Biocompatibility

What is Biocompatibility? The simplest definition of biocompatible is the quality of non-toxicity; however, this definition is problematic as the quality is entirely contextual. Dental amalgam composed of mercury, silver, tin copper is considered biocompatible and safe in the context of filling cavities in teeth but would be toxic if place subcutaneously. Prior to teflon, IV catheters were made of polyvinyl chloride (PVC). IV catheters are left in place no longer than three days and in this context PVC is biocompatible. If left in place longer, PVC causes inflammation. The “The Williams Dictionary of Biomaterials” defines biocompatibility as “the ability of a material to perform with an appropriate host response in a specific application.” The ideal material would exhibit no cytotoxicity or sensitization of cells. It would cause no local irritation or systemic toxicity even after years of exposure.  A perfect biocompatible material would have no effect on reproduction or development and it would be non-carcinogenic. Unfortunately, there is no known material with all of these characteristics. Instead, materials used in implants are chosen according to their ability to perform in the context of location, function, and duration.

The story of biocompatibility begins in a rather unexpected place and time. The Battle of Britain pitted Luftwaffe Messerschmitt fighters against the Royal Air Forces Hurricane. The Messerschmitt was 40 mph faster and armed with both 7.925mm machine guns and 20mm cannons. Hurricanes had only 7.7mm machine guns; it was common for Messerschmitt craft to be struck hundreds of times during combat and still limp back to base. The Luftwaffe had over 900 Messerschmitt while the RAF had only 650 Hurricanes. Despite a significant numerical and technological advantage the Battle of Britain marked the Nazis first major defeat in WWII. This victory was in part due to the RAF’s Flying Aces. 5% of the combat pilots in this altercation were responsible for the vast majority of victories. For example, RAF ace Paddie Finucane downed 32 enemy planes; this equated to 4% of the German Air Force. The first modern studies into biocompatibility and physiological responses to foreign materials resulted from an ace pilot injured in this battle. In May of 1940, Ace Mouse Cleaver was shot down and parachuted to safety. He was  blinded in the right eye and lost most of the vision in his left after the canopy of his Hurricane shattered leaving shards in his face and eyes. The canopy was made of Perspex, a material which had only become available four years prior. Most RAF crafts were still using standard glass and it’s this retrofit which saved his vision. Physician Harold Ridley expected Cleaver to require removal of his eyes because shards of glass cause inflammation and rejection. To his surprise, Mouse had no inflammatory response and retained his vision in his left eye. Ridley proposed that perspex could be used to make artificial intraocular lenses. Decades later, Mouse Cleaver received one of these perspex implants intraocular lenses. His vision was saved by perspex twice.

This work is organized into three sections. The first explains how the body responds to foreign bodies. The second section explores testing methods used to determine biocompatibility. The third discusses a number of materials with potential for use in implanted devices.

Section 1: Foreign Body Reactions and Rejection

Rejections of implants come in a variety of flavors. The most common reasons for failed implants are poor implantation technique or aftercare; however, our focus here is on reactions to non-biocompatible materials. Foreign body reactions occur either acutely or chronically. Before discussing what happens when an implant goes wrong let’s discuss the healing process when an implant goes right. Assuming proper sterilization and good technique, normal healing begins with platelet aggregation and the formation of a fibrin clot which stop bleeding. Damaged cells release inflammatory mediators such as histamine resulting in the clinical signs of inflammation. Local blood vessels dilate in order to bring in immune cells and causing the area to appear red. The temperature of the area is increased assisting the immune system in fighting possible infections. Dilated vessels allow fluid to move into the area causing swelling and dead or damaged cells are removed via phagocytosis. As the inflammation begins to subside, cells begin proliferating. A clean incision can close overnight although it takes weeks for collagen fibers to return. It’s these fibers that give structural integrity and prevent a wound from reopening. This is followed by blood vessels and nerves growing back into the area. Although small incisions may appear to be healed within a month, full maturation can take as long as a year.

Acute reactions are those that occur during the initial month of healing. Local cells proliferate and attempt to heal the wound but cytotoxic materials making up the implant kill these cells. As they die, inflammatory mediators continue to be released causing  a progression of swelling, redness, and pain. Phagocytes consume dead cells producing a pus filled abscess around the implant. Unfortunately, it’s nearly impossible to differentiate between a rejection resulting from infection and a rejection due to the material implanted. Treatment is the same for both as well. The wound must be incised and drained for the site heal and remain free of infection.

