Stem cells and nanotechnology in regenerative medicine and tissue engineering

STEM CELLS AND NANOTECHNOLOGY IN
REGENERATIVE MEDICINE AND TISSUE
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CONTENTS
■ INTRODUCTION
■ TISSUE ENGINEERING AND REGENERATIVE MEDICINE
STRATEGIES
■ Cell Therapy
■ Tissue Engineering and Biomaterials
■ Bioactive Factors in Tissue Engineering
■ NANOTECHNOLOGY IN REGENERATIVE MEDICINE AND TISSUE
ENGINEERING
■ Introduction to Nanotechnology
■ Nano-Based Cell Tracking
■ 2D Nano topography
■ 3D Nano scaffolds (Phase Separation, Self-Assembly, Electrospinning)
■ Growth Factor Delivery
■ CONCLUSIONS

INTRODUCTION
■ Alexis Carrel, winner of the Nobel Prize in Physiology or Medicine in 1912 and the father of
whole-organ transplant, was the first to develop a successful technique for end to end
arteriovenous anastomosis in transplantation.
■ The limitations of organ transplant because of immune reaction were recognized early.
■ To eliminate the need for immunosuppression, regenerative medicine and tissue engineering
approaches have generally focused on using donor-derived autologous cells or cells that will
not elicit an immune response.
■ However, terminally differentiated cells are typically limited in their ability to proliferate, and it is
therefore difficult to obtain sufficient cell number for regeneration of tissues. In addition, cells
from tissues to be treated or replaced are likely to have undesirable defects that require
structural repair.
■ The characteristics that define stem cells- capacity for self-renewal, long-term proliferation, and
the ability to differentiate into several different cell types—make them the optimal cell source for
the development of new tissues and organs.

General schematic of tissue engineering strategy.
(1) Cells are isolated from the patient and
(2) expanded in 2D culture.
(3) Expanded cells are then combined with
various natural or engineered bioactive
molecules (e.g., growth factors,
nanoparticles, or DNA) into
biocompatible scaffolds and
(4) cultured in vitro under specific culture
conditions to promote tissue formation.
(5) Finally, functional tissue-engineered
constructs are implanted into the
donor to replace the damaged tissue.

Cell Therapy
■ With the rising popularity of stem cell research, cell therapy has evolved as a
potential treatment method for a variety of conditions. Cell therapy involves
delivery of cells either into the bloodstream or directly into the tissue of
interest. Although tissue engineering strategies combine cells with scaffold
materials and bioactive factors before implantation, cell therapy relies
critically on the interaction of the donor cells with host tissues to restore
function.
■ The greatest limitations in stem cell therapy are the low survival rates of the
injected cells and the inability to closely control the location of those cells that
do survive. Studies examining the effects of the delivery of MSCs after
myocardial infarction have shown that the majority of cells injected
intravenously were eventually found in the lungs, spleen, and liver, with only
a small percentage of the cells engrafted into the injured heart wall.

Tissue Engineering and Biomaterials
■ Today, in addition to being biocompatible, biomaterials in tissue engineering
applications have become increasingly sophisticated and are designed to
meet several criteria.
■ First, they should provide appropriate mechanical strength to ensure that the
tissue can withstand the normal forces it experiences or perform its physical
functions in vivo.
■ Second, they must provide a compatible surface for cell attachment and
appropriate topographic information.
■ Third, they should ideally be designed to degrade over a length of time that is
appropriate for the specific application, such that ultimately, the engineered
tissue is able to approximate its native state.

Bioactive Factors in Tissue Engineering
■ In addition to the cell source and scaffolds, the use of bioactive factors is
important for the optimization of tissue-engineered constructs. Although
the ECM provides the structural component of the native tissue, it also
contains soluble bioactive factors, such as growth factors and cytokines,
whose signals direct aspects of cell behavior, including survival,
proliferation, migration, and differentiation.
■ A large number of in vitro studies have successfully used growth factors
to direct differentiation of stem cells down specific cell lineages.
However, it may be difficult to effectively control differentiation in vivo.
Specifically, intravenous administration of growth factors may be
undesirable and largely ineffective since repeated infusions of high
concentrations of growth factors would be required due to the short half-
life of the factors, which may lead to negative systemic effects.

