Liu Nanobionics Lab

Tissue Engineering Nanotechnology and applications

SUBMITTED BY : VISHWAMITRA KUMAR ROLL NO : CH8276

Outline
What is Tissue Engineering What is Nanotechnology Why we apply Nanotechnology to Tissue Engineering How is Nanotechnology applied to Tissue Engineering
Different nanofabrication techniques Applications

Tissue Engineering
Expand number in culture Remove cells from the body. Seed onto an appropriate scaffold with suitable growth factors and cytokines

Re-implant engineered tissue repair damaged site Place into culture

Tissue Engineering
Differentiated cells Adult stem cells Embryonic stem cells
Cells Scaffolds

Hydrogels Nanofibrous scaffolds Self-assembling scaffolds Solid freeform fabricated scaffolds

Tissue Engineering

Biorectors

Signals

Dynamic cell seeding Improved mass transfer Mechanical stimuli

Small molecules Growth factors/polypeptides Nucleic acids (DNA, siRNA, and antisense oligonucleotides)
Nanotechnology and Tissue Engineering: The Scaffold, CRC Press; 1 edition (June 16, 2008)

Nanotechnology Overview
Nanotechnology is a branch of science and engineering which deals with structures and devices in nanometer scale.

View & Characterize

Optical Microscopy Electron Microscopy Atomic Force Microscopy Create & Manipulate Nanofabrication: (Top down & Bottom up) Lithography (Optical, E-beam, NIP, DPL) Etching (Wet Etching, Plasma Etching) Deposition (Evaporation, PECVD, Electrochemical Deposition, Sputtering, etc.) Epitaxial growth (MOCVD, MBE, etc.)

Why we apply Nanotech in TE?

Cells on microfibrous scaffolds have a polarized relationship, with one side of the cell attached to the scaffold, the other exposed to physiological media. In comparison, it is likely that cells are more naturally constrained by nanofibrous scaffolds.

Nanofibrous Scaffold
Electrospinning Self-Assembly

Electrospinning

This process involves the ejection of a charged polymer fluid onto an oppositely charged surface. Multiple polymers can be combined Control over fiber diameter and scaffold architecture

Research on Parameters of Electrospinning Process

Solution properties
Viscosity Conductivity Surface tension Polymer molecular weight Dipole moment Dielectric constant Flow rate Electric field strength Distance between tip and collector Needle tip design Collector composition and geometry

Controlled variables

Ambient parameters
Temperature Humidity Air velocity

Tissue Engineering. May 2006, 12(5): 1197-1211.

Research on Materials
Polyglycolic acid (PGA) Polylactic acid (PLA)
Highly crystalline, hydrophilic, byproduct is glycolic acid Hydrophobic, lower melting temperature, byproduct is lactic acid Highly crystalline

Polydioxanone (PDO)

Elastin Gelatin collagen Fibrillar collagen Collagen blends Fibrinogen

Polycaprolactone (PCL)

Blends

Semi-crystalline properties, easily co-polymerized, byproduct caproic acid PGA-PLA PGA-PCL PLA-PCL PDO-PCL

Synthetic polymers

Natural polymers

PGA, PLA and PLGA most commonly used PDO most similar to Elastin collagen blend (limited by shape memory) PCL most elastic and mixed frequenlty with other material s Provide nanoscale physical features
Collagen Type I & III + PDO: best possible match for blood vessels
Advanced Drug Delivery Reviews Volume 59, Issue 14, 10 December 2007, Pages 1413-1433

Self Assembly

Figure 1: Fabrication of various peptide materials.

Figure 2: Self-assembling peptides form a three-dimensional scaffold woven from nanofibers ~ 10 nm in diameter.

(a) Representation of self-assembling peptide. (b) Electron micrograph of three-dimensional scaffold formed in vitro. (c) Rat hippocampal neurons form active nerve connections; each green dot represents a single synapsis. (d) Neural cells from a rat hippocampal tissue slide migrate on the three-dimensional peptide scaffold. Cells on the polymer membrane (left) and on the peptide scaffold (right) are shown. Both glial cells (green) and neural progenitors (red) migrate into the three-dimensional peptide scaffold. (e) Brain damage repair in hamster. The peptide scaffold was injected into the optic nerve, which was first severed with a knife. The cut was sealed by the migrating cells after 2 days. A great number of neurons form synapses. (f) Chondrocytes from young and adult bovine encapsulated in the peptide scaffold. These cells not only produce a large amount of glycosaminoglycans (purple) and type II collagen (yellow), characteristic materials found in cartilage, but also a cartilage-like tissue in vitro53. (g) Adult rat liver progenitor cells encapsulated in the peptide scaffold. The cells on the two-dimensional dish did not produce cytochrome P450type enzymes (left). However, cells in three-dimensional scaffolds showed cytochrome P450 activity (right).
Nature Biotechnology 21, 1171 - 1178 (2003)

Self Assembly

Figure 3: Lipid, peptide and protein scaffold nanowires.