Chronic reactions after the acute phases of healing. Over time, molecules and atoms begin adhering to the surface of the implant. Once enough fouling has occurred proteins begin adhering as well. Some material are designed to facilitate this protein adsorption such as those used to repair bone. These materials facilitate the bonding of osteoblast cells leading to the implant being integrated into bone. Under most circumstances however, protein adsorption is undesirable. The fouling on an implant attracts and binds to macrophages. The macrophages then fuse together becoming foreign body giant cells. These giant cells sequester the foreign implant and begin releasing a number of degrading agents such as reactive oxygen species, enzymes, and acids. These agents are highly effective and can even break down metals. If the implant material is susceptible to break down toxic substance such as heavy metal can be released resulting in systemic poisoning. Often this degradation process leads to a breach in the coating of an implant exposing cells to the more cytotoxic substances within. Dying cells begins a far more rapid rejection process akin to an acute reaction resulting in the formation of a pus filled abscess.

If the implant material is able to survive the chemical onslaught, the giant cells begin secreting proteins which promote the formation of fibrin. The fibrin forms a fibrous capsule which isolates the implant from the rest of the body. These capsules can be thick and may affect function of an implant but generally no further immune response occurs and the implant is not rejected.

Section 2: Biocompatibility Testing

Guidelines for biocompatibility testing are provided by the International Organization for Standardization (ISO) in document 10993 and in FDA memorandum #G95-1. According to these guidelines risk assessment should evaluate the finished device and not just the materials used. The purpose of this is to evaluate not just the materials used but also the processing and manufacturing. A great example of why this is important is found in early attempts at producing implantable magnets. Although the coatings being applied were biocompatible for the intended purpose manufacturing defects proved to be an immense hurdle. That said, the FDA does make some exceptions in the testing they require when a product is made of materials with a long history of safety. Grinders are almost universally limited in resources and thus selection of material is most often based on faith in reviewed literature rather than extensive testing.

Biocompatibility testing isn’t looking at immunological response alone either. An improperly designed implant may fail due to the forces it exerts on surrounding tissue, thus implants designed in the grinding community come with additional risk in that we don’t necessarily know how the body will respond to new stimuli. Implanted bone conduction speakers may eventually be shown to cause bone necrosis. Implanted electrodes may be found to damage the nervous system. Even seemingly benign stimuli such as the LED lighting from devices like the NorthStar may have unforeseen consequences. This certainly isn’t meant as a discouragement but should be taken into consideration. Another risk unique to grinding is the possible use of materials which characteristics not previously encountered in implants. For example, submicron sized particle of otherwise benign substances have been shown to have properties such as immunogenicity and toxicity. I won’t be surprised if the first in vivo applications of nanotechnology occurs in some grinders workshop, but when working with such technology any prior biocompatibility data is worthless.

Biocompatibility testing should be performed not only during the initial production of a device but also whenever changes are made in the manufacturing process. There have been cases in which medical devices harmed patients after a seemingly minor change. Although an acid bath may be intended for passivation of a surface it may also affect the surface in other ways such as reducing the amount of pyrogenic material present. If another method of passivation is used, the device may begin causing fevers after implantation. A change to pay particular attention to is resin and polymer suppliers. If one changes suppliers they may end up with a product full of processing solvents such as formaldehyde. A previously biocompatible product has now become a toxin.

TESTS:

Cytotoxicity

Cytotoxicity testing is performed using cell culture assays. These assays use isolated cells to assess the toxicity of materials in order to screen a material prior to implantation tests. Qualitative tests are excellent for screening new materials. Quantitative tests provide a more rigorous analysis as so how cytotoxic a material may be.

Qualitative Cytotoxicity Tests

Three qualitative tests are used to determine cytotoxicity. These include Direct Contact tests, Agar Diffusion Assays, and MEM Elution Assays.

Direct Contact tests are used to test thin low density materials such those used in contacts lenses. The material is placed directly onto the cell culture and then incubated. Cytotoxicity is determined by observing the cells for lysis, morphogenic changes, and degeneration.

An Agar Diffusion assay is used on larger denser materials and consists of agar being placed over cultured cells. The material is then placed on top of the agar and the cells are incubated. If the material is cytotoxic, a region of lysed, malformed, or degenerated cells  becomes apparent.

A MEM Elution assay consists of washing the material to be tested with solvents in order to extract possible cytotoxic compounds. The solution is then transferred into a cell culture which is incubated. Cytotoxicity is determined by assessing these cells for morphological changes, lysis, and degeneration.