Introduction to Nanotechnology
■ As more is learned about the mechanisms that control tissue growth and
formation, it has become clear that the answer most likely lies in controlling the
cell behavior at the nanoscale. Although complete in vitro recreation of the in
vivo environment remains a somewhat distant target, this may not be
necessary for success. Instead, it is important that we aim to understand the
interactions between cells and their native environment and apply this
knowledge to the development of constructs that will jumpstart tissue
formation down the correct path before allowing the most effective incubator—
the human body—to take over and remodel the engineered cells or constructs
into the optimal structure.

Nano-Based Cell Tracking
■ Magnetic nanoparticles are commonly composed of superparamagnetic iron
oxide (SPIO) particles 50–500 nm in diameter, which show enhanced contrast
under magnetic resonance imaging (MRI). SPIO particles have been
successfully used for cell tracking in stem cell therapy studies in animals and
humans. Although initially it was thought that SPIO particles did not affect cell
behavior, recent studies suggest that exposure to magnetic fields after
labeling may alter adipogenic and osteogenic differentiation capacity. Further
studies examining the effects on cells with SPIO particles under MRI are
important before their clinical use.
■ Traditional fluorescent cell-labeling methods are limited by their lack of
photostability, narrow excitation range, cell and tissue autofluorescence, and
broad emission spectra. Quantum dots, or Q-dots, are fluorescent
nanoparticles with diameters that typically range from 2 to 5 nm and can be
synthesized in any color. Q-dots have greater photostability and a narrower
emission profile than traditional fluorescent dyes, and they can be linked to
specific proteins or DNA sequences to monitor specific cell behaviors.

2D Nanotopography
■ One of the most common integrin binding sequences is the arginine-
glycineaspartic acid (RGD) sequence. By modifying a substrate with the RGD
peptide, cell adhesion, proliferation, and migration can be altered. Because
obtaining substantial quantities of purified proteins is difficult and may cause
adverse host immune responses, the use of short synthetic peptides that are
not recognized by the host’s immune system but still contain functional
domains to modulate cell adhesion to scaffolds is an attractive method for
tissue engineering.
■ Studies using surfaces modified to have nanoscale features or express
specific cell-adhesion peptides provide a highly organized way of examining
cell behavior with topographic and biochemical cues and give important
insights into the interactions of cells with textured surfaces. However, these
substrates are limited in their complexity compared to the native tissue
environment.

3D Nanoscaffolds
■ Extensive studies have shown that there are clear differences in cell behavior
and tissue formation on flat surfaces compared with 3D ECM scaffolds and
that these differences occur at the protein and subprotein level.
■ The goal in successful application of 3D tissue engineered scaffolds is to identify
and enhance key components that will provide the appropriate signals to cells to
generate a functional tissue that can be translated into clinical use.

Phase Separation
■ Thermally induced phase separation (TIPS) is a technique that is particularly
useful for generating scaffolds with a specific pore size. In this method, the
temperature of the polymer solution is adjusted to a point at which a “polymer-
rich” and a “polymer-poor” phase is generated. The solvent is removed, and
the polymer-rich phase solidifies, forming a porous solid structure, which is
then freeze dried. Nanofibrous scaffolds with varying fiber diameters and pore
sizes can be generated by adjusting the polymer concentration, the type of
solvent, and the phase separation temperature. Specifically, fibers ranging
from 50 to 500 nm in diameter—similar to the size of native collagen—can be
produced. Additionally, highly controlled interconnected macroporosity can be
introduced by pouring the polymer solution over a negative wax mold, which is
removed after solidification of the polymer.

Scaffolds created using thermally induced
phase separation derived from three
dimensional reconstructions of computed
tomography (CT) scans. (a) Human ear
reconstructed from histological sections and
(b) the resulting nanofiber scaffold (scale
bar¼10mm). (c) Human mandible
reconstruction from CT scans and
(d) resulting nanofiber scaffold (scale
bar¼10mm).
(e) Scanning electron micrographs showing
interconnected spherical pores within
mandible segment (scale bar¼500mm) and
(f) nanofiber pore morphology within a single
pore (scale bar¼5mm).