Figure 4: Microlenses and fiber-optics fabricated from protein scaffolds.

Nature Biotechnology 21, 1171 - 1178 (2003)

Self-Assembling Peptide Scaffolds for Regenerative Medicine

SAPNS heals the brain in young animals.

SAPNS allows axons to regenerate through the lesion site in brain.

PNAS March 28, 2006 vol. 103 no. 13 5054-5059

Phase separation
This process involves dissolving of a polymer in a solvent at a high temperature followed by a liquidliquid or solidliquid phase separation induced by lowering the solution temperature Capable of wide range of geometry and dimensions include pits, islands, fibers, and irregular pore structures Simpler than self-assembly
a) powder, b) scaffolds with continuous network, c) foam with closed pores

SEM of nanofibrous scaffold with interconnected spherical macropores

Advanced Drug Delivery Reviews Volume 59, Issue 14, 10 December 2007, Pages 1413-1433

Carbon Nanotube
Cell tracking and labeling Sensing cellular behavior Augmenting cellular behavior Augmenting cellular behavior Cytotoxicity

Murine myoblast stem cells incubated with DNA-encapsulated nanotubes

neuron bridging an array of carbon nanotubes thereby creating neural networks.

Biomaterials Volume 28, Issue 2, January 2007, Pages 344-353

Block Coploymer
Synthetic scheme of block copolymers.

In vitro release profile of FITClabelled dextran (Mr 20,000) from PEOPLLAPEO (Mr 5,0002,040 5,000) triblock copolymer. Injectable drug-delivery system

Gelsol transition curves.

Science 30 May 1997: Vol. 276. no. 5317, pp. 1401 - 1404

Nature 388, 860-862 (28 August 1997)

Printing Technology
Nanoimprinting Lithography Organ Printing Contact Printing

Nanoimprinting Lithography
Prof. Stephen Y. Chou

Thermal-sensitive Polymer Optical-sensitive Polymer

Nanopattern-induced changes in morphology and motility of smooth muscle cells

Alignment and elongation characterization

Wound healing assay for cell motility

SMC morphology

BrdU cell proliferation assay

Biomaterials Volume 26, Issue 26, September 2005, Pages 5405-5413

Organ printing: computer-aided jet-based 3D tissue engineering

Fig. 1. Fusion of embryonic myocardial ring. Myocardium rings were cut from Stage 1516 HH chick ventricle, containing only myocardium, endocardium and some intervening matrix. Isolated rings beat steadily for several days; (a) adjacent apposed rings fused overnight and (b) beat as one. (c). Schematic representation of principle of organ printing technology: placing of cell aggregates layer by layer in solidifying thermo-reversible gel with sequential cell aggregate fusion and morphing into 3D tube.

Fig. 2. Cell printer and images of printed cells and tissue constructs.

Fig. 3. (a) Printed bagel-like ring that consists of several layers of sequentially (layer-by-layer) deposited collagen type 1 gel. (b) Manually printed living tube with radial branches from the chick 27stage HH embryonic heart cushion tissue placed in 3D collagen type 1 gel.
Trends Biotechnol. 2003 Apr;21(4):157-61.

Contact Printing

J. Am. Chem. Soc., 2005, 127 (48), pp 1677416775 Advanced Materials Volume 19 Issue 24, Pages 4338 - 4342

Summary
Nanofibrous Scaffold
Electrospinning Self-Assembly

Nanoporous Scaffold
Phase Separation

Carbon Nanotube Block Copolymer Printing

Nanoimprinting Lithography Organ Printing Contact Printing

Lab Chip, 2004, 4, 98 - 103

1)Coronary Heart Disease Myocardial Infarction Congestive Heart Failure Dysfunctional Heart Valves Peripheral Vascular Disorders Abdominal Aortic Aneurysms 2)Neurological Stroke Parkinsons Disease Alzheimers Disease Epilepsy Traumatic Brain and Spinal Cord Injury Multiple Sclerosis 3)OrthopedicNon-union Fractures Cartilage Damage and Repair Ligament Damage Vertebral Disc Damage Bone Graft Materials 4)UrologicalIncontinence Kidney Disease Bladder 5)Skin/IntegumentaryBurns Diabetic Ulcers Venous Ulcers lastic Surgery

