A Simple Device Grows Some Amazing Bone Cell Tissue
A collaboration between Penn State materials scientists and biologists is creating a better way to study breast cancer.
Materials Ph.D. student Ravi Dhurjati
with a 6-month bone cell culture
It is estimated that one out of every eight women will develop breast cancer in her lifetime. Breast cancers that metastasize to bone are particularly pernicious, and there is little in the way of treatment at the present time. But studying the interaction of breast cancer cells with bone is difficult and typically requires costly animal studies. The interdisciplinary work of Penn State materials scientists and biologists in the Materials Research Institute and the Huck Institutes of the Life Sciences offers hope for an effective tool in the fight against cancer and other bone diseases.
These days, Ravi Dhurjati, Ph.D. student in the Materials Science and Engineering Department, thinks about little else than the bone cells growing in a special device in the incubator near his office. Within these simple cell culture devices, called bioreactors, bone cells are flourishing far beyond the ordinary life span of bone cells in a standard culture dish. Using conventional technology, bone cell cultures can be sustained for only 15 to 30 days. Yet Ravi’s cell cultures are going strong after more than six months, with no end yet in sight.
Bone is dynamic, constantly being remodeled within the human body. It is why tennis players develop a larger arm, through bone as well as muscle growth. Cells called osteoblasts form new bone tissue, while other cells, called osteoclasts, are called in to destroy bone, for instance to clean up bone fragments in a fracture. However, bone remodeling is a slow process, and that is why Ravi’s innovation is so important – bone cells can now be grown in the laboratory for months, not days.
Ravi’s detour into cell biology came about from a conversation at the lunch table with Erwin Vogler, associate professor of materials science & engineering and bioengineering. "I’ve always had great conversations with Dr. Vogler," Ravi says. "It’s fun to work with someone with such an agile mind as his. He was describing to me one of his pet projects from the 1980s, a little culture device he invented for DuPont. From that conversation grew my Ph.D. project."
The device, called a compartmentalized cell culture chamber, was issued a patent at that time, which has since expired. "I guess it was just not the right time or place for the idea then," Vogler speculates. "Ravi had earned a Master’s degree in ceramics, and he was making simple orthopedic devices out of ceramic materials. We decided to test his materials with bone cells in the bioreactor because we could use long–term exposure. We’ve had remarkable results."
a. Diagram of simultaneous-growth-and-dialysis device
b. The Vogler bioreactor
c. Artist's drawing
"The first thing we did," Ravi recalls, "was to look at the design and go into the machine shop and begin fabricating the device. The films we developed had to be very exact, with highly specific surface engineering characteristics. But we were using relatively crude academic methods that were available in the laboratory. We’d go to the engineering lab and get some polymer and press the polymer between two plates. We optimized the heat and the pressure. Eventually, through trial and error, we had something that worked. Once the device was made, I injected some bone cells into it."
The first two cultures did not grow well. The cells failed to attach to the polymer film, and Ravi returned to Erwin Vogler for advice. "Dr. Vogler has an enormous background in basic science and knows a great deal about what makes cells attach. So he solved the first problem of attaching cells."
Vogler also encouraged Ravi to look more deeply into the literature of cell growth outside the body. "I did this huge review that went back 100 years and looked at all the studies by people involved in this sort of work," Ravi says. "I asked ’Where did this fascinating idea come from that we would take cells out of our bodies and put them into a dish?’ Because I was a materials scientist and not a biologist, I felt free to ask this kind of basic question. When you go back 100 years, you will find a lot of these relevant questions being asked, and there are still basic questions unanswered in biology. For all its sophistication, biology still has a frontier element."
"You really need to meet this guy in materials."
Drs. Carol Gay and Andrea Mastro are faculty members in the Department of Biochemistry and Molecular Biology and in the Huck Institutes of Life Sciences at Penn State. They have collaborated on a number of projects involving bone-seeking breast cancer – one of the group of deadly cancers that includes breast, prostate, myeloma, and to a lesser extent, lung cancers that seek bone. "Erwin contacted me by e–mail as an expert on bone cells," Dr. Gay explains, "and I brought in Andrea. I didn’t know Erwin, but I ran into Channa Reddy, the Director of the Huck Institutes, on the sidewalk one day, and he said you really need to meet this guy in materials. You need to get together."
