MME 4506Biomaterials

Protein adsorption and biological responses to biomaterial surfaces

Millions of medical devices are implanted into humans each year with reasonable levels of success

FDA and other regulatory agencies ‘‘stamp’’ these medical devices as biocompatible

However there is still no clear understanding of the origins of biocompatibility

Materials that do not leach biologically reactive substances will heal in the body in a manner now considered biocompatible.

Not all non-leaching materials are equally biocompatible irrespective of surface properties

The body reacts similarly to nearly all materials that we call biocompatible and walls them off in an avascular, tough, collagenous bag, roughly 50–200 micrometers thick

After implantation of a biomaterial in a living system, proteins have been observed on the surface in a short time, less than 1 second later.

A monolayer of protein adsorbs to most surfaces in seconds to minutes

This event occurs well before cells arrive at the surface, therefore they see primarily a protein layer rather than the actual surface of the biomaterial.

Since cell respond specifically to proteins this interfacial protein film may be the event that controls subsequent bioreaction to implants

Cells arrive at an implant surface after proteins adsorb, by diffusive, convective or active locomotion mechanisms

On the surface of the biomaterial cells adhere, differentiate, release active compounds to communicate with other cell types, multiply and organize themselves into tissues comprised of one or more cell types.

Extracellular matrix is secreted by cells to fill the spaces between cells and serve as attachment structure for proteins and cells. Formation of blood vessels that is critical to provide this new tissue with nutrition and to remove wastes is done by cells. They finally react distinctively to irritation and injury.

All cells communicate by the release and detection of signaling agents (cytokines) through a network made up of multiple and interactive signaling pathways.

The state of a cell (shape, structure, biological activity, etc.) depends on the signals it receives from its biological environment.

For example, platelets normally circulate in the bloodstream in a passive state.

Upon vascular injury a signaling cascade is initiated that activates the platelets for their role in healing the vascular injury.

For this reason, whole blood must be treated (e.g., heparinized) when removed from the body to keep it from coagulating

In a normal wound, the macrophage cell responsible for ‘‘orchestrating’’ wound healing is activated.

In the presence of an uncomplicated wound, the macrophage turns on the pathways leading to normal healing by first cleaning up the wound site and then secreting the appropriate cytokine messenger molecules.

These soluble messengers activate processes in the cell types needed for healing (fibroblast, keratinocyte, osteoblast, etc.).

The surfaces of today’s biomaterials present in the wound site, turn this normal healing processoff.

After protein adsorption the macrophages adhere to the biomaterial

The proteins adsorbed on the material surface are non-specific and of high variety

Macrophages do not recognize the surface of a biomaterial because of the non-specific proteins

As a result macrophages spread on the surface as they try to phagocytose it. They cannot digest or engulf this large mass, so, to increase their effectiveness, they fuse together to form multinucleatedgiant cells.

Of course, these cells still cannot engulf a macroscopic medical device.

The giant cells signal to the body that there is a large foreign body to be walled off. The fibroblasts arrive and generate the collagen capsule

While biomaterials developed in vitro are highly bioactive, (induce cell adhesion and differentiation), they do not heal normally in the body

Uncontrolled biological encapsulation directly confounds the performance of many implanted devices. The presence of this capsule seriously degrades the performance of implant electrodes, drug delivery systems, and breast implants by preventing intimate contact between device and tissue.

The reaction associated with this foreign body response (long term, low level inflammation and macrophage activation) may also

• inhibit the healing of vascular grafts, • trigger capsular opacification found with intraocular lenses, • lead to the extrusion of percutaneous devices, • increase device calcification,• induce contact lens discomfortSo it generally leads to complications and less than desirable outcomes associated with today’s medical devices.

In vivo biocompatibility tests done on 10 common biomaterials shows that they are all found to heal essentially identically after one month implantation in mammals.

gold, polyurethane, silicone rubber, polytetrafluoroethylene (PTFE), polyethylene (PE), poly(methyl methacrylate) (PMMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(ethylene terephthalate) (PET), titanium, alumina

These materials are hydrophilic, hydrophobic, hard, soft, polymeric, ceramic and metallic and exhibit wide range of properties

Each material adsorbs different proteins, and shows substantially different cell attachment and cell growth behavior in vitro.

