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By Rishi Gupta, Aaron Geisberger, and Dr.
Gareth Hughes
(Zyvex Corporation) and Dr. Brad Layton (Drexel University)
Download a printable copy here.
Collagen is the most abundant protein in
the mammalian anatomy, manifesting itself in a wide variety
of forms, functions, and types. In biological systems, collagen
fibers provide the major mechanical support for cell attachment,
thus developing the shape and form of tissues1.
Collagen is used as scaffolding material in tissue engineering,
but must first be characterized for mechanical strength. Traditional
methods for fiber characterization are not suitable for sub-micron
size collagen. The Zyvex L100 Manipulator System is the instrument
of choice for characterizing fibers on these length scales.
This application note presents a brief description of collagen,
its applications, and describes how the Zyvex L100 can be
used to manipulate microscopic collagen fibers for mechanical
characterization.
Collagen proteins are comprised of polypeptide
chains (alpha-chains) that form a unique triple-helical structure
that is 300 nanometers long and 1.5 nanometers in diameter.
Each a-chain has a repeating amino acid structure of Gly-Xaa-Yaa,
where Gly is glycine and Xaa-Yaa can be any amino acid pairing,
though most often are proline and hydroxyproline1,2.
There are more than twenty disparate collagen types that exist
in animal tissue, five of which are known to form fibers:
Types I, II, III, V, and XI. These types tend to self-assemble
into periodic, cross-striated fibers, which can reach centimeters
in length and tens of microns in diameter. Type I collagen
is the predominant fiber-forming collagen type and is found
in bones, skin, teeth, and tendons. Type II collagen, considered
the second most abundant, is found in cartilaginous tissue,
developing cornea, and vitreous humor1.
Type I collagen is regenerated in response
to tissue injury and as a result of fibrotic disease1,.
However, many processes do not have efficient regenerative
capabilities, if any at all. For example, the anterior cruciate
ligament (ACL) has little innate ability to heal itself. Current
medical treatments for damaged or lost tissue include transplantation,
reconstruction, drug therapy, prosthesis, and medical devices.
Tissue engineering has been investigated as an alternative
to these treatments. Tissue engineering is a multidisciplinary
field with goals of replicating the functions of living tissues
both in vivo and in vitro. Appropriate scaffolding
material is needed to provide a basis of support and encourage
cell regeneration. Because collagen proteins are a major structural
element in so many of the body’s tissues and organs, collagen
fibers are a logical choice for scaffolds3.
The mechanical and material properties
of any implanted biological substance must be ascertained
before deemed as an acceptable therapy. The scaffold should
be biocompatible, biodegradable, suitable for cell attachment,
and have a 3-dimensional, porous structure. The scaffold must
also have mechanical properties that duplicate the tissue
being replaced4.
The implanted scaffold must promote the development
of mechanically stable structural support, thus fulfilling
its inherent role as a scaffolding element. For this reason,
mechanical properties of the collagen fibers must be characterized
before they can be considered as a scaffolding material. Upon
application of a load, the collagen fiber must return to its
original length (within some small percent error) to be considered
an effective load bearing support structure. The mechanical
characterization involves tensile and compression testing3.
Though elastic, most collagen exists in a crystalline-polymer
form that is susceptible to molecular defects upon loading
and compression. The ability to gauge the molecular response
of collagen fibers during mechanical testing is invaluable
for understanding the mechanisms and processes involved in
protein assembly and fiber formation. This information will
affect the methods by which scaffolds are synthesized.
Current techniques for measuring mechanical
properties of collagen involve using custom built3
or proprietary tensile loading systems5,6. These
instruments clamp onto the fibril and apply a tension. The
established methods for characterizing collagen scaffolds
are ineffective for structures with lengths below one centimeter
and diameters below ten microns. Collagen fibers are made
up of a hierarchy of structures, starting with amino acids
that form proteins that are 300 nanometers in length and 1.5
nanometers in diameter. The proteins further assemble into
fibrils of varying lengths and diameters. The fibrils form
fibers that can reach centimeters in length and hundreds of
microns in diameter7. One major limitation of the
instruments mentioned above is that they can only accommodate
macroscale fibers and fibrils, and are not applicable to fibers
on length scales that require optical microscopes.
For any test of fibers that are on these length
scales, handling the fibers is the biggest barrier to success.
Zyvex Corporation’s L100 Manipulator System is the solution
to the problems limiting the science of collagen fiber characterization
(Figure 1). The L100 can be used under an
optical microscope to grasp, pull, and twist hydrolyzed and
dry collagen fibers for mechanical characterization.
In addition to limitations in dexterity, current
techniques cannot replicate fatigue-inducing conditions experienced
by ligaments in vivo, such as muscles in a state
of exercise. A collagen fiber experiences continuous loading
and relaxation as ligaments support flexing and relaxing of
muscles during constant periods of activity such as running
or swimming. Additionally, the elastic limits for bending
are relevant when attempting to replicate ligaments that support
bending of the extremities. Pronation and supination cause
ligaments to feel a tensile and torsional stress, which tensile
loading equipment cannot replicate. The Zyvex L100 can be
used to facilitate mechanical characterization of collagen
fibers by applying both tensile and torsional stresses.
