Extreme Zoom In...
Alan Fincham, Janet Oldak and Haibo Wen take a closer look at enamel biomineralization with the CCMB’s atomic force microscope.
Extreme Zoom In...Alan Fincham, Janet Oldak and Haibo Wen take a closer look at enamel biomineralization with the CCMB’s atomic force microscope. |
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Haibo Wen, a post-doctoral research associate with Drs. Alan Fincham and Janet Oldak, points to an image derived by atomic force microscopy. "It's beautiful," notes Wen. The image is of collagen from human bone. The collagen is magnified to the point where periodic, perpendicular banding can be seen along the length of the fibrils. Each band represents a distance of less than 100 nanometers. Wen,
Oldak and Fincham use atomic force microscopy (AFM) to analyze the
structure The
power of AFM in the analysis of For the biomineralization group at CCMB, the technology of AFM was especially appealing. "Amelogenin has interesting aggregation properties that we'd seen with dynamic light scattering. But we wanted to know if amelogenin molecules had any specific shape or substructure," recalled Oldak. With the AFM, Oldak and the others were able to demonstrate that amelogenin underwent a very specific assembly process rather than a random precipitation or aggregation process. They named the bubble-like structures they viewed with the AFM, nanospheres. The recognition of the basic morphologic form of the amelogenin nanospheres provided Oldak, Fincham and Wen with a jump-off point to explore more functional aspects of amelogenin assembly. Access
to AFM technology allowed the group to resolve an intriguing issue
surrounding the assembly of amelogenin. For decades biomineralization
researchers had observed that Collaborations with investigator and CCMB faculty member, Dr. Malcolm Snead, allowed the biomineralization group to assess the functional importance of particular regions or even base mutations within the amelogenin molecule. Oldak and the others used the AFM to analyze strategically mutated versions of the amelogenin protein produced by members of Snead's research team. Some of these mutations mimic the disease causing, amelogenesis imperfecta mutation. Other engineered amelogenins were truncated in specific regions. "We found that removal of some portions of the molecule affected the assembly, creating tiny nanospheres with heterogeneous distributions. When other regions were removed we actually saw fusion of the nanospheres," said Oldak. "The images we got from the recombinant amelogenins were even more beautiful than the ones from the gel extracted from the developing teeth. You can see how the amelogenin gel is built with 20 nanometer nanospheres. You can see the individual nanospheres and how the gel can induce mineralization," said Wen. Future experiments that Oldak, Fincham and Wen plan to perform surround the exploration of force plots that can be created between various AFM tips and surface materials. The researchers want to bind amelogenin molecules to a tip and then scan different surfaces. "We can have an apatite surface, a mica surface, a calcite surface, graphite, gold, materials with varying hydrophobicity andhydrophilicity. We can study how amelogenin nanospheres interact with any of those materials," said Oldak. Oldak,
Fincham and Wen view the AFM as a powerful tool that allows them to
address fundamental issues in biomineralization. For their purposes,
these questionsrevolve around how apatite crystals and amelogenin
nanospheres Despite numerous successes with the AFM technology, Oldak, Fincham and Wen have identified areas in need of improvement. "All techniques have their limitations. For AFM it's the sample preparation," said Wen. One such area the group would like to improve upon is image resolution. When the biomineralization group is able to further increase the AFM's resolution, they will be able to study the substructure of amelogenin nanospheres. With maximum resolution they can determine if repetitive structures exist on a single nanosphere or if the nanospheres are hollow. Expansion
of the types of media used for sample analysis will also have beneficial
outcomes. Thus far, most of the group's AFM studies have been performed
in air. Ideally, the group would like to observe the nanospheres in
liquid so that they can more closely mimic in vivo conditions, but the
optimization of buffering conditions is technically challenging. Oldak
and The AFM applications Oldak and Wen enumerate are extensive. "It's important to communicate to the Dental School and USC in general that we have the AFM and that people are welcome to use it or to collaborate with us," emphasized Oldak. Clinical and basic scientists could use the AFM to look at DNA morphology, DNA-protein interactions, polymers, cellular events, cell attachments or even the surface topography of the tooth itself. Currently, Wen, Oldak and Fincham are the only CCMB researchers sufficiently trained to operate the AFM. Training is time consuming. Operation of the equipment requires a substantial time investment for sample preparation, image optimization and force image analysis. 'The AFM needs a full time person to be dedicated to it," agreed Oldak and Wen, "We take advantage of it as much as we can but we'd still like it to be used more often." A selection of recent publications:
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Last Updated: 04/19/07 |
and assembly properties of amelogenin, the protein essential
for the biomineralization of teeth. Primarily due to the initiative of
Oldak and Fincham, CCMB was able to acquire an atomic force microscope
in 1998. "We're in the beginning stages with the AFM technology.
Right now we're looking at the surface topography. AFM is much better
than scanning electron microscopy for biological samples. With SEM, I
can reach approximately a 30,000-fold magnification. With AFM, I can
reach close to nanometer resolution," said Wen. 
biological samples came with the
invention of an alternate mode for force measurement called tapping.
Traditional force microscopy uses a probe that travels directly over the
surface of a highly polished inorganic material. "Force mode is a
relatively aggressive method that would cause the tip and the biological
sample to be damaged," said Oldak. With the tapping mode, the probe
is designed to bounce with controlled, rapid periodicity, only lightly
touching the surface. The amplitude of the tap is kept constant.
"It's like a person walking over a flat surface whose height you
measure. When the person encounters a step, the measured height
increases," explained Oldak. 
when amelogenin gel extracted from
developing dental enamel matrix was taken from a near freezing
temperature to one near room temperature, the gel went from clear to
optically opaque. As Wen explained, "When the temperature is
increased, aggregation increases, creating some spaces. The gel
structure isn't homogeneous anymore and appears opaque. When the gel is
cooled, these aggregates can dissociate, recover their homogeneous
structure and the gel becomes clear again." In vivo, mineralization
occurs at a physiologic temperature. "Obviously, it is always 37°C.
But in vivo, the local pressure probably effects the properties of the
amelogenin assembly," said Wen. 
interact with one another to form highly organized and
durable mineralized enamel. Each researcher hopes that the AFM will
allow them to address whether amelogenin nanosphere assembly is
dependent on the nature of the apatite crystal surface. "If
amelogenin adsorbs specifically on certain crystals it will inhibit
perpendicular to that surface and promote the growth of other surfaces.
That. could explain how the morphology and orientation are controlled in
vivo," said Oldak. 
Wen both note that that once the nanospheres are put in liquid they
tend to float away. Identification of an adhesive agent that binds the
nanospheres to the surface without destroying the integrity of the
protein is now under way. 