A surface coating comprises a primer coat that permits adhesion of eukaryotic cells thereto, and a plurality of macromolecular structures attached to the primer coat. At least some of the macromolecular structures have a cell-resistant character, meaning that cells generally will not adhere to them. The macromolecular structures are distributed across an area of the primer coat so that the surface coating permits adhesion of the eukaryotic cells to the primer layer and resists the adhesion of non-eukaryotic cells. Typically, the primer coat comprises a self-assembled polymeric monolayer and the macromolecular structures comprise nanoscale hydrogels. Such surface coatings may be formed on articles of manufacture for insertion into the body, such as orthopedic devices.

Description of the Invention

FIELD OF THE INVENTION

This invention is related to the field of surface coatings, particularly those having cell-resistant properties. It is also related to the field of biomedical devices, particularly those, such as orthopedic devices, that are intended for implantation in the human body.

BACKGROUND OF THE INVENTION

Surfaces have been extensively modified by chemical, biochemical, and topographic means to render them either adhesive or resistant to cells, and the resulting knowledge base has had a very substantial impact both on the basic scientific understanding of cell-material interactions as well as on important applications associated with biomedical devices and tissue-engineering constructs. Because of the many varied specific and non-specific mechanisms involved in cell adhesion, however, a surface that is adhesive to one cell type is usually also adhesive, to varying degrees, to other cell types. The surfaces of orthopedic implants are no exception. The oxidized metal or hydroxyapatite-coated surfaces used in most implant applications satisfy the critical design criteria of being osteoinductive, but they are also adhesive to bacteria. A number of materials modifications have been made to render such surfaces resistant to bacteria--PEGylation, for example, has been used--but these bacteria-resistant surfaces also resist adhesion of eukaryotic cells.

Surface coatings having submicron features offer a solution to the problem of creating a surface that is differentially adhesive to osteoblasts and bacteria. This solution is based on the modulation of surface adhesiveness using nanoscale hetero-features organized on surfaces in two dimensions at submicron length scales. Such patterning is being explored in several contexts, including control of cell adhesiveness. However, the idea of modulating nanoscale adhesiveness to achieve differential cell adhesion based on fundamental differences in the length-scale properties of bacteria and eukaryotic cells is new.

SUMMARY OF THE INVENTION

This invention provides a new surface treatment that permits the adhesion of one or more types of eukaryotic cells on a surface while simultaneously resisting adhesion by one or more types of non-eukaryotic cells (e.g., bacteria). This differential adhesiveness has applicability toward reducing the risk of infection associated with articles to be implanted in living bodies.

In one embodiment, the invention provides a surface coating that is differentially adhesive to bacteria and eukaryotic cells, and comprises a primer coat that permits eukaryotic cells to adhere thereto, and a plurality of macromolecular structures, such as nanohydrogels, attached to the primer coat. At least some of the macromolecular structures are cell-resistant (i.e., they resist the adhesion of cells thereto), and are sufficiently distributed across an area of the primer coat so as to permit the adhesion of eukaryotic cells thereto, and repel bacteria therefrom.

In another embodiment, the primer coat of the surface coating described above, is provided with a factor to promote the adhesion of eukaryotic cells to the primer coat.

In yet another embodiment, the surface coating is provided with both cell-resistant macromolecular structures and macromolecular structures that include a factor to promote the adhesion of eukaryotic cells thereto.

In a further embodiment, the primer coat comprises a self-assembled monolayer of polymer formed by electrostatic self-assembly of the monolayer on a substrate, and the macromolecular structures are deposited on the primer coat by electrostatic self-assembly on the monolayer.

In a yet further embodiment, an article is provided having the surface coating assembled upon it, and, in a variation of the yet further embodiment, the surface coating is self-assembled on a continuous, topographically complex surface of the article.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of the present disclosure, the following definitions are to be applied:

A macromolecular structure is any particle having submicron lengths along at least two dimensions, preferably in the range of about 50 nm to about 250 nm, and having a cell-resistant or cell-adhesive macromolecule, preferably a gel, exposed at a surface thereof. A nanohydrogel is an example of a macromolecular structure.