APPLICATIONS

6)Dental Missing teeth Periodontal disease 7)Organ Transplantation Liver Heart Kidney Pancreas 8)Ophthalmology Cornea Retina 9)Gastrointestinal Esophagus Stomach Small Intestine Colon 10)Ear, Nose and Throat/Respiratory/Cardiopulmonary Trachea Respiratory Epithelial Cells (Nasal Turbinates)

11)Cancer Urology (Bladder, kidney/Renal cell, prostate) Neurology (Brain/Glioblastoma/Glioma, CNS, head/Neck) Obstetrics/Gynecology (Breast, pelvic, ovarian, endometrial) Orthopedic (Chordoma/Bone) Gastrointestinal/Gastroenterology (Colorectal, gastric, pancreas) ENT (Esophageal, oral, pharynx, olfactory) Hematopoietic (Leukemia, lymphoma) Respiratory (Lung/Mesothelioma) Dermatology (Melanoma/Skin)

Carbon nanotubes are among the numerous candidates for tissue engineering scaffolds since they are biocompatible, resistant to biodegradation and can be functionalized with biomolecules. However, the possibility of toxicity with nonbiodegradable nano-materials is not fully understood.

References
Nanotechnology and Tissue Engineering: The Scaffold, CRC Press; 1 edition (June 16, 2008) Quynh P. Pham, Upma Sharma, Ph.D., Dr. Antonios G. Mikos, Electrospinning of Polymeric Nanofibers for Tissue Engineering Applications: A Review, Tissue Engineering. May 2006, 12(5): 1197-1211. Catherine P. Barnes, Scott A. Sell, Eugene D. Boland, David G. Simpson, Gary L. Bowlin, Nanofiber technology: Designing the next generation of tissue engineering scaffolds, Advanced Drug Delivery Reviews, Volume 59, Issue 14, Intersection of Nanoscience and Modern Surface Analytical Methodology, 10 December 2007, Pages 1413-1433, ISSN 0169-409X, DOI: 10.1016/j.addr.2007.04.022. Shuguang Zhang, Fabrication of novel biomaterials through molecular self-assembly, Nature Biotechnology 21, 1171 - 1178 (2003) Rutledge G. Ellis-Behnke, Yu-Xiang Liang, Si-Wei You, David K. C. Tay, Shuguang Zhang, Kwok-Fai So, and Gerald E. Schneider, Nano neuro knitting: Peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision PNAS 2006 103 (13) 5054-5059 Benjamin S. Harrison, Anthony Atala, Carbon nanotube applications for tissue engineering, Biomaterials, Volume 28, Issue 2, Cellular and Molecular Biology Techniques for Biomaterials Evaluation, January 2007, Pages 344-353, ISSN 0142-9612, DOI: 10.1016/j.biomaterials.2006.07.044. Miri Park, Christopher Harrison, Paul M. Chaikin, Richard A. Register, Douglas H. Adamson, Block Copolymer Lithography: Periodic Arrays of ~1011 Holes in 1 Square Centimeter, Science 30 May 1997: Vol. 276. no. 5317, pp. 1401 - 1404 Byeongmoon Jeong, You Han Bae, Doo Sung Lee and Sung Wan Kim, Biodegradable block copolymers as injectable drugdelivery systems, Nature 388, 860-862 (28 August 1997) Evelyn K.F. Yim, Ron M. Reano, Stella W. Pang, Albert F. Yee, Christopher S. Chen, Kam W. Leong, Nanopattern-induced changes in morphology and motility of smooth muscle cells, Biomaterials, Volume 26, Issue 26, September 2005, Pages 5405-5413, ISSN 0142-9612, DOI: 10.1016/j.biomaterials.2005.01.058. Mironov V, Boland T, Trusk T, Forgacs G, Markwald RR. Organ printing: computer-aided jet-based 3D tissue engineering. Trends Biotechnol. 2003 Apr;21(4):157-61. Yu, A. A.; Stellacci, F., Contact Printing beyond Surface Roughness: Liquid Supramolecular Nano-Stamping, Advanced Materials, 19, 4338-4342, 2007 Yu A.A., Savas T., Cabrini S., diFabrizio E., Smith H.I., Stellacci F., High resolution printing of DNA features on poly(methyl methacrylate) substrates using supramolecular nano-stamping, J. Am. Chem. Soc., 127, 16774-16775, 2005

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