biological signals and biophysical stimulation [7–8]. Therefore, there is a need to develop suitable biomaterial platforms in which hMSCs can be expanded and stimulated to lineage-specific differentiation, or maintained in an undifferentiated state [9–10]. In this context, cell encapsulation within hydrogels has many advantages for regenerative medicine, providing a hydrated three-dimensional environment with excellent permeability to nutrients and oxygen necessary for tissue healing or regeneration [11]. Silks are versatile protein biomaterials with useful physico-chemical and mechanical properties that undergo biodegradation in vivo, exhibit long-term stability and biocompatibility and have been successfully used in a range of tissue engineering applications [12–17]. In particular, native silk fibroin and its derivatives have been extensively studied as starting materials for hydrogels due to their self-assembly via physical beta sheet crosslinks, avoidance of chemical crosslinkers, and the mechanical properties and slow biodegradability of the hydrogels [12,18]. However, the gelation of silk aqueous solutions obtained by protein self-assembly occurs slowly under physiological conditions, often limiting cell encapsulation [19]. Therefore, we have developed more rapid modes of silk gelation useful for cell encapsulation, including vortexing [20] and ultrasonication [21]. As previously reported, we have developed two silk ionomer derivatives, silk fibroin-poly-L-lysine (SF-PL) and silk fibroin-poly-L-glutamic acid (SF-PG) [22], which physically interact via charge complexation to form reversible silk-based hydrogels suitable for cell encapsulation [22]. Furthermore, using layer by layer techniques, these silk ionomers can be used for the formation of pH-sensitive shell microcapsules for the encapsulation and delivery of macromolecules or cells [23]. In previous studies, we have also investigated the impact of biophysical signaling in control of hMSC function and differentiation [24–25]. These results indicate that the bioelectrical properties of stem cells, such as the transmembrane potential (Vmem), can strongly influence the differentiation state in hMSCs, and that the modulation of cell Vmem could be a useful tool to control stem cell differentiation [24–25]. In particular, the differentiating cells exhibited a hyperpolarized Vmem compared with undifferentiated hMSCs and improved osteogenic differentiation upon hyperpolarization [24]. The aim of the present study was to develop a new method for the encapsulation of hMSCs within charged silk-based hydrogels in order to investigate how stem cells fate is affected by the net charge in the three-dimensional environment. Part of the motivation for such studies was based on our prior studies of the role of stem cell membrane potential on hMSCs fate and function as mentioned above [24–25]. For the present study, ultrasonication was used to induce fast polymer gelation and in situ hMSCs encapsulation. The cells were cultured within the hydrogels in adipogenic, osteogenic or maintenance growth media for up to 56 days to assess differentiation.