A hydrogel is a type of gel (i.e., an intramolecularly cross-linked macromolecule that can swell in the presence of a solvent) that specifically interacts with water and water-based solutions, including physiological media. It may be noted that both cell-resistant and cell-adhesive hydrogels are known, and can be formed by appropriate selection of the polymer used, and the degree of cross-linking present.

A nanohydrogel is a hydrogel whose diameter in the swollen state is less than about one micron and, preferably, whose diameter in the fully-hydrated state is less than about 250 nm.

A material is considered to be cell-resistant if cells, including both eukaryotic cells and bacteria, generally will not form adhesive contacts with a surface of the material. It is known that cells generally will not form adhesive contacts with hydrogels that are formed from any of a number of polymers, including, but not limited to poly(ethylene oxide), certain poly(ethylene oxide) acrylates, and other poly(ethylene glycol) derivatives. Other examples of such materials are known to those of ordinary skill in the art.

A material is cell-adhesive if a cell will form an adhesive contact with a surface of the material. A cell that has adhered to a material will not be removed from that surface by mechanical stress such as that associated with rinsing the material with water or a buffer solution.

A cell-adhesion factor is any chemical entity that promotes or mediates adhesion of a cell to another material. Such factors include, but are not limited to, compounds or fragments of compounds such as antigens, antibodies, or extracellular matrix molecules (e.g., laminin, fibronectin, collagen, integrin, serum albumin, polygalactose, sialic acid, lectin-binding sugars, synthetic oligopeptides, or carbohydrates). Other such chemical entities include compounds having functional groups such as, but not limited to, hydrophobic groups or alkyl groups having charged moieties. Other examples of such entities and moieties are known to those of ordinary skill in the art, as well as methods of modifying various materials to include such entities or moieties.

Specific adhesion is adhesion that is mediated by reversible bonds between specific, complementary molecules including, but not limited to: antibodies, or fragments of antibodies, and their antigens, cell surface receptors and their ligands, or lectins and carbohydrates. In the present invention, mechanisms that are known to promote specific adhesion among cells may be adapted to promote adhesion between cells and polymeric structures, or between polymeric structures, by linking the complementary molecules to the polymers. Other examples of such molecules are known to those of ordinary skill in the art.

Non-specific adhesion includes mechanisms of adhesion such as electrostatic attraction, hydrogen bonding, covalent bonding, and other mechanisms of adhesion that do not rely on the reversible bonding between complementary molecules.

A differentially adhesive surface, with respect to cell adhesion, permits the adhesion of one type of cell to the surface, while resisting the adhesion of a different type of cell.

A primer coat comprises one or more layers that are applied to a substrate and form a base to which a macromolecular structure may adhere.

A substrate is considered to be the foundation on which molecules, particularly, but not exclusively, those in the primer layer, may be immobilized.

Electrostatic self-assembly refers to a process by which two different structures, such as, but not limited to, a polymer molecule and a substrate, or a macromolecular structure and a primer coat, attract each other by means of opposite charge or oppositely aligned dipoles, and bind by some form of secondary bonding.

Self-assembled monolayers include, but are not limited to, layers of polymer molecules that adsorb onto a substrate by electrostatic self-assembly. Other methods of forming such self-assembled monolayers are known to those having ordinary skill in the art.

The term "topographically complex" describes surfaces that extend in three dimensions and have been made rough by means such as mechanical abrasion, cutting, or etching, or have been manufactured in such a condition. The term especially applies to the visible and hidden surfaces of articles comprising masses of sintered particles.