There is also no established technique for integrating
atomic force microscopy into the mechanical characterization
apparatus. Atomic Force Microscopy (AFM) has been used to
image collagen fibers8, and can be used to learn
about changes in their molecular structure upon loading and
compression. A means of integrating an AFM with the L100 is
being investigated for imaging fibers under stress.
Most collagen macromolecules and fibers are
highly crystalline polymers that exhibit large relaxation
and retardation times, attributing to their function as the
stress-bearing extra-cellular matrix of biological tissue.
The major collagen classes (I and II) are often found as co-polymers
with the remaining fibril-forming types. For example, the
Type I–III collagen pairing is found in arterial walls1.
Because collagen fibers are polymeric, their mechanical strength
can be gauged using standard polymer fiber characterization
techniques.
The Zyvex L100 is a piezo-motor actuated
manipulation tool with sub-micron resolution for use with
either scanning electron or optical microscopes.
Figure 1 shows the L100 System head.
Figure 1: The L100 Assembly System in the
Optical Docking Station.
The modular head unit consists of two four-degrees-of-freedom
(DOF) positioners and one three-DOF positioner. Figure 2 is
a schematic describing the nomenclature for the positioning
directions.
Figure 2: A schematic of a four-degree-of-freedom
positioner, highlighting the axial nomenclature.
The axial direction (or Y-axis) is defined as
being motion towards the center of the sample stage. All actuators
have 100 nanometer precision, which is more than sufficient
to manipulate collagen fibers.
The L100 is interfaced via the Zyvex Optical
Docking Station*, which consists of an optical stereomicroscope,
X-Y-translation stage, connector harness, and ring light.
All of these components are mounted onto an optical breadboard
for easy adjustment. The stereomicroscope can be connected
to a charge coupled device (CCD) camera for signal acquisition.
* Zyvex Corporation offers a wide range
of accessories for the Nanomanipulator suite of instruments,
including the Optical Docking Station and NanoEffector™ tools.
For more information, contact Zyvex at 877.998.3999 ext. 271
or email sales@zyvex.com.
The L100 System achieves its manipulation capabilities
with quick-disconnect tools that attach to the end of the
positioners. Zyvex’s NanoEffector™ tools includes static probes
such as wires, and more dynamic tools, such as microelectromechanical
systems (MEMS) based, thermally actuated grippers.
The loop test is an alternative to tensile
tests and is particularly applicable to short fibers. Monofilaments
for composite materials have been tested for mechanical strength
using the loop test9. In this test, a fiber is
contorted to form a loop. The L100 can apply a torsional stress
to a collagen fiber, thereby creating a loop. The radius of
curvature of the loop is diminished by pulling the fiber in
opposite directions, and the bending strength can be measured
at the point just before the fiber breaks. When used in the
Zyvex Optical Docking Station, real-time image capturing can
be performed for analysis. Loop test theory and applications
are discussed in subsequent sections of this note.
The L100 can be used to mimic tensile fatigue
testing on collagen fibers. For sub-micron scale collagen
fibers, the L100 can be used to position the collagen fiber
onto a Zyvex MEMS-based oscillator with a preload. The MEMS
oscillator can replicate continuous stress/relax states of
the fiber, simulating muscle exercise.
There are two key steps to follow when
preparing a collagen sample for manipulation with the L100.
The first step is rehydration, where the moisture in the dried
collagen fiber is replenished. The second is binding the collagen
fiber to a probe. This can be done using cyanoacrylate-ester
based glue. These steps are discussed below.
Collagen will lose water over time, and
must be rehydrated in order to maintain its intrinsic elasticity.
Hydrating collagen fibers can be done with phosphate-buffered
saline (PBS) in a spray or soak, or with an aqueous bath.
The soak is performed before any manipulation work. The collagen
is placed in a bath or in soaked gauze for an hour5.
The PBS spray is administered during manipulation to maintain
hydration, and should be done every 60 seconds3.
This will ensure the collagen maintains it elasticity during
the test.
The L100 uses MEMS grippers to strip collagen
fibers from the fibril sample. Thus, the fiber must be strongly
bound to the sample holder. After rehydrating the fiber, dip
the probe in a cyanoacrylate-ester based glue, and place the
fiber onto the probe with a pair of tweezers. The glue must
be dry before attempting any manipulation. Figure
3 shows a 250 micron diameter tungsten probe (the
sample holder) with a 10 micron diameter collagen fiber (rat
tail tendon) affixed to it using a cyanoacrylate-ester based
glue.
Figure 3: The probe with a collagen fiber
glued to it.
The first task in any manipulation experiment
is to set-up the L100. To perform such experiments as the
loop test, place the probe with the collagen fiber into the
three-degree-of-freedom (DOF) positioner. This positioner
should be located between two four-DOF positioners. These
positioners have MEMS gripper NanoEffectors and are initially
positioned at the extreme ends of their axial rotation. The
grippers should be “powered-closed” so that that the applied
force can be varied. As a powered-closed gripper encloses
a fiber, it can squeeze the fiber beyond the contact point
by increasing the actuation voltage.
