Figure 5: (A) Histogram of pore sizes (n=137). (B) Atomic force micrograph of tubule exterior...
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Figure 5: (A) Histogram of pore sizes (n=137). (B) Atomic force micrograph of tubule exterior showing smooth surfaces and pores. We have developed a novel melt spinning technique for fabricating biomimetic hollow fibers and tubule bundles. Fibers with high aspect ratios may be achieved by caramelizing sucrose and rapidly drawing sugar strands from the mixture. Tubules are created by encapsulating this core fiber with a thin layer of degradable polymer and selective dissolution of the sucrose core. Porous scaffolds may thus be fabricated for tissue engineering without specialized equipment and using only minimal quantities of organic solvents. These scaffolds are shown to be viable for peripheral nerve regeneration by inducing nerve and glial cell alignment in vitro, and may be customized for other tissue/cell culture systems. Imaging and Data Analysis Fluorescence microscopy was conducted on a Nikon Diaphot 300 microscope. Fluorescent images were focus (z)stacked to combine data from multiple image planes. Digital post-processing and analysis was conducted with ImagePro, ImageJ, PhotoAcute and Photoshop CS3. The external surface topography of hollow PLLA fibers was analyzed with a PSIA XE-120 atomic force microscope. Scanning electron microscopy was performed on a JEOL JSM-840 SEM using a 5 kV acceleration voltage at 100-5000X magnification after sputter coating with Au/Pd. Statistical Methods All data are reported in the form of mean ± SD. A p- value of less than 0.05 was considered significant. The authors would like to thank the Indiana Clinical Translational Sciences Institute (TL1RR025759), the National Science Foundation (GK12), and the State of Indiana for their generous support. We would also like to thank Michel Schweinsberg for the graphic illustrations. •Melt spinning sugar fibers and dip coating them in polymer can be used to create tissue engineering scaffolds using a minimum of equipment and materials in a rapid and simple fabrication process •The resulting hollow tubules demonstrate appropriate aspect ratios, surface characteristics, and porosity to serve as a scaffold in nerve regeneration. •Cell cultures of neurons and glial cells demonstrate that these scaffolds are biocompatible and encourage neuronal alignment. •These scaffolds are appropriate for use as nerve regeneration scaffolds and should be considered for use in other areas of tissue engineering. Tubule Formation Fibers were prepared by caramelizing gran- ulated sucrose before cooling to the glass transition temperature (~110°C). Glass micro pipettes were used to draw the viscous fluid at a rate of ~0.35 m/s. Pulled sucrose fibers were then dip coated in poly-l-lactic acid (PLLA) in chloroform (2% w/v) singly or in bundles. Fibers were heated to 37C over- night to allow solvent evaporation. PLLA-sugar composite fibers were cut to length and their cores dissolved with diH 2 O. The resulting hollow tubules were sterilized in 70% EtOH overnight. Cell Culture and Immunocytochemistry Constructs were incubated in laminin overnight. Schwann Cells (RT4-D6P2T) were seeded on the scaffolds (10 4 cells/cm 2 ) and Figure 2: OVERVIEW METHODS RESULTS METHODS (Continued) CONCLUSIONS ACKNOWLEDGEMENTS Sweet Science: Biomimetic Tissue Engineered Scaffolds with Intraluminal Microchannels Todd Rickett 1,2 *, Jianming Li 1 , Riyi Shi 1 1 Purdue University Weldon School of Biomedical Engineering 2 Indiana University School of Medicine *Contact: [email protected], 765-496-3018 cultured in DMEM. Chick dorsal root ganglia were cultured at 5,000 cells/cm 2 in F12 neuronal media. To differentiate intra-luminal cell growth, PLLA -sucrose fibers were embedded in PDMS prior to sucrose dissolution, and cells were pippetted into the tubules. Cells were fixed and permeabilized. After incubation with RNase A, DNA was labeled with propidium iodide (PI). Actin microfilaments were marked with phalloidin conjugated AlexaFluor 488 (AF). Neurofilament staining was accomplished with monoclonal rabbit anti- neurofilament antibody, and AF GOAL To develop a novel tissue engineering scaffold for nerve regeneration that may be simply fabricated with little cost or equipment. RESULTS (Continued) 25 μm 25 μm 40 μm A B C A B C Figure 1
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Transcript of Figure 5: (A) Histogram of pore sizes (n=137). (B) Atomic force micrograph of tubule exterior...