Quantitative Cytotoxicity

Quantitative cytotoxicity test is performed via colorimetric methods using dyes or substances which are normally sequestered within cells. Dead cells are unable to keep dies out or sequester substances and thus can be identified via color changes. These types of tests can quickly screen a large volume of sample at different concentrations and quantify the percentage of living cell remaining in a culture. The disadvantage of a quantitative assay is that is doesn’t assess for morphological changes in cells; it only differentiates between cells which are alive and those that are dead.

Sensitization Assays

Sensitization assays identify if a material causes adverse effects after prolonged exposure. After extended exposure some material can cause either hypersensitivity reactions or allergic reactions. There are three primary tests used to identify such materials.

The Guinea Pig Maximization test consists of the test material being administered to a guinea pig along with an adjuvant. The adjuvant is a compound that modifies immune response maximizing the response to the material being tested. The animal is then later exposed again to the test material and any allergic reactions that occur are measured. This is a similar process to that used in vaccines. Those against vaccines often cite the presence of substances such as aluminum as having dangerous side effects such as causing an immune; however, this is the exactly the point of the aluminum. It’s act as an adjuvant maximizing the creation of antibodies and in the doses administered no harm has as of yet been demonstrated.

A Closed Patch test is similar to the patch testing performed on patients to determine what allergens are causing their dermatitis. Multiple topical doses of the test substance are placed on a subjects skin which is then observed for inflammation. This type of test is only useful for devices used outside of the body.

Murine Local Lymph Node Assays consists of a test material being secured to the animal’s skin. If the material initiates sensitization then allergens will be transported to a lymph node. Immune cells in the lymph node proliferate in response. The assay determines sensitivity based on measuring this increase.

Acute Systemic Toxicity

An Acute Systemic Toxicity test utilizes extracts from a material which are then injected into mice. The intent is to detect if systemic toxicity occurs in response to substances which may leach out of an implant over time. This type of test looks for systemic responses such fever and changes in activity of enzymes from the liver which are often involved in chemical metabolism.

Subchronic Toxicity

Subchronic toxicity testing consists of exposing animals to a material for 10% of their lifespan. These animals are then observed for adverse effects. While unnecessary in devices intended for short term use, this type of testing is essential for permanent and long term implants.

Genotoxicity and Carcinogenesis Bioassay

Genotoxicity evaluations are intended to detect mutagens which can cause genetic damage. Mutagens can increase both the risk of cancer and inheritable disorders. The most common type of test to detect mutagenicity is the Ames test. Some strains of Salmonella have lost the ability to produce histidine due to mutation. They require histidine to grow but cannot produce it on their own. The genes responsible for the histidine producing mechanism still exist but can’t be expressed. The Ames test exposes such strains to substances and if they are mutagenic they reverse this mutation allowing the Salmonella to survive in a culture free of histidine. Other genotoxicity tests include chromosal abberation testing and comet assays.

Carcinogenesis assays are used to determine the tumorigenic potential of materials by implanting them and  them observing for abnormal growths over the total lifespan of an animal. Testing using dogs often takes as long as seven years resulting in this being a particularly costly type of test as well as a controversial one.

Reproductive And Developmental Toxicity

Reproductive toxicity testing determines is a substance will affect reproductive function in an organism or the development of its offspring. These toxic effects can be subtle such as in the case of endocrine disruptors or blatant such as with teratogens like thalidomide. This test consists of dosing a pregnant animal and waiting for it to come to full term at which time the animal is killed and both the animal and its fetus are examined for detrimental effects. The test is then performed on a second animal which is allowed to give birth. Its progeny is then allowed to become pregnant. Once at full term this animal and its progeny are killed and examined to determine any effects on the second generation.

Implantation Tests

Implant studies consist of a completed device being implanted under the skin of an animal in order to evaluate not only safety of the composition of a device but also the safety of the function of a device. For example, a new model of pacemaker may require implantation testing in order to determine if the functioning device poses a risk as well as a final assessment of the biocompatibility of a device using a particular manufacturing method. Implantation testing is essentially the last step of a series of biocompatibility tests. Unfortunately, it’s also the only test easily performed by those attempting to develop implants in the biohacking community.

How can we assess the biocompatibility of coating materials?

Were a person intending to develop a medical device then there really wouldn’t be any shortcuts available. The FDA makes a few exceptions for situations such as when a device is entirely composed of substances which have a long track record of safety; however, many of the tests are still necessary and the costs can be phenomenal. Fortunately, the majority of projects being undertaken aren’t intended for medical treatment. As such, they aren’t under the scrutiny of the FDA. For example Nickel is known carcinogen and toxin and yet is commonly found in cheap jewelry. Because implants developed by grinders aren’t medical in nature they simply aren’t regulated. This is no way means we shouldn’t pay close attention to what we use but it does allow us to utilize a shortcut.