The surface coating disclosed herein presents surfaces whose cell adhesiveness is spatially modulated at cellular and subcellular-scale lengths. This spatially modulated adhesiveness provides a mechanism by which such a coating becomes differentially adhesive to different cell types. For example, such a structure has the ability to control the differential adhesion between eukaryotic cells and bacteria because these two classes of cells have characteristic sizes that differ by a factor of 5 to 50, depending upon the specific cell types involved. This ability is not present in surfaces which have been made cell-adhesive by chemical or topographic methods alone, since such surfaces are usually adhesive to many cell types. The surface coating described herein further has direct application to the modification of orthopedic implants, and other objects or devices to be implanted in the human body, where there is a pressing need to preserve the ability of these implants to promote osteointegration via the adhesion and proliferation of osteoblasts (which are a type of eukaryotic cells) while simultaneously reducing the threat of infection by resisting the adhesion of bacteria to the implant. As disclosed herein, the surface coating may be assembled on the surface of such an article by electrostatic self-assembly, enabling the surface coating to be deposited on topographically complex surfaces typical of, for example, modern implants, the surfaces of which are created by masses of sintered beads.

FIG. 1 (see Original Patent) presents a schematic cross-sectional view of one embodiment of a surface coating 10 disclosed herein. In this particular embodiment, a primer coat 12 has been formed on a substrate 14. In this particular example, the primer coat 12 comprises a self-assembled monolayer of a cationic polymer assembled on the substrate 14 by electrostatic deposition. For applications in which the surface coating 10 is to be used in biological applications (e.g., as a coating on a device to be implanted in the human body) the polymer should be able to maintain a positive (+) charge at the surface 16 of the primer coat 12 in physiological media. The primer coat 12 has a cell-adhesive character. The material used to form the primer coat 12 may itself be cell-resistant or cell-adhesive, with the adhesiveness provided or enhanced, respectively, by the addition of one or more cell-adhesion factors 18 to the surface 16 of the primer coat 12. The design of the primer coat 12 is important to the control the structure, heterogeneity, and stability of the self-assembled monolayer of nanohydrogels 20. In the embodiment of FIG. 1, the particular type of nanohydrogels 20 are cell-resistant hydrogels. A suitable polycation primer coat 12 may based on polymers such as poly(L-lysine) (PLL), poly(allylamine hydrochloride) (PAH), or linear/branched polyethyleneimine (PEI), or their copolymers.

It should be noted that the charge and conformation of the polymer in the primer coat 12 plays a significant role in the surface organization of the nanohydrogels 20 deposited upon it. The charge and polymer conformation can be controlled by copolymer composition, pH, and ionic strength, and other properties. It must be practical to controllably deposit the primer coat 12 on both planar (e.g., silicon) and topologically complex (e.g., beaded-metal coupons) substrates in order to mediate subsequent deposition of the nanohydrogels 20. Such substrates typically are coated with oxides of the substrate material, and can maintain a negative (-) charge that will electrostatically attract cationic polymer molecules.

A series of PLL, PAH, or PEI copolymers can be used to give control over the thickness of the primer coat 12 and the molecular conformation of the polymers in the primer coat 12. Importantly, the different adsorption characteristics of different polycation polymers can be used to control surface coverage by the nanohydrogels 20. For example, the thickness of PAH films can be manipulated by varying deposition pH. The fact that the deposited thickness increases with increasing pH reflects the electrostatic nature of interactions between positively-charged PAH segments and the negatively charged substrate. By decreasing pH below the PAH pK.sub.b value of 10.5, the linear charge density of PAH decreases and loopy chain conformations are observed. Such a conformation is important, because it leads to greater deposition of a nanoparticles. Similar control can be achieved by varying the ionic strength during polymer deposition. Polymers adsorbed from low-salt solutions form relatively flat conformations due to the repulsions between charged segments, whereas polymer chains have more loops and tails when adsorbed from high-salt solutions. Analogous arguments can be made concerning the salt and pH-dependent deposition behavior of PEI and PLL.

