May 2005
Many dental researchers dream of one day stepping into the laboratory, pulling out a detailed set of instructions, and engineering a replacement tooth. This decades-old dream has gained momentum recently as scientists have identified more of the molecules that nature employs to make a tooth. Yet, even as these molecular parts are identified, scientists must begin to solve the larger puzzles of how they self assemble to form the tooth’s various specialized tissues, such as enamel and dentin.
Further complicating matters, the solution to each puzzle will be different. Take dental enamel. It begins to form in the tooth bud not as the hard, white substance so familiar to us in the mirror, but as a soft, protein-rich matrix specked with mineral crystals called calcium hydroxy apatite. When a tooth finally erupts through the gums, the original protein matrix is gone. In its place is a tissue that is nearly 100 percent calcium hydroxy apatite, raising the question: What happened to the protein matrix? Researchers know that the matrix and its dominant protein amelogenin initially serve as a template that nature has designed to orchestrate the spatial and temporal dynamics of the mineralization process. Left unanswered is the next question: How do the amelogenins actually orchestrate the process? In the March 4, 2005 issue of the journal Science, a team of NIDCR grantees and colleagues offer an intriguing possibility based on a series of laboratory studies. Like so many important discoveries in biology, the group made its big find through sheer serendipity. Recently, the Inside Scoop talked with Dr. Janet Moradian-Oldak, a scientist at the University of Southern California and an author on the paper, to hear more about this chance discovery and what it might mean in learning to make enamel in the laboratory.
You set out to crystallize amelogenin, the dominant matrix protein. Is that correct?
Exactly. We wanted to crystallize amelogenin in solution and essentially lock it into a structure in which all of its constituent molecules have ordered orientation. This would allow us to analyze the precise secondary and tertiary structure of amelogenin and ask questions such as: How do full length amelogenins aggregate into spherical structures called nanospheres that seem to be so essential to the structure of the matrix? Which parts of the protein interact to form these nanospheres? And what is their spatial orientation? While such questions might sound technical, answering them is absolutely essential to understand how enamel is formed in the tooth bud.
How long have you wanted to crystallize amelogenin?
Since the late 1990s when I was working with Dr. Alan Fincham, a Research Professor with more than 25 years experience working on amelogenin protein. But we faced a huge problem. One of the prerequisites for crystallization is that your protein of interest must not aggregate. Well, amelogenin is one of those proteins that aggregates easily. So much so, it almost seems like fiction to even attempt to crystallize it. But we forged ahead to understand its solubility and assembly properties in various solvents and under various temperatures. The availability of a recombinant form of amelogenin, which is expressed in the bacterium E. coli, has allowed such studies to be pursued in a systematic manner.
What happened next?
I decided that if we really wanted to crystallize the protein, we had to produce a much larger supply. You can’t send a small amount of the protein to crystallographers and expect them to get the job done in one try. Amelogenin is just too tough to work with. In 2000, I hired a full-time technician to produce recombinant amelogenin. He did a great job, and we soon had an ample supply - tens of milligrams - to enable the research.
With more of the protein now at your disposal, you needed someone to help crystallize amelogenin. Is that when Dr. Gieseppe Falini entered the picture?
Yes, I ran into Giuseppe at a scientific conference in Orlando, Florida that was held in 2001. We hadn’t seen each other for a couple of years.
He has a rather unique research background, I understand.
Giuseppe started his scientific work in the biomineralization field, specifically in the study of the controlled crystallization of calcium carbonate crystals formed in shells. In this contest he was also involved in projects on art preservation. He has taken advantage of biomineralization principles to design substrate for protein crystallization.
When we talked in Orlando, Giuseppe happened to mention that one of his latest research specialties was crystallography. He said he was pursuing an approach in which he used substrates, such as siliconized mica, that recognize domains within a protein and initiate the crystallization process. I thought that was a very original idea, and we talked about applying it to amelogenin. Usually these conversations are informal and quickly forgotten. But I was eager to find a crystallographer who was interested in working with amelogenin. So, a few weeks later, I sent five milligrams of recombinant amelogenin to his laboratory at the University of Bologna.
