The musculoskeletal system comprises multiple tissue types including bone, muscle, tendon, ligament and cartilage as well as their respective tissue interfaces such as bone-to-tendon entheses and muscle-to-tendon junctions. The maintenance and repair of these multi-tissue structures involves the spatial control of stem cell differentiation toward tissue-specific cells, such as osteoblasts, tenocytes and myocytes [1
]. This process is regulated by physical and biochemical microenvironmental cues imparted by the interactions of cells with their extracellular matrix (ECM), neighboring cells, and secreted local and systemic signaling molecules, including growth factors (GFs) [1
]. Signaling molecules, regulate the pericellular environment where they reside in both the ‘liquid-phase’ (freely diffusing) form and the ‘solid-phase’ (immobilized) form that exists in an equilibrium state between desorption from and adsorption to the ECM and cell surfaces [3
]. The unique architecture and biochemical composition of the ECM allows it to sequester (immobilize) and release GFs at picogram to nanogram levels [3
], and can negatively or positively regulate GF bioactivity and bioavailability [3
]. As such, GF sequestration by the ECM immobilizes GFs to specific locations, which in turn imparts the temporal and spatial cues required for directing cell behaviors such as cell adhesion, migration, proliferation, differentiation and apoptosis, which are vital for orchestrating complex processes such as development, maintenance and wound healing [3
]. Therefore, developing toolsets that can be used to selectively control the physical placement and dosage of multiple exogenous GFs in a physiologically-relevant manner in order to spatially direct a stem cell population toward multiple cell fates simultaneously is a logical consideration for studying stem cell behaviors and may also have direct applications in regenerative medicine.
Prior work reported by our group and by others has shown that ECMs patterned with solid-phase GFs can be engineered to control various aspects of stem cell behavior, including proliferation, migration and differentiation in vitro
] as well as differentiation in vivo
]. We previously demonstrated that a GF-patterned fibrin ECM created using an inkjet-based bioprinting technology can drive a single stem cell population toward osteoblast and myocyte fates simultaneously, in registration to printed patterns in vitro
]. In the work presented here, we report on the extension of this approach to spatially drive stem cell differentiation towards a tendon fate simultaneously with osteoblast and myocyte differentiation.
Using solid-phase GFs to direct stem cells to tenocytes in vitro
has not been previously reported in literature. Therefore, prior to studying multi-lineage patterning, candidate tendon-promoting GFs had to be identified and validated. Candidate GFs were screened against mouse C3H10T1/2 mesenchymal fibroblasts, C2C12 myoblasts and primary muscle-derived stem cells (MDSCs) using both liquid- and solid-phase immunofluorescence staining for the tendon marker Scleraxis (Scx) [24
]. Quantitative PCR studies were subsequently performed to elucidate the mechanism by which stem cells differentiated towards a tendon lineage. Following this, solid-phase presentation of FGF-2 and/or BMP-2 on fibrin-coated glass coverslips using either coarse hand-printing or high resolution, low-dose inkjet bioprinting was used to demonstrate spatial control of stem cell differentiation towards multiple cell fates simultaneously.