Carol Gay watches as Andrea Mastro
peers through the confocal microscope.
Vogler experienced the same type of push to collaborate. "We are all busy, and I didn’t get an immediate response from Dr. Gay. Left on my own, I probably would not have followed up on the original e–mail and the collaboration would have died. But Channa kept saying, ’Erwin, you need to talk to Andrea and Carol, and I’ll say as much to them.’ To me that is true sponsorship. In a more rigid system this wouldn’t have happened. The materials people would stay in their box and the biology people in theirs. Although most universities have free access between disciplines, Penn State administration actively sponsors and facilitates cross-disciplinary research. In fact, the Materials Research Institute and Huck came up with seed grants to get this team really working together in the lab. Carlo (Pantano, MRI Director) and Channa have a broad overview of Penn State research and are effective match makers. Partly as a consequence of their personal efforts, Penn State has a truly unique environment that fosters cross-disciplinarity."
There was some skepticism among the biologists at first. Andrea Mastro recalls that Ravi would come by the lab to pick the brains of one of her grad students, bringing by micrographs of bone tissue taken from the bioreactor. It was some time before they saw the images themselves. The osteoblasts they saw were robust, with a cuboidal shape, making lots of protein. In contrast, dish culture cells are flat and make little protein. They are unlike normal cells in the body.
Inside the body, bone cells are in layers, a community of cells that interact. In the culture dish, cells remain in a monolayer with little interaction. With the bioreactor, cells are growing into a matrix of tissue six to eight cells deep. "The design makes wonderful sense," says Carol Gay. When I saw this and saw Erwin’s enthusiasm and the results of the micrographs, well, from a biologist’s point of view, this bioreactor is a wonderful new tool."
Layers of collagen with embedded
osteoblasts seen in an optical micrograph.
According to Vogler, the device works on a principal called "simultaneous-growth-and-dialysis" invented by G.G. Rose in the early 1960s and discarded for 20 years. In Vogler’;s version, a growth space for the cells is enclosed by gas permeable films that allow oxygen and nutrients to filter through from above while allowing built up waste to dialyze through the growth medium into a reservoir below. Access to the growth space and the reservoirs were provided by "taper ports" through which pipettes could be inserted to take samples, or add or remove fluids. This solves the problem of "culture shock," the strain cells experience when they are removed from the body. This shock is compounded every three to five days in normal cell cultures as the growth fluid is replaced to refresh nutrients and wash out waste products, such as lactic acid.
"In conventional cell culture, the cells go from feast to famine, feast to famine every few days between refeedings," Vogler says. "At the same time, waste products go from high to low. All the while cells are trying to make a comfortable environment by excreting extra cellular matrix, which enhances cell growth, only to have this effort washed away with the waste products. In the bioreactor, you don’t have to perturb the cells with medium exchange. In fact, we refresh the bioreactor with growth medium only every 30 days instead of every 3 days, as is traditionally done."
The Army Idea Award
The United States Military Health System spent approximately $56M on direct breast cancer treatment in 2003, with an expected rise in annual costs to $75M by the end of the decade. The Army Idea Awards are a highly competitive initiative of the Department of Defense Breast Cancer Research Program that fund innovative peer–reviewed research into treatments and cures for breast cancer. Recently, the Penn State collaborators were awarded just under $500,000 to pursue their study of breast cancer in bone using the bioreactor.
Laurie Shuman, graduate student in
immunobiology, collaborates across disciplines.
Laurie Shuman is a graduate student in immunobiology advised by Andrea Mastro. She and Ravi have worked together since she joined the team in January of 2005. "This is a very close collaboration," she says. "I’ve enjoyed it. It’s a whole new world working with materials science. Ravi and I help each other look for protocols and try to understand what’s going on. I get him osteoblast and cancer cells for his bioreactors. After the osteoblasts are 3 to 6 months along, we add the cancer cells tagged with a green fluorescent dye. We found that the breast cancer cells were altering the morphology of the osteoblasts. It would be great to see if we can form bone and see how cancer cells are effecting the bone environment. We’d like to be able to test anticancer drugs without having to use mice."
