The identification, and subsequent production using recombinant DNA techniques, of growth factors that regulate proliferation, differentiation, and biosynthetic activities of cells has provided biomaterials engineers with an important set of tools for controlling cell and tissue responses to implants.
Bone is a reservoir for many biomolecules that regulate bone metabolism. Some bone growth factors are synthesized elsewhere, but most are produced locally and become incorporated in bone matrix during bone formation. Bone growth factors exert their effects locally through paracrine and autocrine mechanisms. Because growth factors are often produced and act locally and because they have short systemic half-lives, localized application to tissues is needed. Biomolecular, or biochemical, surface modification seeks to control tissue-implant interactions by delivering biomolecules directly to the interface. By delivering one or more type of osteotropic molecule directly to the tissue-implant interface, bone formation may be promoted. The simplest way to deliver biomolecules to the tissue-implant interface is by dipping the device in a solution of protein before inserting it. Although some encouraging results have been reported, a major drawback with the adsorption method is that it provides little, if any, control over the delivery, including retention and orientation, of molecules. Proteins are initially retained on the surface by weak physisorption forces, and then, depending on the implant microenvironment, which varies among anatomical sites and among patients, the molecules desorb from the surface in an uncontrolled manner to interact with cells. Considering the necessity of specific receptor-ligand interactions for activity of many relevant biomolecules, appropriate orientation of immobilized protein may also be needed. An alternate approach is to chemically attach biomolecules to surfaces. Adhesive peptides, such as the Arg-Gly-Asp (RGD) sequence that binds to cell surface receptors of the integrin superfamily, have been the focus of much attention. Because of redundancy in the affinity of integrins for adhesive proteins and because a variety of cells possess the same integrins, nonselective attachment of cells to RGD-modified surfaces can occur. Some groups are attempting to circumvent this problem by using longer peptides, having a particular conformation, rather than use short tetra-, penta-, or hexapeptides. Others are examining non-RGD peptides that may be more specific for bone cells. A lack of specific interaction with cells of the osteoblastic lineage remains a concern, however.
Immobilization of proteins, such as growth factors, on the surface may provide more control over cell-biomaterial interactions. Whereas RGD-peptides primarily mediate adhesion of cells to substrates, immobilized growth factors may be able to modulate subsequent cell functions, such as proliferation, differentiation, and activity, on biomaterial surfaces. Compared to studies with peptides, few reports regarding chemically immobilized growth factors, especially osteotropic molecules, are available. This approach mimics the way growth factors are immobilized on or in the extracellular matrix, which acts to present the growth factors to cells in a more favorable orientation.
In our previous research, we developed methods for immobilizing proteins on “bioinert” biomaterial surfaces while retaining the bioactivity of the molecules . More recently, we demonstrated that bone morphogenetic protein 4 (BMP-4) immobilized on the surface of Ti-6Al-4V retained its ability to promote osteoblastic differentiation of pluripotent cells. Whereas BMP-4 simply adsorbed on the surface is effective, albeit with great variability, the protein is completely lost from the surface during an overnight incubation in cell culture medium. Consequently, cells cultured on the preincubated adsorbed surfaces do not exhibit osteoblastic activity; only the surfaces with immobilized BMP-4 stimulated alkaline phosphatase acitivity, which is an indicator of early osteoblastic differentiation.
Other projects have focused on more rational strategies for immobilizing proteins. For example, properly designed immobilization schemes can control presentation/orientation of immobilized growth factors to cells. The figure at the top of this page illustrates the difference between randomly immobilized growth factor molecules and those bound to the surface in a controlled, oriented manner. With the random approach, some biomolecules will present the receptor-binding site to approaching cells, but other molecules will be inaccessible. In contrast, a rational immobilization scheme can be used to present each growth factor molecule in an orientation that makes it available for binding to cell surface receptors.