In vivo all materials quickly acquire a layer that contains many proteins (possibly comprised of 200 or more proteins) in many states of orientation and denaturation so that they adsorb a complex, non-specific layer of proteins.

Nature’s use of proteins as signaling agents comes from one (or a few) specific proteins in fixedconformations and orientations so they optimally deliver signals.

The hypothesis for the slow healing of biomaterials suggests that the body views the non-physiologicproteinaceous foreign body capsule layer as something with which it has no experience and reacts to it as an unrecognized foreign invader that must be walled off

The main strategy for development of healing biomaterials is the synthesis of surfaces of biomaterials that present to the body the same signaling groups as the surface of a clean, fresh wound

Properties of the surface strongly influence the composition and recognizability of the adsorbed protein layer, which in turn affects the subsequent cellular interactions.

The adsorption of proteins onto a biomaterial surface from the surrounding fluid phase is rapid, with the surface properties of the biomaterial determining the type, amount, and conformation of the adsorbed proteins.

The composition of the adsorbed protein layer (i.e., the type and concentration of the proteins present in the adsorbed film) can differ from the fluid phase composition and can change with time adsorbed

Research to inhibit non-specific protein adsorption is driven by such questions:

How resistant to protein pickup can such surfaces be made? Why are they resistant to protein adsorption? How long can they remain resistant to protein fouling? Can they be functionalized with organic groups permitting the immobilization of active biomolecules on a smooth background?

Surfaces that interact with precision with biological systems will be complex:multicomponentmultilayer orientated patterned

Given the complexity of the molecular structures that make up the individual biomolecules comprising these surfaces, fabrication and characterization of such surfaces requires extreme skill in surface science and engineering.

Nonfouling or protein resistant surfaces refer to surfaces that resist the adsorption of proteins and adhesion of cells.

It is generally accepted that:• Surfaces that resist protein adsorption will also resist cell adhesion• Hydrophilic surfaces are more likely to resist protein adsorption • Hydrophobic surfaces usually adsorb a monolayer of tightly adsorbed proteins.

Nonfouling surfaces have medical and nonmedical applications such as

Blood compatible materials,Implanted devices,Urinary catheters,Diagnostic assays,Biosensors,Intravenous syringes and tubing,Heat exchangers,Ship bottoms

In addition to their medical and economic importance, research on nonfouling surfaces provide important experimental and theoretical insights into the phenomenon of protein adsorption

Surfaces containing poly ethylene glycol (PEG) make up the majority of materials researched as nonfouling surfaces.

One important area of research is on resistance to bacterial biofilms

Bacteria adhere to surfaces via a conditioning film of proteins that adsorb first to the surface

Bacteria stick to this conditioning film and begin to secrete a gelatinous slime layer (the biofilm) that aids in their protection from external agents like antibiotics

Such biofilms cause inflamatory reaction to infected devices such as urinary catheters, endotracheal tubes, vascular grafts, hip joint prostheses, heart valves

If the conditioning film can be inhibited, bacterial adhesion and biofilm formation can be reduced

Literature on protein adsorption and cell interaction with nonfouling surfaces reveals many aspects of protein adsorption on biomaterial surfaces:

• Hydrophobic surfaces have a strong tendency to adsorb proteins irreversibly. The driving force for this is the unfolding of the protein on the surface, accompanied by the release of many hydrophobically structured water molecules from the surface. As a result the entropy of the system increases.

• Cationic proteins bind to anionic surfaces and anionic proteins bind to carionic surfaces. The driving force is a combination of enthalpy decrease due to ion-ion bondings and entropy increase due to the accompanying release of counterions with their waters of hydration.

• It has been commonly observed that proteins adsorb in monolayers (they do not adsorb nonspecifically onto their own monolayers.

