Nanofrontiers

Diamonds are not only a girl’s best friend, but also humankind’s best answer for delivery of lifesaving and disease-destroying drugs. Nanodiamonds are associated with a drug-delivery platform created from diamond-dust waste material collected from the byproducts of conventional mining and refining operations. Shaped like tiny balls, the 5-nanometer-diameter diamond particles offer promise for patients with life-threatening diseases or for promoting bone growth at implant sites or combating osteonecrosis of the jaw. This is the finding of a study led by Dean Ho, PhD, professor of oral biology and medicine at the University of California, Los Angeles and codirector of the Jane and Jerry Weintraub Center for Reconstructive Biotechnology.

Coated with drugs or proteins and delivered in a larger complex that the body cannot eject or in sites that require slow-release drug therapy, the diamonds rapidly bind to proteins in the body or bone and can remain intact for long periods in order to treat the affected area. “This is sustainable nanomedicine that can be used for soft tissue engineering and tissue regeneration, as well as for treating other serious oral health diseases, such as periodontitis and even oral cancer,” says Ho, whose earlier research involved investigating the effects of nanodiamonds in cancer therapy. “There is a great opportunity to merge nanotechnology with dentistry. Oral disease is an area where this technology can make a big impact.”

One of the areas in which Ho and his team believe a significant impact can be realized is in promoting bone growth for patients who have periodontitis or bone-loss diseases. In conventional bone-loss treatment modalities, which are often costly and time-consuming, a sponge-like material is inserted surgically into the site to locally administer therapeutic bone morphogenetic proteins that promote bone growth. One of the biggest drawbacks to conventional approaches is what Ho calls the “burst release” of the drug. “When the medication is delivered conventionally into a moist or wet environment like the oral environment, the drug targets the site briefly but dissipates in less than an hour and is absorbed into the patient’s system,” Ho explains. “Ideally, you want to keep a drug focused and working for a longer period in order for that drug to be effective.” The more serious problem with conventional drug-delivery therapies, he says, is the potential for adverse events. If too much of a therapeutic protein is administered, for example, the patient might develop an infection or other negative medical reactions.

Drug therapy delivered using nanodiamonds, on the other hand, provides hours, even days, of therapy to a specific site. The tiny particles bind quickly to both bone morphogenetic proteins and fibroblast growth factor and slowly release the medicament due to the unique electrostatic and chemical properties of the diamond-particle surface. This allows the proteins to be delivered over hours or even days, providing a more effective treatment of the affected area for a longer period. In addition, nanodiamonds can be delivered noninvasively, such as by syringe or oral rinse. “You are reducing the number of injections for the patient, reducing the risk factor for toxicity, and increasing the effectiveness of the therapy as well as the efficacy of delivery, without wasting any of the drug to dissipation,” Ho says.

Another aspect of key interest to the research team is dental implants. Bone loss is inevitable around implant sites, possibly leading to the implants becoming loose or even failing. Key to implant success is creating a sound structural and functional connection between the bone and the load-bearing implant. Ho believes nanodiamonds coated with bone-growth proteins will not only aid in the integration of the implant with surrounding living bone at the outset of implant treatment but could also be used to promote bone growth in patients for whom the bone has begun to recede around an implant site.

“When you develop a vehicle to carry a specific drug to a specific disease site, you want a vehicle that contains as many of the benefits as possible from economics, safety, the scalability of the drug in terms of production costs to its ability to be tolerated in the body,” Ho says. “We believe we have discovered the mechanism that can do that and help improve patient care.”

For scientists, the human mouth remains one of the body’s most secretive and elusive areas to research. More than 50% of the species of bacteria living in the mouth—and billions of cells comfortably exist in this self-contained tropical environment—refuse to grow in a Petri dish. That means that until recently, scientists could not classify, name, or even study them. In the present era, researcher have discovered the presence of these bacteria when they were able to sequence genes from them. However, until recently, the roles that some of these mystery bacteria play in healthy and disease-ridden mouths have been unclear. Now with the ability to study the DNA makeup of cells from what is termed the mouth’s “biological dark matter,” scientists can gain new insight into how these bacteria may interact in the mouth, and what diseases they may cause, and perhaps find ways to counteract the negative impact they play in causing diseases, such as periodontitis.

“We can sequence the DNA from single oral bacterium and see how they might live even though they have never been grown in culture,” says Clifford Beall, PhD, research assistant professor of oral biology at The Ohio State University and lead author of a recent study published in the journal PLOS ONE. “We have found that hundreds of species of bacteria are living in the mouth.” Working in conjunction with Oak Ridge National Laboratory, Beall collected 12 individual cells from the healthy mouth of one individual to begin sequencing the genome of the Tannerella BU063 bacterium. BU063, which has not been successfully cultured, is closely related to Tannerella forsythia, one of multiple bacteria linked to periodontal disease. The DNA of the BU063 genome has been identified by the federal Human Microbiome Project as one of the “most wanted” genomes for sequencing. Unlike its “cousin” T forsythia, BU063 is in healthy gingiva.