Spatial modulation of the cell-adhesive properties of the surface coating 10 is provided by cell-resistant macromolecular structures 20 on the surface 16 of the primer coat. In the particular embodiment represented in FIG. 1, the cell-resistant macromolecular structures 20 are cell-resistant nanohydrogels 20 comprising a cross-linked anionic polymer. Similar to the selection of a polymer for the primer coat, selection of the polymer used to synthesize nanohydrogels 20 is important to the structure of the surface coating 10. When a polycationic primer coat 12 is used, the polymer used to synthesize the nanohydrogels 20 should be one that will maintain a negative (-) charge in physiological media (i.e., a polyanionic monomer), while retaining the nanohydrogels' 20 cell-resistant character in such media. The positive (+) charge of the primer coat 12 and the negative (-) charge of the nanohydrogels 20 enable electrostatic self-assembly of the nanohydrogels 20 on the primer coat 12, in an unpatterned lateral distribution across the primer coat 20 (i.e., distributed across two-dimensions). The nanohydrogels 20 adhere to the primer coat 12 by electrostatic attraction and, thus, cannot readily removed from the primer coat 12 by mechanical stress. It may be noted that any type of macromolecular structure 20 may be deposited on a primer coat 12, as long as both the macromolecular structure 20 and primer coat 12 maintain opposite electrical charges, or present oppositely aligned dipoles, in the same media. Further, a macromolecular structure 20 and primer coat 12 may be designed so that they attach to each other by other mechanisms of adhesion, whether specific or non-specific. Such mechanisms can be used to fix the two-dimensional organization of the nanohydrogels 20 after their deposition.

FIG. 2 (see Original Patent) presents a schematic cross-sectional view of a larger portion of the surface coating 10 of FIG. 1, illustrating the adhesion of a eukaryotic cell 22, such as an osteoblast, and the absence of adhesion of a bacterium 24, such as a staphylococcal bacterium. Both the size and spacing of the cell-resistant nanohydrogels 20 are important to controlling the differential adhesion of eukaryotic cells 22 and bacteria 24 to the surface coating 10. Significantly, osteoblasts are typically on the order of 5-10 .mu.m in diameter, have flexible cell walls that can conform to a substrate, and adhere to surfaces in the presence of cell-adhesive factors. In contrast to osteoblasts, bacteria such as Staphylococcus epidermidis (S. epi) and Staphylococcus aureus (S. aureus) tend to be spherically shaped and do not easily conform to a substrate. In contrast to osteoblasts, staphyloccoci are only about 0.6-0.9 .mu.m in diameter, and their surface adhesion is mediated by both specific and non-specific binding mechanisms. Thus, the cell-resistant nanohydrogels 20 must be spaced such that a bacterium 24 will not have an adequate opportunity to bind to the surface coat 12, while allowing eukaryotic cells 22 to bind to the spaces between the cell-resistant nanohydrogels 20. Accordingly, the cell-resistant nanohydrogels 20, or other macromolecular structures 20 that may be used in their place, should have submicron dimensions (i.e., lengths of less than one micron) in at least two dimensions, and, preferably, have nano-scale dimensions. The cell-resistant nanohydrogels 20 of the embodiment of FIGS. 1 and 2 should have diameters in a preferred range of about 50 nm to about 250 nm. In that size range, the preferred average spacing between the cell-resistant nanohydrogels 20 would be in the range of about 0.2 .mu.m to about 5 .mu.m. Such a spacing would allow the eukaryotic cells 22, with their flexible walls, to adhere to the primer coat 12 within the spaces 26 between the cell-resistant nanohydrogels 20, while bridging such nanohydrogels. The bacteria 24 would be, effectively, repelled from the surface coating 10. The desired differential adhesion of eukaryotic cells 22 relative to bacteria 24 can be achieved when as little as 2-20 percent of the surface area of the primer coat 12 is covered by cell-resistant nanohydrogels 20. However, such differential adhesion can also be achieved when as much as 80-98 percent of the primer coat 12 is so covered. This greater coverage would correspond to a surface coating 10 that is presents an effectively continuous surface that is resistant to bacterial adhesion, but has micron-sized windows for eukaryotic adhesion.

FIG. 3 (see Original Patent) presents a schematic cross-sectional view of a second embodiment of a surface coating 28. In this particular embodiment, a primer coat 30 has been formed on a substrate 32. The primer coat 30 and the substrate 32, as well as the surface 34 of the primer coat 30, may have the same features and characteristics of the respective primer coat 12, substrate 14, and surface 16 of the surface coating 10 of FIGS. 1 and 2, except for the differences that are noted herein. In this second embodiment, the primer coat 30 may be provided with one or more cell-adhesive factors (not shown) exposed at its surface.