Figure 5: (A) Histogram of pore sizes (n=137). (B) Atomic force micrograph of tubule exterior showing smooth surfaces and pores.
We have developed a novel melt spinning technique for fabricating biomimetic hollow fibers and tubule bundles. Fibers with high aspect ratios may be achieved by caramelizing sucrose and rapidly drawing sugar strands from the mixture. Tubules are created by encapsulating this core fiber with a thin layer of degradable polymer and selective dissolution of the sucrose core. Porous scaffolds may thus be fabricated for tissue engineering without specialized equipment and using only minimal quantities of organic solvents. These scaffolds are shown to be viable for peripheral nerve regeneration by inducing nerve and glial cell alignment in vitro, and may be customized for other tissue/cell culture systems.
Imaging and Data AnalysisFluorescence microscopy was conducted on a Nikon Diaphot 300 microscope. Fluorescent images were focus (z)stacked to combine data from multiple image planes. Digital post-processing and analysis was conducted with ImagePro, ImageJ, PhotoAcute and Photoshop CS3. The external surface topography of hollow PLLA fibers was analyzed with a PSIA XE-120 atomic force microscope. Scanning electron microscopy was performed on a JEOL JSM-840 SEM using a 5 kV acceleration voltage at 100-5000X magnification after sputter coating with Au/Pd.
Statistical MethodsAll data are reported in the form of mean ± SD. A p-value of less than 0.05 was considered significant.
The authors would like to thank the Indiana Clinical Translational Sciences Institute (TL1RR025759), the National Science Foundation (GK12), and the State of Indiana for their generous support. We would also like to thank Michel Schweinsberg for the graphic illustrations.
•Melt spinning sugar fibers and dip coating them in polymer can be used to create tissue engineering scaffolds using a minimum of equipment and materials in a rapid and simple fabrication process
•The resulting hollow tubules demonstrate appropriate aspect ratios, surface characteristics, and porosity to serve as a scaffold in nerve regeneration.
•Cell cultures of neurons and glial cells demonstrate that these scaffolds are biocompatible and encourage neuronal alignment.
•These scaffolds are appropriate for use as nerve regeneration scaffolds and should be considered for use in other areas of tissue engineering.
Tubule FormationFibers were prepared by caramelizing gran-ulated sucrose beforecooling to the glass transition temperature (~110°C). Glass micropipettes were used todraw the viscous fluidat a rate of ~0.35 m/s.Pulled sucrose fibers were then dip coatedin poly-l-lactic acid (PLLA) in chloroform (2% w/v) singly or in bundles. Fibers wereheated to 37C over-night to allow solvent evaporation. PLLA-sugar composite fibers were cut to length and their cores dissolved with diH2O. The resulting hollow tubules were sterilized in 70% EtOH overnight.
Cell Culture and ImmunocytochemistryConstructs were incubated in laminin overnight. Schwann Cells (RT4-D6P2T) were seeded on the scaffolds (104 cells/cm2) and
Figure 2:PI (nuclei, red) and AF (cytoskeletal, green) staining of cells growing on (A) and in (B) hollow PLLA tubules.
OVERVIEW
METHODS
RESULTS
METHODS (Continued)
CONCLUSIONS
ACKNOWLEDGEMENTS
Sweet Science: Biomimetic Tissue Engineered Scaffolds with Intraluminal Microchannels
Todd Rickett1,2*, Jianming Li1, Riyi Shi11 Purdue University Weldon School of Biomedical Engineering2 Indiana University School of Medicine*Contact: [email protected], 765-496-3018
cultured in DMEM. Chick dorsal root ganglia were cultured at 5,000 cells/cm2 in F12 neuronal media. To differentiate intra-luminal cell growth, PLLA -sucrose fibers were embedded in PDMS prior to sucrose dissolution, and cells were pippetted into the tubules. Cells were fixed and permeabilized. After incubation with RNase A, DNA was labeled with propidium iodide (PI). Actin microfilaments were marked with phalloidin conjugated AlexaFluor 488 (AF). Neurofilament staining was accomplished with monoclonal rabbit anti-neurofilament antibody, and AF conjugated goat anti-rabbit IgG.
GOALTo develop a novel tissue engineering scaffold for nerve regeneration that may be simply fabricated with little cost or equipment.
RESULTS (Continued)
25 μm
25 μm
40 μm
A B
C
A
B
C
Figure 1