A number of companies produce adhesives and coatings which have undergone extensive biocompatibility testing and if requested they will often send out their results. It’s important to analyze these results as a substance may be totally biocompatible for certain functions.. for example in the production of a catheter meant to stay in a vessel for only 3 days, but inappropriate for another use such as in a permanently implanted subcutaneous device. The biggest problem with this type of approach is that the more expensive and time intensive tests are seldom performed. These documents will most often include some form of assay for cytotoxicity, acute systemic effect, and implantation testing but seldom looks into mutagenicity, carcinogenicity, and reproductive effect.

Despite these shortcomings, choosing a coating by analysis of testing performed by a manufacturer is the best approach available short of having a full biocompatibility test series performed. Understanding as to the implications of this information is best acquired through examples. Each suggested biocompatible material will be analyzed according to their biocompatibility tests.

Surface Integrity Testing

No matter how amazingly biocompatible a coating is, if applied poorly an implant will fail. There are many reasons why this may occur. Many resin coatings contain solvents which prevent crosslinking from occurring until the product is used. While the final product may be biocompatible the solvent most certainly aren’t. It’s important to read the documentation and become familiar with a coating before use in order to guarantee that any such solvents are removed during the curing process.  Some substances such as silicone are inert in the body but have a degree of permeability to water. This slow movement of liquid can allow leaching of enclosed toxins eventually leading to rejection. Some resins bond poorly to each other and thus if applied in multiple layers can leave channel through which fluids can move once again allowing leaching to occur. Coatings applied by sputtering such as titanium nitride have their own issues such as pinholing. Although appearing perfect under a microscope, sputtered coatings can have pinhole gaps which over time result in failure.

There are number of tests which can be used. For example, the paint industry uses crosshatch testing in which a number of cuts are made on a surface and an adhesive used to pull away any paint which hasn’t adhered properly. The reason a test like this won’t work for us is that it destroys the coating during the test and does nothing to expose microscopic flaws. We need a test that can be performed which leaves the device capable of being implanted and demonstrates even the smallest of flaws.

The best type of test for this is immersion testing. I’ll describe two methods of immersion testing: nickel exposure and saline. Nickel exposure testing is excellent for general testing of a coating although isn’t appropriate for final testing of a device as nothing implanted should really contain any significant quantities of nickel. Saline testing is great for testing a final implant for coating failures as it involves nothing toxic, although it’s ability to demonstrate failure is somewhat limited. I’ve worked with a number of grinders who attempted other methods of testing coatings and I always emphasize doing a saline test as well. A run of titanium nitride coated magnets looked especially promising at one point. They survived a number of weeks immersed in sulfuric acid without fail. Despite this, they all failed after a period immersed in saline. This was a anti-intuitive result. There may be some reason for this failure that has nothing to do with pinholing but saline is the type of environment these implants will be exposed to. If they can’t survive a little salt water then they shouldn’t be implanted.

A nickel exposure test requires a solution of dimethylglyoxime ethanolammoniacal. It’s pretty simple to mix up. All that’s required is household ammonia and dimethylglyoxime. Dimethylglyoxime is inexpensive and easy to purchase. In theory, the solution only has to be 1% to be effective; however, exact measurement isn’t too important here. A half teaspoon in 400ml of ammonia will more than suffice. Test the solution by applying a few drop to a piece of nickel. The nickel should turn a pretty pink color. On it’s own dimethylglyoxime isn’t particularly toxic; however, after exposing it to nickel compounds form which can be strongly carcinogenic. Be very cautious not to contact the solution and dispose of it safely.

A piece of nickel is then enclosed in the coating material to be tested and cured appropriately. Place the coated nickel in a container with the dimethylglyoxime ethanolammoniacal solution and seal it. Changes in color are usually immediate if the coating has any flaws. If the nickel doesn’t change color keep the vessel sealed for at least one month. This will show if the solution is able to permeate through the coating over time. If a coating does fail a test, it’s prudent to repeat the test a number of times to see if the failure could be due to a flaw created during application. I was surprised to find one substance only failed if the coating was applied in multiple steps but if applied all at once it was fine.

Saline testing is an even simpler process. Buy or make a 0.9 Nacl solution and add a few drops of dish soap. Dish soap is amphipathic meaning that it bonds to both hydrophobic and hydrophilic substances. The point of it in this case is to prevent surface tension. Because flaws in coatings such as pinholing are so small, surface tension can prevent the saline from entering the flaw. Dish soap prevents this. After coating your device immerse it in this saline solution and observe over time for changes. A good period is 3 months. If the material within is totally unaffected by water this test may not provide any relevant results. However, it’s useful in the case of electronic devices which may fail in function or in the case of magnets which degrade and crack apart if the coating is flawed.

