science/technology pair of natural tissues," Grainger says. "Most people have assumed that you could make a material and force it on the body if you sewed it, hammered it, or glued it in and that the body basically had to accept it. We've never really questioned what the host itself wants to respond to in order to optimally integrate the implanted material or to regenerate itself." Now, using advances in cellular and molecular biology and surface character ization techniques, researchers are prob A. Maureen Rouhi mind. They were developed from a mate ing both synthetic and native biomaterials rials perspective, with a concern for me with more rigor than ever before. And C&EN Washington chanical properties, physical properties, they are applying the insights provided by epending on your point of view, and ease of fabrication. But as far as biol biology to better engineering and design. biomaterials research in the past ogy is concerned, these materials are all How the surfaces of biomaterials interact 30 years has been either a fabulous foreign objects, not very different from a with the body is receiving much scrutiny. Grainger says that's because a lot of the bi success or a miserable failure in terms of bullet or a splinter." clinical impact. On the one hand, materi Lacking this necessary biological per ological response to any implanted materi als that interface intimately with the spective, "we have been miserable failures al is determined by both the chemistry body to help restore vital functions and in mimicking structure, function, and re- and the morphology of the surface. At a workshop on "Contemporary Bio structure have a long history of use for materials through Precise Control of Macheart valves, blood vessel prostheses, hip romolecular Chemistry and Architecture" and knee joint replacements, pacemak Surfaces cue cell coorganized by Grainger and Ratner last ers, intraocular lens implants, dental im response November in Williamsburg, Va., they and plants, and contact lenses, among others. others described research aimed at under They have saved millions of lives and en standing events on surfaces where chemis hanced the quality of life for many more. try and biology literally meet at an inter But their performance "is far from face. Grainger's group uses molecular and that of the body part they are intended R = COOH cell biology methods to study how cells to replace," says Buddy D. Ratner, a pro respond to surfaces with carefully con fessor of chemical engineering and direc trolled chemistry. Ratner and coworkers tor of the University of Washington Engi are engineering surfaces for biological rec neered Biomaterials (UWEB) Engineering ognition and specificity. Research Center, Seattle. "And that leads Meanwhile, at the University of Illi to tremendous cost to the health care nois, Urbana-Champaign, research in the system and the patient in reoperations R = CH laboratory of Deborah E. Leckband, an and complications." assistant professor of chemical engineer A diverse assortment of materials cur ing, aims to identify the molecular basis rently are used in clinical applications. of adhesion and molecular recognition at They include metals such as stainless steel, membrane surfaces through direct force titanium, and platinum; synthetic poly measurements, as well as surface, analyt mers ranging from different varieties of sil ical, biochemical, and cell biophysical icone rubber to polyurethanes and biode methods. And at the University of Heidel gradable polyesters; natural polymers such berg, Germany, physical chemistry pro as various proteins; and ceramics such as fessor Michael Grunze and colleagues are bone-mimicking hydroxyapatite, calcium working on a general model to explain carbonates, and phosphates. and predict the nonspecific interactions "The current array represents the same ' γ, γ ;•( ^ .Λ. < γ )i / χ . ν ·. of surfaces with proteins and other bio basic menu that was around after World logically relevant molecules. Gold War Π," says David W. Grainger, an associ In addition to being physically and ate professor of chemistry at Colorado Spreading of a Swiss 3T3 fibroblast mechanically apt for the specific use, im State University, Fort Collins, who current cell plated on a COOH-terminated selfplanted biomaterials also must be com ly is serving as chief executive officer at assembled monolayer (top) indicates patible with the body. What the body re Gamma-Α Technologies, Herndon, Va. effective signaling on the surface. The gards as foreign, it will reject and try to "Despite thousands of patents on new ma fibroblast cell on a determinated destroy or wall off. What the body recog terials intended for clinical use over the monolayer curls away from surface. nizes as native or part of itself, it accepts past 30 years, their clinical impact has The cells were stained with a rhodaand integrates. mine-tagged toxin that binds to fila been relatively insignificant." mentous actin and then were imaged Whether an implanted material is per The problem, Ratner notes, is that underfluorescentlight ceived as foreign or native depends to a "materials now used in medicine were large extent on what its surface presents. never really developed with biology in
CONTEMPORARY BIOMATERIALS
Understanding surfaces is key to the design of clinically useful materials
D
JANUARY 18, 1999 C&EN 51