• At least 50% of the surface should be covered before resistance to protein adsorption is observed

• Adsorbed protein films are nonfouling surfaces for other proteins but not to cells

• Surfaces coated with physically or chemically immobilized high molecular weight PEG are highly resistant to protein adsorption. This is due to a combination of 2 factors:

i. The resistance of the polymer coil to compression due to its desire to retain the volume of a random coil (entropic repulsion)

ii. The resistance of the PEG molecule to release bound and free water from the hydrated coıl. (osmotic repulsion)

The size of the adsorbing protein and its stability in the folded state are important factors determining the extent of adsorption on any surface

The thermodynamic principles governing the adsorption of proteins onto surfaces involve various enthalpic and entropic terms favoring or resisting adsorption:

The overall conclusion is that resistance to protein adsorption at biomaterial interfaces is directly related to resistance of interfacial groups to the release of their bound watersWater may be bound to surface groups by both hydrophobic and hydrophilic interactions.

Favoring adsorption Opposing adsorption

(-) ΔHadsorption (+) ΔSadsorption (+) ΔHadsorption (-) ΔSadsorption

Short range VdW bonds

Long range ion-ion bonds

Desorption of structured

water

Unfolding of proteins

Dehydration of the

interface between

surface and protein

Unfolding of protein

PEG chain compression

Adsorption of proteins

PEG chain compression

PEG osmotic repulsion

Hydrophilic surfaces have more resistance to the release of their bound waters

So the most common approach to reducing protein and cell binding to biomaterial surfaces have been to make them more hydrophilic

The approaches to make surfaces more hydrophilic are:

Chemical immobilization of a hydrophilic polymer such as PEG, on the biomaterial surface bya. Using UV or ionizing radiation to graft copolymerize a hydrophilic monomer onto surface groupsb. Depositing such a polymer from the vapor of a precursor monomer in a gas discharge process

Other approaches are physical adsorption of surfactants, chemical derivatization of surface groups polar groups like hydroxyls or negatively charged groups like carboxylic acids since most proteins and cells are negatively charged.

In addition coating with hydrogels and natural biomolecules like albumin, casein, hyaluronic acid exhibit resistance to nonspecific adsorption of proteins.

Research on bacteria shows that the nonfouling surfaces provide weak resistance to bacteria and biofilm buildup, only the best nonfouling surfaces slowing down the adhesion and colonization.

PEG groups on nonfouling surfaces are susceptible to oxidative damage and this reduces their effectiveness in bacteria resistance.

Defects on nonfouling surfaces such as pits, uncoated areas also provide footholds for bacteria and cells to begin colonization.

Resistance to bacterial adhesion remains an unsolved problem in the surface science.

Surfaces of biomaterials are modified in order to influence the biointeraction while the key physical properties of the material are retained.

The two categories of surface modification are

1. Chemically or physically altering the atoms, compounds or molecules in the existing surface by chemical reactions, etching, mechanical roughening

2. Overcoating the existing surface with a material having a different composition by coating, grafting, thin film deposition.

The modified zone at the surface of the material should be as thin as possible. Modified surface layers that are too thick can change the mechanical and functional properties of the material.

Thick coatings are also more subject to delamination and cracking.

Ideally alteration of only the outermost molecular layer (0.3-1 nm) should be sufficient.However thicker films than this are necessary in practice since it is difficult to ensure that the original surface is uniformly covered .

Extremely thin layers may be subject to surface reversal and mechanical erosion.

Some coatings like PEG protein resistant layers require a minimum thickness to function that is related to the molecular weight of chains.

In general minimum thickness needed for uniformity, durability, and functionality is determined experimentally for each system.

The surface-modified layer should be resistant to delamination and cracking.

This is achieved by • covalently bonding the modified region to the substrate• intermixing the components of the substrate and the surface film at an interfacial zone like an

interpenetrating network• applying a compatibilizing layer at the interface• incorporating appropriate functional groups for strong intermolecular adhesion between a substrate

and an overlayer

Surface rearrangement can occur in metallic and organic materials by the driving force for minimization of interfacial energy.

Sufficient atomic or molecular mobility must exist for the surface changes to occur in observable periods of time.

Surface chemistries and structures can switch because of diffusion or translation of surface atoms or molecules in response to the external environment.

A newly formed surface chemistry can migrate from the surface into the bulk or molecules from the bulk can diffuse to cover the surface.

Surface reversal must be prevented or inhibited by cross-linking, sterically blocking the ability of surface structures to move, incorporating a rigid, impermeable layer between the bulk material and surface modification.

The surface modification reaction should be monitored by surface analysis techniques to ensure that the intended surface is formed.

