Fabrication of micropatterned PCL-collagen nanofibrous scaffold for cellular confinement induced early osteogenesis

The intricate interaction of the scaffold's architecture/geometry and with the cells is essential for tissue engineering and regenerative medicine. Cells sense their surrounding dynamic cues such as biophysical, biomechanical, and biochemical, and respond to them differently. Numerous studies have recently explored and reported the effect of contact guidance by culturing various types of cells on different types of micropatterned substrates such as microgrooves, geometric (square and triangle) micropattern, microstrips, micropatterned nanofibers. Amongst all of these micropatterned polymeric substrates; electrospun nanofibers have been regarded as a suitable substrate as it mimics the native ECM architectures. Therefore, in the present study; stencil-assisted electrospun Grid-lined micropatterned PCL-Collagen nanofibers (GLMPCnfs) were fabricated and its influence on the alignment and differentiation of pre-osteoblast cells (MC3T3-E1) was investigated. The randomly orientated Non-patterned PCL-Collagen nanofibers (NPPCnfs) were used as control. The patterns were characterized for their geometrical features such as area and thickness of deposition using surface profiler and scanning electron microscopy. A 61 % decrease in the overall area of GLMPCnfs as compared to the stencil area demonstrated the potential of electrofocusing phenomenon in the process of patterning electrospun nanofibers into various micron-scale structures. The MC3T3-E1 cells were confined and aligned in the direction of GLMPCnfs as confirmed by a high cellular aspect ratio (AR = 5.41), lower cellular shape index (CSI = 0.243), and cytoskeletal reorganization assessed through the F-actin filament immunocytochemistry (ICC) imaging. The aligned cells along the GLMPCnfs exhibited elevated alkaline phosphatase activity and enhanced mineralization. Furthermore, the gene expression profiling revealed upregulation of key osteogenic markers, such as ALP, OCN, OPN, COL1A1, and osteocyte markers DMP1, and SOST. Consequently, the research highlights the impact of GLMPCnfs on the cellular behaviour that results to the pre-osteoblast differentiation and the potential for stimulant-free early osteogenesis. These results offer an extensive understanding and mechanistic insight into how scaffold topography can be modified to influence cellular responses for effective bone regeneration strategies.