The fact that scientists can now DNA-sequence genes within a mystery bacterium will bring them ever closer to finding which genes trigger periodontal disease. Although BU063 and T forsythia are each other’s closest known relatives, hundreds of different genes are between them. Because many bacteria are associated with periodontitis, Beall and his coworkers looked for genes that were common to the periodontitis bacteria and T forsythia, but missing in BU063. “We found some genes that are present in these bacteria and T forsythia but not present in BU063 to further narrow down the search,” Beall says. “We can predict genes that might be important, and if they are confirmed by further experiments, we might be able to find ways to combat the disease.”

At present no antibiotics or other drugs are effective against diseases such as periodontitis because multiple bacterial complexes are involved in the infection and the biofilm that the bacteria live in is highly resistant to antibiotic treatment. Periodontitis affects not only the gingiva but also the bone surrounding the tooth and can lead to eventual tooth loss. The current treatment modality involves surgery or deep cleaning to remove the bacterial infection from the pockets around the gingiva. Beall hopes his research and that of others will lead to methods to combat the disease on a molecular level. “When we have studied the bacteria associated with periodontitis, we have seen that some bacteria like to be together and some others don’t,” Beall says. “There is the possibility that we may be able to use these bacterial relationships to positively influence the disease-causing bacteria to not associate in pockets around the gingiva and cause periodontal disease.”

Beall admits this is but one step in identifying the culprits behind periodontal disease. “There are many of us working on similar projects focused on solving periodontitis,” Beall says. “Unraveling the complexity of the oral microbiome will take many years but should result in important breakthroughs.”

Microbial cells living in the human body, like those Clifford Beall, PhD, is studying, outnumber human cells 10 to 1. Because many of these organisms cannot be studied using conventional means, The Human Microbiome Project was established by the National Institutes of Health to use DNA sequencing technology to identify, characterize, and analyze the role these microbial cells play in human health and disease. This project has not only generated a new breed of scientists and researchers but also stimulated a new field of research called metagenomics to study these complex microbial communities and how they interact.

Growing replacement parts for the body is no longer science fiction. Artificial heart valves, ears, noses, and even teeth can now be cultured and grown in the laboratory or living cells that are 3D printed into three-dimensional scaffolds of human organ shapes that are patient specific. The hope is that one day the standard of care will call for harvesting a patient’s own stem cells from various sources and programming them to grow into a replicate organ or used to replace a diseased or damaged body part. As revolutionary and pioneering as these bioengineering advances are, growing organs outside of the body and then surgically attaching them to areas on or inside the body, opens the door to the possibilities for medical complications and risks. That is why a researcher at Harvard is taking another approach. What if, instead of growing replacement body parts in the laboratory, these organs could be replicated inside the body using the complex developmental processes that naturally occur inside the human embryo during gestation? It was this question that led Don Ingber, MD, PhD, founding director of the Wyss Institute for Biologically Inspired Engineering at Harvard University, and his team to discover an artificial gel-like material that could mimic the power of the embryo to shape organs and enable the engineering of new teeth, bone, or other tissues to repair or replace damaged tissues and organs. “We studied the embryo to understand how organs in the body are formed and then devised design and engineering criteria and guidelines as we attempted to replicate that process,” Ingber explains. They discovered that embryonic tissues use a physical process called mesenchymal condensation that initiates the formation of many bodily organs. During condensation, two adjacent tissue layers comprised of connective tissue cells (called mesenchyme) that are loosely distributed in the tissue space, and the layer of epithelial cells that cover it exchange the biochemical signals that trigger the formation of a specific organ. The mesenchymal cells are then induced to migrate toward the epithelium, and to pack tightly together, or “condense,” into a small mass just below where the tooth will form. Ingber’s group recently discovered that the physical compression of these mesenchymal cells stimulates them to activate genes that begin the tooth-forming process. When the team members first made this discovery, they postulated that if one could find a way to artificially compress these mesenchymal cells at some other site in the body, then these cells would activate and form a tooth. However, there was no way to accomplish this goal.

Recently, the research team set out to develop a sponge-like material in which they could encapsulate these cells, which would be tissue friendly and behave like “shrink wrap,” causing the cells to compress as they do in the embryo without injuring them. The team turned to a gel-forming polymer being used by scientists to deliver drugs to body tissues and then chemically modified the gel to promote attachment of the cells. What resulted was a unique polymer gel material in which the cells could be cultured and would also shrink and compress when warmed to body temperature.

“We have successfully shown that if you encapsulate mesenchyme cells in the gel and implant the gel beneath a mouse kidney capsule where the blood supply is rich, the implanted gel contracts and the cells begin laying down the mineralized tissues of a natural tooth,” Ingber says. However, mesenchyme cells alone can’t build teeth; they must be combined with epithelial cells to form the complex tooth layers. “It’s an exciting new frontier for organ regeneration because this research opens up a new pathway that could potentially be used for engineering many organs in the body,” Ingber says.