Shuman says that she and Ravi would also like to be able to explore growing bone for prosthetics. Currently, implants such as titanium have a short life expectancy as osteoblasts react to the foreign material and die off, which allows the implant to loosen and tear away in the body. Implants made of a patient’s own bone tissue could be permanent, she says. With eight to ten new bioreactors being fabricated, they will have a line of bone cell cultures to work with by early 2006.
Bone cells are just the beginning.
Carol Gay says, "Erwin’s first idea, and it was a good one, is that if you can grow bone in vitro, you can replace worn out bones or repair nonunion fractures. Say you have a broken collarbone that won’t heal; you can take bone cells from another part of the body and grow your own sheet of tissue that won’t be rejected. These cultures need very little attention. You could use them in space to do the kinds of experiments NASA is doing on bone. Bone tissue is highly affected by space flight."
The time that was not right for the bioreactor is finally here, Vogler believes. "We have an aging demographic that is developing bone disease, especially osteoporosis, in which bone growing osteoblasts become less efficient while bone destroying osteoclasts increase. It’s estimated that orthopedic healthcare will cost the U.S. economy $600 billion a year by 2030."
Ravi’s experiments with the bioreactor have already yielded results never before seen in the lab. Using high resolution transmission electron microscopy and confocal microscopy, the researchers have observed the first osteoblasts undergoing a "phenotypic transition" to osteocytes, a process never before seen in vitro. They have recorded the important hallmarks of cancer in bone – metastasis and tumor formation – , which likewise has never been observed in the lab. Finally, Ravi has grown the longest-living culture of bone cells, by several times over, ever reported in the literature.
Erwin Vogler, inventor of the compartmentalized
bioreactor, in the biomaterials lab.
"This thing keeps going on and on," Vogler remarks. "The other day Ravi stopped me in the hall and showed me a sliver of white chip in the culture that looked a lot like bone. My gosh, we are growing bone you can see with the naked eye. Now, we want to grow osteoclasts in a bioreactor and bring them together with the osteoblasts to observe the bone remodeling process in the lab. Then we want to see how different types of drugs affect the process. In this way, maybe can try to learn how to interrupt the cancer process. Once we have a system with all the important components, we can challenge that against the cancer. Then we can go on to other degenerative bone diseases, and then to other kinds of cells. Neuroscience is a growing field at Penn State. Maybe we can grow some brains in this thing – we’ll always have a shortage of that!"
The group, which also includes recently graduated materials science Ph.D. Xiaomei Liu, is writing up its results in a paper to be submitted to a peer-reviewed journal in January 2006. In it they will give detailed specifications for Ravi Dhurjati’s and Erwin Vogler’s compartmentalized bioreactor, foregoing any attempt to commercialize the device. "I have no interest beyond pure science," Ravi explains. "After we publish our paper, any biology lab in the country will be able to build such a device on their own."
Vogler agrees, adding, "We could make much more elaborate devices, but this is something we can use every day. It’s an ideal compromise for what we want to do. We can take it apart, clean and sterilize it, and reuse it."
Of her colleagues in the biology lab, Carol Gay remarks, "Once people catch on to this device, I doubt many of them will ever go back to the old way of growing cultures."
This work was supported, in part, by a grant from the Pennsylvania Department of Health. The Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions. This work was also supported by the Pennsylvania State Tobacco Settlement Formula Fund and NIH AG13087–10. The authors appreciate additional support from the Huck Institutes of Life Sciences, Materials Research Institute, and Departments of Bioengineering and Materials Science & Engineering of the Pennsylvania State University.
Contacts:
Erwin Vogler, Validate to view address
Ravi Dhurjati, Validate to view address
Carol Gay, Validate to view address
Andrea Mastro, Validate to view address
Laurie Shuman, Validate to view address
