Tissue Engineering Nanotechnology and

applicationsSUBMITTED BY :

VISHWAMITRA KUMARROLL NO : CH8276

Liu Nanobionics Lab

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

Remove cells from the body.

Expand number in culture

Seed onto an appropriate scaffold with suitable growth factors and cytokines

Place into culture

Re-implant engineered tissue repair damaged site

Tissue Engineering

Tissue Engineering

Cells Scaffolds

Biorectors Signals

Differentiated cellsAdult stem cellsEmbryonic stem cells

Dynamic cell seedingImproved mass transferMechanical stimuli

HydrogelsNanofibrous scaffoldsSelf-assembling scaffoldsSolid freeform fabricated scaffolds

Small moleculesGrowth factors/polypeptidesNucleic acids (DNA, siRNA, and antisense oligonucleotides)

Nanotechnology and Tissue Engineering: The Scaffold, CRC Press; 1 edition (June 16, 2008)

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

Create & Manipulate

View & Characterize Optical Microscopy Electron Microscopy Atomic Force Microscopy

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

• Controlled variables Flow rate Electric field strength Distance between tip and collector Needle tip design Collector composition and geometry

• Ambient parameters Temperature Humidity Air velocity

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

Research on Materials

• Polyglycolic acid (PGA)– Highly crystalline, hydrophilic,

byproduct is glycolic acid • Polylactic acid (PLA)

– Hydrophobic, lower melting temperature, byproduct is lactic acid

• Polydioxanone (PDO)– Highly crystalline

• Polycaprolactone (PCL)– Semi-crystalline properties,

easily co-polymerized, byproduct caproic acid

• Blends– PGA-PLA– PGA-PCL– PLA-PCL– PDO-PCL

• Elastin • Gelatin collagen• Fibrillar collagen• Collagen blends• Fibrinogen

• Synthetic 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

• Natural polymers Collagen Type I & III + PDO: best

possible match for blood vesselsAdvanced 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.

Nature Biotechnology 21, 1171 - 1178 (2003)

(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 P450–type enzymes (left). However, cells in three-dimensional scaffolds showed cytochrome P450 activity (right).

Figure 3: Lipid, peptide and protein scaffold nanowires.

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

Self Assembly

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 liquid–liquid or solid–liquid 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-assemblya) 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 labelingSensing cellular behaviorAugmenting cellular behaviorAugmenting cellular behaviorCytotoxicity

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.

Science 30 May 1997:Vol. 276. no. 5317, pp. 1401 - 1404 Nature 388, 860-862 (28 August 1997)

Gel–sol transition curves.

In vitro release profile of FITC-labelled dextran (Mr 20,000) from PEO–PLLA–PEO (Mr 5,000–2,040–5,000) triblock copolymer.

Injectable drug-delivery system

Printing Technology

• Nanoimprinting Lithography• Organ Printing• Contact Printing

Nanoimprinting LithographyProf. Stephen Y. Chou

Thermal-sensitive PolymerOptical-sensitive Polymer

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

SMC morphology

Alignment and elongation characterization

Wound healing assay for cell motility

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 fromStage 15–16 HH chick ventricle, containing only myocardium, endocardium andsome intervening matrix. Isolated rings beat steadily for several days; (a) adjacentapposed rings fused overnight and (b) beat as one. (c). Schematic representationof principle of organ printing technology: placing of cell aggregates layer by layerin solidifying thermo-reversible gel with sequential cell aggregate fusion andmorphing 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 withradial branches from the chick 27stage HH embryonic heart cushion tissue placedin 3D collagen type 1 gel.

Trends Biotechnol. 2003 Apr;21(4):157-61.

Contact Printing

J. Am. Chem. Soc., 2005, 127 (48), pp 16774–16775

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

APPLICATIONS1)Coronary Heart Disease •Myocardial Infarction•Congestive Heart Failure•Dysfunctional Heart Valves•Peripheral Vascular Disorders•Abdominal Aortic Aneurysms2)Neurological Stroke•Parkinson’s Disease•Alzheimer’s Disease•Epilepsy•Traumatic Brain and Spinal Cord Injury•Multiple Sclerosis3)OrthopedicNon-union Fractures•Cartilage Damage and Repair•Ligament Damage•Vertebral Disc Damage•Bone Graft Materials4)UrologicalIncontinence•Kidney Disease•Bladder5)Skin/IntegumentaryBurns•Diabetic Ulcers•Venous Ulcers•lastic Surgery

6)DentalMissing teethPeriodontal disease

7)Organ TransplantationLiverHeartKidneyPancreas

8)OphthalmologyCorneaRetina

9)GastrointestinalEsophagusStomachSmall IntestineColon

10)Ear, Nose and Throat/Respiratory/CardiopulmonaryTracheaRespiratory Epithelial Cells (Nasal Turbinates)

11)CancerUrology (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 non-biodegradable 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 drug-delivery 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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