Any luck?
He tried to crystallize amelogenin but with no success. We agreed he should keep trying under various conditions, so I sent him more protein and have advised him on different solution conditions that migh work. No luck. I mailed additional protein. Nothing. And so the process went. In the meantime, I worked with other collaborators to try to discover the types of substrates to which amelogenin has an affinity. One way to think of this is to imagine a grain initiating the formation of a pearl. That’s what we were trying to do. Find that substrate, or grain of sand, that allows the crystallization process to occur. So, this was really a team effort.
Then, about a year later, you received an e-mail.
That’s right. Giuseppe had made another attempt to crystallize amelogenin, and he was puzzled by the results. He mentioned that he could not obtain crystals, bu instead some elongated fibers appeared to form in the crystallization solution. He sent me an email with a photo of his latest attempt. To give you a mental image, the structures in the photo looked like long strands of fettuccine. They reminded me of the long calcium hydroxyapatite crystals that one sees in mature enamel.
If my suspicion was correct, Giuseppe had quite serendipitously prompted amelogenin nanospheres to self-assemble into highly ordered structures. The question then was: is there any relation between these fiber-like protein structures and the highly ordered and long apatite crystals of enamel?? It also meant that if appropriate experimental conditions could be replicated, we would have a window from which to view this key step in the mineralization process. True, this window would be under artificial laboratory conditions. But it would provide the first glimpse of the process and show us what is technically possible, information that will be extremely helpful in future efforts to engineer enamel.
What happened next?
Chang Du, a post doc in my laboratory, replicated the conditions. We immediately noticed the formation of spherical balls of amelogenin, or nanospheres, that had assembled into long beaded chains. Because of their thin, ribbon-like appearance, I called them “microribbons.” So, in collaboration with Giuseppe, we went back and systematically studied their assembly using the best possible laboratory instruments, computer software, and imaging techniques. Thereafter, we determined the microribbons were indeed real and, as previously suspected, the nanospheres serve as the subunits of the microribbons.
That led to the critical question: Do these microribbons serve as scaffolds for the mineralization process? So, Chang mineralized the microribbons by dipping them into a calcium-phosphate solution. The result: The microribbons yielded ordered hydroxy apatite crystals with similar orientation to those formed in enamel.
The implication being?
This was the first time that anyone could actually assemble amelogenin into ordered structures, mineralize them, and say that the nanospheres were responsible for the ordered growth. Sure, there were strong suggestions in the scientific literature that this might be the case. But we were actually the first to show it.
Every discovery leads to new questions. What are some of the questions that your work raises?
Well, the first question is: Are these microribbons relevant biologically. Because our work took place in the laboratory and not the tooth, it may be that microribbons cannot form in enamel.
Why's that?
Because their dimensions might not be physically possible in the tooth. But we never claimed that these same microribbons are formed in enamel. What we claim is the beaded subunits form in enamel. And the fact that these beaded chains have a tendency to grow in length is significant. What we also learn is the assembly is a highly organized. It is not a random aggregation, which some thought might be the case.
What about tissue engineering and the idea of creating an artifical tooth?
We now know a few basic principles involved in making enamel - how protein chains self assemble into little balls, how these beaded chains can actually serve as a initiator of a very organized and controlled growth of crystals. There definitely are ways that we can apply these principles. I should add, though, that one lab cannot possibly engineer a tooth. What we’re doing is a very small piece of the puzzle. To really address the different aspects of tooth formation, it will a require a much greater team approach that includes cell and molecular biologists, dentists, pathologists, material scientists, chemists, and others.
Last question: What about the crystallization of amelogenin? Any success? Is it still a work in progress?
Yes, we are continuing our team effort with Dr. Falini, and it is work in progress. We are working on some smaller isoforms, or subtypes, that have less tendency to aggregate.