At least two types of macromolecular structures 36, 38 are provided at the surface 34 of the primer coat 30. In this second embodiment, the respective macromolecular structures, 36, 38 are cell-repellant nanohydrogels 36 and second family of nanohydrogels (i.e., cell-adhesive nanohydrogels 38), both of which may have the same features and characteristics as the cell-resistant nanohydrogels 20, except for the differences that are noted herein. With respect to the nanohydrogels 36, 38, as well as for macromolecular structures 36, 38 in general, it may be noted that it is not necessary that they be made of the same materials as each other, as long as the materials that are used create nanohydrogels 36, 38 having the desired properties, particularly the ability to maintain a negative (-) charge in physiological media.

The significant difference between the cell-repellant nanohydrogels 36 and the cell-adhesive nanohydrogels 38 is that the cell-adhesive nanohydrogels 38 include cell-adhesion factors 40. Preferably, such cell-adhesion factors 40 are selected to preferentially bind to eukaryotic cells (e.g., by a mechanism of specific adhesion not available to bacteria).

FIG. 4 (see Original Patent) presents a schematic cross-sectional view of a larger portion of the surface coating 28 of FIG. 3, illustrating the adhesion of a eukaryotic cell 42, such as an osteoblast, and absence of adhesion of a bacterium 44, such as a staphylococcum. Similar to the surface coat 10, both the size and spacing of the cell-resistant nanohydrogels 36 are important to controlling the differential adhesion of eukaryotic cells 42 and bacteria 44 to the primer coat 12. That is, the cell-resistant nanohydrogels 36 must be spaced such that a bacterium 44 will not have an adequate opportunity to bind to the surface coat 28, as with surface coating 10. The spacing between the cell-adhesive nanohydrogels 38 is less important, as long as the cell-resistant nanohydrogels 36 are adequately spaced to allow eukaryotic cells 42 to adhere to the spaces among them, and to have the effect of repelling bacteria 44. It may be noted that the cell-resistant nanohydrogels 36 and the cell-adhesive nanohydrogels 38 will be distributed laterally across the primer layer 30, such that a eukaryotic cell 42 may interact with the resulting groups of cell-adhesive nanohydrogels 38 much the same as it would interact with areas of the primary coat 30 that would otherwise be exposed between the cell-resistant nanohydrogels 36. As is illustrated in FIG. 4, a eukaryotic cell 42 may adhere entirely to the cell-adhesive nanohydrogels 38, while bridging the cell-resistant nanohydrogels 36, without directly contacting any substantial portion of the primer coat 30. As with the embodiment of FIGS. 1 and 2, the desired differential adhesion of eukaryotic cells 22 relative to bacteria 24 can be achieved when as little as 2-20 percent of the surface area of the primer coat 30 is covered by cell-resistant nanohydrogels 36. At such a coverage, it is preferable that the ratio of cell-adhesive nanohydrogels 38 to cell-resistant nanohydrogels 36 be in the range of about 1 to about 10. Further, such differential adhesion may also be achieved with a primer coat that has much-greater coverage by cell-resistant nanohydrogels 36 with smaller areas covered by cell-adhesive nanohydrogels 38.
 

Claim 1 of 13 Claims

1. A surface coating, comprising: a primer coat that permits adhesion of eukaryotic cells thereto; and a plurality of essentially spherical hydrogels attached to a said primer coat such that portions of said primer coat are exposed between said hydrogels, wherein each of said plurality of hydrogels has a diameter of less than one micron, and wherein at least some of said plurality of hydrogels are cell adhesion-resistant hydrogels, said cell adhesion-resistant hydrogels being in an unpatterned lateral distribution across a portion of said primer coat and spaced so as to permit adhesion of eukaryotic cells to said exposed portions of said primer coat, while resisting the adhesion of non-eukaryotic cells thereto.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.