The surface-modified region is thin and consists of small amounts of material. Undesirable contamination can be introduced during modification reactionsThe potential for surface reversal to occur during surface modification is also high.

For these reasons special surface analytical tools are necessary since conventional analytical methods are not sensitive enough to detect surface modifications:

Contact angle methodsElectron spectroscopy for chemical analysisSecondary ion mass spectrometryScanning electron microscopyInfrared spectroscopyScanning tunneling microscopyAtomic force microscopyScanning probe microscopy

Some physicochemically surface modified biomaterials

To modify blood compatibilityOctadecyl group attachment to surface for albumin affinityPlasma fluoropolymer depositionChemically modified polystyrene for heparin-like activity

To influence cell adhesion and growthOxidized polystyrene surfacePlasma deposited acetone or methanol filmPlasma fluoropolymer deposition to IOLs to reduce cell adhesion

To control protein adsorptionSurfaces with immobilized PEG to reduce adsorptionTreated ELISA dish surface to increase adsorptionSurface cross-linked contact lens to reduce adsorption

To improve lubricityPlasma treatmentRadiation grafting of hydrogelsInterpenetrating polymeric networks

To improve wear and corrosion resistanceIon implantationDiamond depositionAnodization

To alter transport propertiesPolyelectrolyte grafting

To modify electrical characteristicsPolyelectrolyte graftingMagnetron sputtering of titanium

All of the surface mentioned modification methods can be applied both as a uniform surface treatment or as patterns on the surface with length scales of millimeters, microns or nanometers.

Proteins and cells are deposited in surface patterns and textures in order to control bioreactions

Photolithographic techniques that were developed for microelectronics have been applied to patterning of biomaterial surfaces when used together with surface modification techniques. For example, plasma-deposited films patterned using a photoresist lift-off method

Microcontact printing is a newer and simpler method that utilizes a rubber stamp made of the pattern which is desired on the biomaterial surface. It can be inked with thiols to stamp gold, silanes to stamp silicon, proteins or polymers to stamp many types of surfaces

Surface irregularities on medical devices such as grooves, hills, pores, steps are expected to guide many types of cells and to aid tissue repair after injury.

Pores or surface roughness repeated periodically are termed texture.

Porous scaffolds used in vivo and in vitro for tissue engineering direct the response of cells by rough and porous surfaces, improving the following:

Organization of the cytoskeletonOrientation of extracellular matrixAmount of produced extracellular matrixAngiogenesis

The exact cellular and molecular mechanisms underlying cellular and matrix orientation around surface irregularities are not yet clear.

For many biomedical applications there is a need for porous implant materials

The specific requirement for a porous material using in bone growth is to have diameter in the optimum range of 75-250 micrometers. For ingrowth of fibrocartilegenous tissue the recommended pore size is in the range 200-300 micrometers. Other important requirements are pore interconnectivity, interconnection size and compressibility,

Porous biomaterials are used for artificial blood vessels, artificial skin, drug delivery, bone and cartilage reconstruction, periodontal repair and tissue engineering.

Porosity is generally considered as a microtexture based on the importance of this type of surface morphology for cell and tissue response.

Contact guidance is the phenomenon that cells adapt and orient to the biomaterial surface microtopography.

Alignment and adhesion of cells to microgrooves with dimensions 1.65-8.96 micron in width and 0.69 micron in depth has been reported and explained due to the mechanical inflexibility of cytoskeletal components that prevents bending of “arms” of the cells over surfaces.

Cells want to achieve a biomechanical equilibrium condition with a resulting minimal net sum of forces. It is found that the anisotropic geometry of surface features establishes stress and shear-free planes that influence the position of cytoskeletal elements.

There are hypotheses relating cell contact site, deposited extracellular matrix, surface microtexture and cell response.

For example, a microtextured surface possesses local differences in surface free energy resulting in a specific deposition pattern of proteins.

More in vivo research has to be done to learn and understand the biological effects of microtexturedsurfaces.

For this, development of techniques that enable the production of surface microtexturing on nonplanar surfaces is required.

In addition, the relationship between the surface topographical design of an implant and tissue compatibility has to be investigated.

Biomaterials that induce normal healing mechanism can be realized as a result of research in these directions