Direct laser machining-induced topographic pattern promotes up-regulation of myogenic markers in human mesenchymal stem cells.

The engineering of tissue is preferably done with stem cells, which can be differentiated into the tissue of interest using biochemical or physical cues. While much effort has been focused on using biological factors to regulate stem cell differentiation, recently interest in the contribution of physical factors has increased. In this work, three-dimensional (3-D) microchannels with topographic micropatterns were fabricated by femtosecond laser machining on a biodegradable polymer (poly(L-lactide-co-ε-caprolactone)) substrate. Two substrates with narrow and wide channels respectively were created. Human mesenchymal stem cells (hMSCs) were cultured on the scaffolds for cell proliferation and cellular organization. Gene expression and the immunostaining of myogenic and neurogenic markers were studied. Both scaffolds improved the cell alignment along the channels as compared to the control group. Microfilaments within hMSCs were more significantly aligned and elongated on the narrower microchannels. The gene expression study revealed significant up-regulation of several hallmark markers associated with myogenesis for hMSCs cultured on the scaffold with narrow microchannels, while osteogenic and neurogenic markers were down-regulated or remained similar to the control at day 14. Immunostaining of myogen- and neurogen-specific differentiation markers were used to further confirm the specific differentiation towards a myogenic lineage. This study demonstrates that femtosecond laser machining is a versatile tool for generating controllable 3-D microchannels with topographic features that can be used to induce specific myogenic differentiation of hMSCs in vitro, even in the absence of biological factors.

Multiscale topological guidance for cell alignment via direct laser writing on biodegradable polymer.

Direct laser writing on biodegradable polymer to create microchannels for aligning cells is presented here. This technique offers the advantages of ease-of-manufacturing, ease-of-design, high-speed single-step fabrication, and noncontacting to the material. In this work, microchannels of 100 microm width, 100 microm depth, and 50 microm intervals were created on a biodegradable polymer film directly using a Ti-sapphire femtosecond pulsed laser. Multiscale topological features were achieved as a result of the laser beam-material interaction. These topological features were used to guide cell alignment in the microchannels. We present results on the morphology of poly(L-lactide-co-epsilon-caprolactone) copolymer micromachined by femtosecond laser and demonstrate the attachment and alignment of C2C12 myoblast cells in the microchannels. C2C12 cells exhibited favorable attachment in the channels after 1 day of seeding. High degree of alignment was observed after 4 days as cells proliferated into a confluent patch inside the channels. This work demonstrated the potential of wavy surface features combined with appropriate channel size for high-density cell alignment using direct laser writing. This method also offers the opportunity to incorporate multiscale topological guidance on other biodegradable polymer implants, such as vascular scaffolds and stents, which require directed cell organization.