Hierarchically Engineered Biomimetic 3-D Scaffolds for Guided Regeneration of Bone Tissue

Considering the complex hierarchical structure of bone, biomimicking the micro and nano level features should be an integral part of scaffold fabrication for successful bone regeneration. In this thesis, we aim to biomimic the micro and nanostructure of bone and study the effect of physical and mechanical cues on cell alignment, proliferation and differentiation. To achieve this, in the first part of our study we have combined effect of anisotropy (micropattern, using photolithography technique) and the isotropy (nanofibers using electrospinning technique) on mesenchymal stem cell (MSC) alignment, proliferation and osteodifferentiation on SU-8 polymer scaffolds. We hypothesize that biomimicking the hierarchical features of bone at nano and microlevel will provide a better niche for MSC alignment and induce early osteodifferentiation of MSCs even in absence of external chemical factors. We divided the scaffolds into groups: electrospun SU-8 nanofibers, electrospun SU-8 nanofibers with UV treatment and micropatterned (20 µm sized ridges and grooves) SU-8 nanofibers by photolithography with UV treatment. Two types of culture conditions were applied: with and without osteoinduction medium. In-vitro cell proliferation assays, protein estimation, ALP osteodifferentiation assay, live dead assay and cell alignment studies were performed on these micro-patterned nanofiber domains. Our findings show that patterned surface induced an early osteodifferentiation of MSCs even in absence of osteoinduction medium. An interesting similarity with the helicoidal plywood model of the bone was observed. The cells showed layering and rotation along the patterns with time. This resembles the in-vivo anisotropic multilamellar bone tissue architecture thus, closely mimicking the sub-cellular features of bone. This might serve as a smart biomaterial surface for MSC differentiation in therapeutics where the addition of external chemical factors is a challenge. With this background, the next set of experiments deals with 3-D cell culture of spheroids on patterned scaffolds. 2-D cell culture has been widely developed with various micropatterning and microfabrication techniques over the past few decades for creating and controlling cellular microenvironments including cell-matrix interactions, cell-cell interactions, and bio-mimicking the in-vivo tissue hierarchy and functions. However, the drawbacks of 2-D culture has currently paved the way to 3-D cell culture which is considered clinically and biologically more relevant. Here we report a 3-D double strategy for osteodifferentiation of MSC spheroids on nano- and micropatterned PLGA/Collagen/nHAp electrospun fiber mats. A comparison of cell alignment, proliferation and differentiation of 2-D and 3-D MSCs on patterned and non-patterned substrate was done. The study demonstrates the synergistic effect of geometric cues and 3-D culture on differentiation of MSC spheroids into osteogenic lineage even in absence of osteoinduction medium. Hence, in this work, we made an effort to successfully combine the strength of 3-D spheroid culture with the hierarchically patterned micro and nanostructures to create a biologically and clinically relevant functional material for regeneration of bone tissue. In the last set of experiment, we have investigated the effect of mechanical strain along with patterned substrates on differentiation of MSCs to osteogenic lineage. We fabricated a thin, stretchable patterned PDMS membrane which warrants high-throughput to study different geometric pattern dimensions in a single sheet of the fabricated membrane. The membrane was integrated at the bottom of a customized 96-well plate device. Our previously developed multiwell stretch device was used for applying uniform strain across individual wells of the developed 96-well patterned stretch plates. We tested the effect of MSC differentiation to osteogenic lineage on 12 different micropattern dimensions at two strain frequencies (0.1Hz and 1 Hz) and a strain magnitude of 7.5%. Hence, we tried to biomimic the physiological conditions found invivo to investigate the effect on MSC differentiation to osteogenic lineage. Hence the findings from this work can be used as a model system to study the effect of topographical parameters and mechanical stimuli not only for bone, but also for other tissues owing to the accuracy and ease of the fabrication technique used. Together, the finding from this study, bio-mimicking the micro and nano features of bone tissue, will improve the efficiency of the currently available scaffolds for bone tissue regeneration and repair.