A final test of coatings is direct observation via microscopy. Observing a coating under a microscope can often reveal cracks and flaws that would otherwise be missed. A traditional light microscope won’t work for this as it requires light passing through a specimen. The correct type of microscope to use is a stereo or dissecting scope. A stereo microscope provides lower magnification but works well on solid top lit objects such as magnets. This process is made even easier with use of a digital camera eyepiece. Pictures can be taken of the unit before and after testing in order to compare and determine if a surface feature is actually a flaw that’s worsening. Each suggested biocompatible coating will be accompanied by immersion testing results and microscopy.

Conclusion:

It was perspex, a material developed for the aerospace industry, which saved Mouse Cleavers vision and began research into biocompatible materials. Advancement in aerospace technology was never as simple as designing bigger engines or more aerodynamic shapes. The capabilities of new crafts were entirely dependant on making thinner, lighter, and yet stronger materials. The advancing of implant technology is equally tied to material science. Methods have emerged allowing the integration of implants with bone. Metals stents are routinely placed in major blood vessels preventing heart attacks and fully functional artificial lungs are nearing approval for clinical testing. The mechanical functions of these devices are simple. It’s advancements in the materials they are made of that make their use a possibility, and unfortunately it’s what keeps such capabilities out of the hands of biohackers.

This work hopes to educate biohackers on biocompatibility and expand the number of materials to be considered for use in devices. I hope this work also functions as a starting place as to where we go next. Many of these biocompatibility tests could be performed in an adequately funded personal lab. A person willing to perform such tests for others in the community would be of incredible value. Furthermore, research and experimentation into new materials or perhaps into DIY versions of existing but patented processes could allow for far more advanced grinds then we are currently working on. I’ll keep compiling and advancing this list of materials as I’m able and I ask that the community help in this endeavor as well.

https://www.nelsonlabs.com/Test/Cytotoxicity

http://www.pacificbiolabs.com/bio_methods.asp#cytotoxicity

https://www.ncbi.nlm.nih.gov/books/NBK144065/

https://www.dojindo.com/Protocol/Cell_Proliferation_Protocol_Colorimetric.pdf

Implantation –

LU:  ISO 10993-6 “Biological evaluation of medical devices – Part 6: Tests for local effects after implantation,”

  1. Genotoxicity Genotoxicity testing may be waived if chemical characterization of device extracts and literature references i

Genotoxicity –

Carcinogenicity –

As described in Attachment A, FDA recommends that carcinogenicity potential be 39 evaluated for devices with permanent contact (i.e., greater than 30 day exposure). This includes devices in contact with breached or compromised surfaces (i.e., wound healing), as well as externally communicating and implanted devices. If novel materials (i.e., not previously used in a legally US-marketed device) are used to manufacture devices in contact with breached or compromised surfaces, externall

H.Reproductive and Developmental Toxicity FDA recommends that reproductive and developmental toxicity be assessed to evaluate the potential effects of medical devices, materials and/or their extracts on reproductive function, embryonic development (teratogenicity), and prenatal and early postnatal development as described in ISO 10993-1.

When additional device or device component chemical information is needed, the following descriptive information should be provided: 1. The identity of the chemical by common name, chemical name, Chemical Abstract Services (CAS) number, and trade name. 2. If known, 44 60 the composition, formula and formula weight, structural information, and manufacturing and purity information for the chemical, such as a detailed description of the manufacturing process (including the substances used, the amounts used in the synthesis, and reaction conditions), specifications for the chemical, analysis of multiple batches of the chemical, and identification of major impurities. 3. The specific amount of each chemical in the formulation by weight percent of the applicable device component and total amount (e.g., µg) in the device. If this information is not available (e.g., from a material supplier), it would be acceptable to use a worst-case estimation approach for the risk assessment. For example, one might assume 100% of the material (e.g., resin pellet) used in the final device formulation is the chemical of concern (i.e., any chemical components of the supplied material). 4. The identity of any other devices marketed in the US (by device name, manufacturer, and submission number) where the chemical entity with direct or indirect tissue contact has been previously used, if known, and comparative information on the composition and amount(s) used. This information is generally available only for components made by the same manufacturer.

Section 3: Biocompatible Materials

Anderson, J. M., Rodriguez, A., & Chang, D. T. (2008). Foreign body reaction to biomaterials. Seminars in Immunology, 20(2), 86-100. doi:10.1016/j.smim.2007.11.004