The L100 is used with the Optical Docking Station
when performing collagen manipulation tasks. The L100 fits
into the X-Y-translation stage with its standard mounting
ring. The cables are connected with the adapter assembly.
Center the L100 with the X-Y-translation stage and bring the
collagen fiber into the focal plane of the optical microscope.
When the collagen fiber is in focus, begin bringing
a MEMS gripper into contact with it. Figure 4a
shows a powered-closed metal-MEMS gripper approaching a collagen
fiber. The gap between the flexure arms of the gripper is
on the order of 6 microns. The gripper is loaded onto a four-DOF
positioner. Figure 4b shows the gripper closed
onto a collagen fiber.
Figure 4a-b: A MEMS gripper grasping onto
a collagen fiber. The gripper opening is six microns.
Grasp the fiber with the grippers and back the
positioner away from the fiber until a smaller fibril is free,
as shown in Figure 5.
Figure 5: A MEMS gripper holding a free collagen
fiber.
Grasp the free end of the fiber with the second
MEMS gripper. A tension is then applied to the fiber by moving
the positioners away from each other
(Figure 6).
Figure 6: The Collagen fiber pulled taut.
To form a loop in the fiber, a torsional stress
is applied by bringing the axial rotators to their opposite
extrema. Moving positioners towards each other relieves the
applied torsion, which causes the fiber to twist. Continue
to position the NanoEffectors until the loop is created. Figure
7 shows the fiber formed into a loop.
Figure 7: The collagen fiber formed into
a loop with the L100.
Zyvex has published an application note on operating
MEMS grippers with the nanomanipulator, which is available
here11.
The L100 can manipulate sub-micron size
collagen fibers for mechanical characterization. Traditional
tensile tests are ineffective at calculating the strength
of small fibers. The loop test is a more effective method
for characterizing small fibers, and the L100 is perfect for
manipulating them.
Because collagen fibers are polymeric,
they can be characterized using polymer fiber characterization
techniques. One such technique is called the loop test. This
test measures the bending strength of fibers by measuring
the smallest radius of curvature a fiber will allow before
breaking. The relationships between the physiometric parameters
of the fiber and the extrapolated information regarding the
compliance are described below.
Figure 8 shows a schematic
of a looped fiber. The fiber has a thickness (diameter) D
and is formed into a loop with radius of curvature p.
Figure 8: The L100 Assembly System in the
Optical Docking Station.
The fiber loop is pulled in the ±y
directions forcing compression and tension about point A,
the point on the bend where breaking will occur9.
The values for critical compressive strain, ecr,
and skin strain, esk, are determined by the following
equations:
esk = Dp/2 (Eq.
1)9,
ecr = D/2p (Eq.
2)11.
The radius of curvature can be measured by analyzing
data collected through the optical stereo-microscope. The
loop test requires adroit manipulation of fibers, especially
at sub-micron length scales. The Zyvex L100 is designed for
manipulating structures on these length scales, and can easily
apply the necessary stress on collagen fibers for mechanical
characterization.
Collagen is a major structural support
element in mammalian organs and tissues. Thus, it is being
used as a scaffolding material in tissue engineering, a field
with an emphasis on tissues and organ regeneration. In order
for collagen to be considered as a candidate for tissue engineering
scaffolds, the mechanical properties of the scaffold must
be determined. The mechanical properties of formed collagen
fibers are assembly dependent, so the molecular response of
the fibrils under load must also be ascertained in order for
tissue engineers to grow site specific collagen in vitro
and in vivo that meet the requirements for bioimplantation.
The L100 Manipulation System can perform the tasks necessary
to fully characterize the fibers and scaffolds grown and used
by engineers for tissue engineering. Integrating Atomic Force
Microscopy into the L100 platform is being investigated as
a tool for molecular analysis. The ability to manipulate sub-micron
size fibers, coupled with Zyvex NanoEffector™ tools and accessories
makes the L100 the premier instrument for collagen fiber characterization.
1. K.E. Kadler, et al., Biochem. J. 316 (1996): 1–11.
2. D.S. Goodsell, www.rcsb.org/pdb/molecules/pdb4_1.html.
3. D.A. Dickerson, et al., J. of You. Invest: Eng. and Appl.
Sci. 8 (2003).
4. D.W. Hutmacher, J. Biomater. Sci. Polymer Edn. 12 (2001).
5. E. Gentleman, et al., Biomater. 24 (2003): 3805–3813.
6. Y.P. Kato, et al., Biomater. 10 (1989): 38–42.
7. T. Ushiki, Arch. Histol. Cytol. 65 (2002): 109–126.
8. H. Wang, et al., Diab. Metab. Res. and Rev. 19 (2003):
288–298.
9. H. Fukuda, et al., Adv. Comp. Mater. 8 (1999): 281–291.
10. K. Tuck, et al., http://www.zyvex.com/Products/CFMC_001a.html.
11. S. Fidan, et al., Comp. Sci. and Tech. 49 (1993): 291–297.
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