Electrospinning
■ Electrospinning has recently become the most commonly used method for
the fabrication of nanofibrous biomaterials. This method involves the
application of a high electric field to a polymer solution delivered at a constant
rate through a needle. At a high enough voltage, the charge on the polymer
overcomes the surface tension of the solution and causes emission of a fine
polymer jet. This jet undergoes a whipping process, and the fibers are further
elongated as the solvent evaporates and fibers are deposited on a grounded
collector.
■ Both natural and synthetic polymer scaffolds have been successfully created
using the electrospinning method. The ability to generate three-dimensional
scaffolds with tailored architecture, mechanical properties, and degradation
characteristics has made electrospinning a popular method in tissue
engineering applications.

Fabrication of electrospun nanofibers.
(a) General electrospinning setup
consisting of syringe filled with polymer
solution that is pumped through a needle
charged with a high-voltage power supply.
When the electrostatic forces between the
collector and the solution overcome the
surface tension of the solution, the solution
is pulled out of the Taylor cone into fine
fibers that are deposited on the grounded
collector. (b) Scanning electron microscopic
image of randomly arranged poly(e-
caprolactone) (PCL) nanofibers formed by
electrospinning (scale bar¼20mm). (c)
Fluorescent image of mesenchymal stem
cells seeded on nanofibers. Green, cells
(membrane label); red, nanofibers; blue,
cell nuclei (DAPI stain); scale bar¼20 mm.

Self-Assembly
■ Another method of generating nanofibrous scaffolds that mimic the structure
of the ECM is through self-assembly of peptide amphiphiles (PAs). This is a
bottom-up approach that mimics natural processes such as nucleic acid and
protein synthesis. PAs are short peptide structures that spontaneously
aggregate into cylindrical micelles approximately 5–8 nm in diameter and
1mm in length. This process occurs through noncovalent bonds under
specifically tailored conditions. The peptides are typically composed of a
hydrophobic alkyl chain tail, which forms the inside core of the fiber, and a
hydrophilic head composed of epitopes, such as RGD, typically found in the
native ECM that face outward, and interact with cells or other components of
the ECM.

General structure of self-assembled peptide
amphiphile (PA). (a) Molecular model of the
PA showing the overall shape of the
molecule. The narrow gray area represents
the hydrophobic alkyl tail and the thicker
head region is composed of hydrophilic
amino acids containing functional groups
that can provide signals to the cells to
influence their behavior.
(b) The PAs self-assemble into nanofibrous
structures upon exposure to physiological
conditions with the hydrophobic tail in the
core and the head region facing the outside
to interact with cells.
(c) Vitreous ice cryotransmission electron
microscopy image of hydrated PA fibers
(scale bar¼200 nm).

Growth Factor Delivery
■ Although the scaffold structure plays an essential role in controlling cell
behavior, chemical or biological modulators of cell activity and phenotype
heavily influence tissue formation both in vitro and in vivo. In native tissues,
growth factors provide specific signals to cells that direct cell activities,
including cell migration, proliferation, and differentiation. The effects of
growth factors are quite complex and are dependent on the concentration of
the growth factor, phenotype of the cells acted on by the growth factor, and
functional characteristics of the specific cell receptor interacting with the
growth factor.
■ Heparin is a sulfated glycosaminoglycan that has a strong affinity for a number of
growth factors, including basic fibroblast growth factor (bFGF), epidermal growth
factor (EGF), vascular endothelial growth factor (VEGF), and transforming growth
factor-b (TGF-b). In one study, low-molecular weight heparin was conjugated to a
poly(ethylene glycol) (PEG) carrier and electrospun with either PEO or
poly(lactide-co-glycolide) followed by successful adsorption of bFGF.

■ The ultimate goal in regenerative medicine and tissue engineering is to
develop technologies to repair or replace tissues without the complication
of chronic immunosuppression and dependence on organ donors. The key
to success is understanding of how native tissues function and applying this
information to establish the proper combination of cellular, structural, and
chemical components that will allow for functional tissue development.
CONCLUSIONS

Micro and Nano engineering approaches to develop
gradient biomaterials suitable for interface tissue
engineering
■ Salt leaching
■ Gas foaming
■ Phase separation
■ Emulsification
■ Solid free form technology
■ Photolithography
■ Microfluidics
■ Microcontact printing
■ Electrospining
■ Nanoimprint lithography

Stem cells and nanotechnology in regenerative medicine and tissue engineering

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Stem cells and nanotechnology in regenerative medicine and tissue engineering