A novel 3D printing method for cell alignment and differentiation

The application of bioprinting allows precision deposition of biological materials for bioengineering applications. Here we propose a 2 stage methodology for bioprinting using a back pressure-driven, automated robotic dispensing system. This apparatus can prepare topographic guidance features for cell orientation and then bioprint cells directly onto them. Topographic guidance features generate cues that influence adhered cell morphology and phenotype. The robotic dispensing system was modified to include a sharpened stylus that etched on a polystyrene surface. The same computer-aided design (CAD) software was used for both precision control of etching and bioink deposition. Various etched groove patterns such as linear, concentric circles, and sinusoidal wave patterns were possible. Fibroblasts and mesenchymal stem cells (MSC) were able to sense the grooves, as shown by their elongation and orientation in the direction of the features. The orientated MSCs displayed indications of lineage commitment as detected by fluorescence-activated cell sorting (FACS) analysis. A 2% gelatin bioink was then used to dispense cells onto the etched features using identical, programmed co-ordinates. The bioink allows the cells to contact sense the pattern while containing their deposition within the printed pattern.The application of bioprinting allows the precision deposition of biological material for bioengineering applications. Here we propose a 2 stage methodology for bioprinting using a back pressure driven automated robotic dispensing system. This apparatus can prepare topographic guidance features for cell orientation and then bioprint cells directly to them. Topographic guidance features generate cues that influence adhered cell morphology and phenotype. The robotic dispensing system was modified to include a sharpened stylus that etched a polystyrene surface. The same CAD software was used for both precision control of etching and bioink deposition. Various etched groove patterns were possible, such as linear, concentric circles and sinusoidal wave patterns. Fibroblasts and mesenchymal stem cells (MSC) could sense the grooves, elongating and orientating themselves in the direction of the features, with the MSCs displaying indications of lineage commitment. A 2% gelatin bioink was then used to dispense cells onto the etched features using identical programmed co-ordinates. The bioink allows the cells to contact sense the pattern while containing their deposition within the printed pattern.

[1]  Mary B. Chan-Park,et al.  Regulating orientation and phenotype of primary vascular smooth muscle cells by biodegradable films patterned with arrays of microchannels and discontinuous microwalls. , 2010, Biomaterials.

[2]  Subbu Venkatraman,et al.  Printing cell-laden gelatin constructs by free-form fabrication and enzymatic protein crosslinking , 2015, Biomedical microdevices.

[3]  R. G. Harrison,et al.  The reaction of embryonic cells to solid structures , 1914 .

[4]  Jason A. Burdick,et al.  Controlling Stem Cell Fate with Material Design , 2010, Advanced materials.

[5]  Peter Hinterdorfer,et al.  Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy , 2005, Journal of Cell Science.

[6]  Jian Shi,et al.  Patterning of two-level topographic cues for observation of competitive guidance of cell alignment. , 2012, ACS applied materials & interfaces.

[7]  Yee Han Kuan,et al.  Scalable alignment of three-dimensional cellular constructs in a microfluidic chip. , 2013, Lab on a chip.

[8]  M. Foss,et al.  Guidance of stem cell fate on 2D patterned surfaces. , 2012, Biomaterials.

[9]  Mauris N Desilva,et al.  Patterning Cells on Complex Curved Surface by Precision Spraying of Polymers , 2010 .

[10]  G. Leitinger,et al.  Comparison of two in vitro systems to assess cellular effects of nanoparticles-containing aerosols , 2013, Toxicology in vitro : an international journal published in association with BIBRA.

[11]  D. Mills,et al.  Influence of channel width on alignment of smooth muscle cells by high-aspect-ratio microfabricated elastomeric cell culture scaffolds. , 2005, Journal of biomedical materials research. Part A.

[12]  Anthony Atala,et al.  3D bioprinting of tissues and organs , 2014, Nature Biotechnology.

[13]  Meifeng Xu,et al.  Laser patterning for the study of MSC cardiogenic differentiation at the single-cell level , 2013, Light: Science & Applications.

[14]  Ali Khademhosseini,et al.  Engineered contractile skeletal muscle tissue on a microgrooved methacrylated gelatin substrate. , 2012, Tissue engineering. Part A.

[15]  Elizabeth G Loboa,et al.  Cytoskeletal and focal adhesion influences on mesenchymal stem cell shape, mechanical properties, and differentiation down osteogenic, adipogenic, and chondrogenic pathways. , 2012, Tissue engineering. Part B, Reviews.

[16]  J. Nyengaard,et al.  The Three‐Dimensional Arrangement of the Myocytes Aggregated Together Within the Mammalian Ventricular Myocardium , 2009, Anatomical record.

[17]  J. Heitz,et al.  Dynamics of spreading and alignment of cells cultured in vitro on a grooved polymer surface , 2011 .

[18]  A Curtis,et al.  Topographical control of cells. , 1997, Biomaterials.

[19]  Evren U Azeloglu,et al.  The guidance of stem cell differentiation by substrate alignment and mechanical stimulation. , 2013, Biomaterials.

[20]  Thomas J Webster,et al.  Improved endothelial cell adhesion and proliferation on patterned titanium surfaces with rationally designed, micrometer to nanometer features. , 2008, Acta biomaterialia.

[21]  Angela W. Xie,et al.  Context clues: the importance of stem cell-material interactions. , 2014, ACS chemical biology.

[22]  L. Stanton,et al.  Nanofiber topography and sustained biochemical signaling enhance human mesenchymal stem cell neural commitment. , 2012, Acta biomaterialia.

[23]  Feng Xu,et al.  Engineering cell alignment in vitro. , 2014, Biotechnology advances.

[24]  C. Wilkinson,et al.  Topographical control of cell behaviour: II. Multiple grooved substrata. , 1990, Development.

[25]  Jonghee Yoon,et al.  Phenotypic Modulation of Primary Vascular Smooth Muscle Cells by Short-Term Culture on Micropatterned Substrate , 2014, PloS one.

[26]  M. Soleimani,et al.  Comparison of random and aligned PCL nanofibrous electrospun scaffolds on cardiomyocyte differentiation of human adipose-derived stem cells , 2014, Iranian journal of basic medical sciences.

[27]  Jeong‐Yeol Yoon,et al.  Linear fibroblast alignment on sinusoidal wave micropatterns. , 2013, Colloids and surfaces. B, Biointerfaces.

[28]  Zhen Ma,et al.  Laser guidance-based cell micropatterning , 2010 .

[29]  Lay Poh Tan,et al.  Micro-/nano-engineered cellular responses for soft tissue engineering and biomedical applications. , 2011, Small.

[30]  L. Germain,et al.  Alignment of Cells and Extracellular Matrix Within Tissue- Engineered Substitutes , 2013 .

[31]  F. Wen,et al.  Direct laser machining-induced topographic pattern promotes up-regulation of myogenic markers in human mesenchymal stem cells. , 2012, Acta biomaterialia.