science/technology When cells contact a surface, receptors on the cell membrane actively search for surface-bound cues that tell the cell whether the surface is native or foreign. The cell response to cues is relayed from receptors in the cell membrane to the inside of the cell. Grainger and his Colorado State graduate student Kristin B. McClary have demonstrated that the response of cultured cells to surface chemistry is genetically determined and can be overcome by genetic manipulations. To monitor cell responses to surface chemistry, Grainger and McClary use various self-assembled monolayers (SAMs) with well-controlled surface chemistries. They present the SAMs, terminated with specific functional groups, to fibroblast cells in the proteinaceous environment of tissue culture medium. They find that the cells attach, grow, and spread substantially better on a carboxylterminated hydrophilic surface than on a methyl-terminated hydrophobic surface. "Somehow, the cells know that they
VISIT
prefer one chemistry and not the other," Grainger says. He and McClary are trying to link the surface chemistry on the outside to events inside the cell that are related to this selection. Proteins have
Grainger (left) and Ratner
much to do with the observed cell behavior. Tissue culture medium represents a mixed bag of more than 200 serum proteins with different compositions and conformations. It's likely that, on the SAM surfaces, the cells seek certain adhesive pro-
US AT B O O T H
teins that are characteristic of the extracellular matrix they see in native tissues, such as fibronectin. Cells will respond well if such proteins are adsorbed at certain critical densities and at conformations that expose their receptor-binding sites. Grainger says successful cell recognition and adhesion involves the binding of surfaceadsorbed fibronectin by its cellular receptor, another membranelocalized protein dimer called an integrin. Docking of the integrin with fibronectin sends a signal through the cell membrane into the cell. That signal then triggers a series of events leading to cell attachment and further spreading on the surface. Studies by McClary and Grainger suggest that the part of fibronectin that binds to cellular integrin receptors is better presented to cells on a hydrophilic SAM surface than on a hydrophobic surface. With more fibronectin-integrin binding on the hydrophilic surface, cells attach and spread better
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science/technology there than on the hydrophobic SAM surface. McClary and Grainger have correlated this surface preference to a cellular en zyme called RhoA, a recently discovered important gatekeeper in this series of bi ological cause-and-effect events leading to cell attachment and spreading. When the signal comes across the membrane indicating that integrin is bound to fibronectin, RhoA—sitting inside the cell near the tail of the integrin—then makes some decisions, Grainger explains. If suf ficient fibronectin-integrin bindings oc cur, enough RhoA is then activated to signal further critical reactions that lead to full adhesion, cell spreading, and ex pression of cell functions. "RhoA families sit as the cornerstones of cell-based events leading to surface at tachment and beyond" Grainger says. "So we want to interrogate the behavior of RhoA as a function of surface chemistry." Using molecular biology techniques, Grainger and McClary find that expres sion of RhoA in cells is modulated by sur face chemistry: Cells growing on a hydrophilic SAM surface have more active RhoA than cells growing on a hydropho bic surface. They also show that the sur face chemistry dependence of RhoA ex pression can be overridden. For example, when cells are geneti cally modified so that the gene encoding RhoA is always turned on, they prolifer ate on a hydrophobic surface to which they otherwise would fail to attach. And when expression of RhoA is turned off by the presence of certain toxic proteins, cells proliferating on a hydrophilic SAM surface shrink and retract, as if trans planted to a hydrophobic surface. "Because RhoA is linked to the expres sion of genes responsible for specific cell functions, surfaces could cue genetic re sponses from cells to encourage their sur face integration and growth and perhaps even tissue regeneration," Grainger says. Correlating gene expression to surface chemistry, "in my opinion, represents the Holy Grail of the biomaterials world," he adds. "We need to understand how gene products responsible for cell behavior are expressed and regulated in response to bio materials. Whatever combinations of genes are up- or downregulated by a local environment are the determinants of a bio compatible response." Like a bullet or a splinter, biomaterials now in clinical use elicit only nonspecif ic responses from their implant environ ment. In the body, they are quickly cov ered with a layer of proteins in all man
ner of conformations and states of denaturation. "But living organisms never use nonspecifically adsorbed proteins for any thing," UWEB's Ratner says. "We believe this nonspecific protein layer is what the body is seeing as foreign. And our goal is to control the nonspecific events occur ring on the surface." The work really is surface engineering guided by the biology of healing, which is based on recognition. "Little progress was made in improving devices because the understanding of the
biology of healing was not yet in place," Ratner says. "Now that this biology is on a more solid footing, we believe that the next quantum jump in progress will have to come from tailoring surfaces so that they rationally work with well-defined bio logical pathways and control biology with precision." One of the first cell types to interrogate foreign objects in the body is the mac rophage. This defense cell is responsible for cleaning up wound sites and then co ordinating healing; that is, signaling other
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