Multiphoton microfabrication and micropatternining (MMM)-based screening of multiplex cell niche factors for phenotype maintenance - Bovine nucleus pulposus cell as an example.

Upon monolayer cultures on flat and rigid plastic dishes, many cells de-differentiate and lose their native phenotype. Technologies able to identify and reconstitute the cell niche factors that best maintain the physiological cellular phenotype in cultures are critical. We have developed a multiphoton microfabrication and micropatterning (MMM) technology, a robust 3D micro-printing platform capable to fabricate protein microstructures and micropatterns with quantitative, spatial and independent control of the mechanical, topological and extracellular matrix properties. Here, using bovine nucleus pulposus cells (bNPCs) as an example, we aim to reconstitute a spectrum of individual cell niche factors (2 mechanical, 9 topological and 4 matrices) in vitro for multiplex cell niche factor screening, and fabricate the optimal combinations of a series of shortlisted cell niche factors that best maintain the bNPC phenotype. Among all factors screened, two topological (micropillar array; fiber-bead structure) and two matrix (laminin; vitronectin) factors were shortlisted and the combinatory cell niche factors reconstituted from the shortlisted factors were found to synergistically augmented the expression of selected bNPC phenotype markers (Col II, SNAP25 and Keratin 8) and maintained their morphology and phenotype. These optimal cell niches can be micro-printed on culture dishes for physiologically relevant cultures and contribute to biomimetic scaffold design for intervertebral disc tissue engineering.

Collagen microencapsulation recapitulates mesenchymal condensation and potentiates chondrogenesis of human mesenchymal stem cells - A matrix-driven in vitro model of early skeletogenesis.

Mesenchymal condensation is a critical transitional stage that precedes cartilage or bone formation. A microencapsulation technique was previously established to entrap mesenchymal stem cells (MSC) in nanofibrous collagen meshwork. We hypothesize that collagen microencapsulation of MSCs mimics the mesenchymal cell condensation process. Specifically, human MSCs at different concentrations were microencapsulated in collagen for different time points before evaluation for early skeletogenesis markers. A transient upregulation of mesenchymal condensation markers including peanut agglutinin, fibronectin, integrins α5 and αv, an enhanced nuclear localization of SOX9 and binding interactions with COL2A1, and other changes in chondrogenic, hypertropic and osteogenic marker were demonstrated. Collagen microencapsulation upregulated both the chondrogenic and the osteogenic transcription factors and the encapsulated hMSCs hold the potential to differentiate towards both chondrogenic and osteogenic lineages. We also hypothesize that collagen microencapsulation potentiates MSC chondrogenesis. Particularly, chondrogenic differentiation of hMSCs were induced at different time post-encapsulation before evaluation for chondrogenesis outcomes. Sustained SOX9, ACAN and COL2A1 expression were noted and the timing to induce supplement chondro-inductive factors matters. This study reports an extracellular matrix-based in vitro model of mesenchymal condensation, an early stage in skeletogenesis, contributing to rationalizing development-inspired tissue engineering.