This review is aimed at investigating the biocompatibility of synthetic materials used in joint replacements. Investigation essentially involves testing and hence, it is as important to test the biomaterial in-vivo along with in-vitro studies. Various testing procedures exist to ensure that biomaterials are accepted by the body. Also, the cellular response to foreign materials in particulate dimensions and bulk is considered. The body's reaction, when it responds to a biomaterial, is described in detail as a sequence of events.
Joint replacements use biomaterials to substitute the natural materials of the body, anatomically & functionally. Total joint replacement prosthesis differs from implants, since it consists essentially of articulating surfaces. Biomaterial testing is generally done in the bulk form to assess its biocompatibility however, this is not sufficient. Implants produce wear particles, especially from the bearing surfaces, articulating surfaces and other anchoring devices (bone cement), which causes unwanted tissue reactions (1). Thus, testing for biocompatibility, when the material is in its particulate form is also important to determine how exactly it will behave when implanted.
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Synthetic materials have been around since a long time, and when used within the human body, it is known as ''biomaterials''. A biomaterial can be defined as ''a non-viable material used in a medical device and intended to interact with a biological system'' (2). Biocompatibility can be defined as ''ability of a material to perform, with appropriate host response, in a specific application'' (3). Biocompatibility is different from inertness, where there is absence of any response. Earlier, this was the property desired in any implantable material, but in present times its usage is no longer advised.
In total joint replacements, biomaterials are commonly used to replace the hip, knee etc. Its selection is generally limited to those materials which can withstand cyclic loading applications. Metals are widely used in manufacturing implants & are known to provide appropriate material properties such as; strength, resilience to fractures, durability, formability, resistance to corrosion and most importantly, biocompatibility (4).
I BIOCOMPATIBILITY INVESTIGATION OF SYNTHETIC TJR MATERIALS
There is a need to determine if biomaterials used are biocompatible and will function appropriately in an in-vivo environment. In-vitro testing provides helps to predict how a material might behave within the body. Biocompatibility of both bulk biomaterial and particulates can be assessed.
To evaluate biocompatibility of materials, cultured cells have been used for many decades. In-vitro testing minimizes the use of animal-testing to an extent. It is time saving, inexpensive and determines if any further testing is required. The experimental duration, variability of results and statistical analysis should be considered. The below four conditions are essential in standardizing & enabling the repeatability of data (5).
Cytotoxicity is ability to induce toxic effects at the cellular level (alterations, enzymatic inhibition etc). The potency of the chemical is indicated by the number of cells that have been affected.
Dosage is of two types i.e. delivered and exposed dosage. Delivered dosage is the amount of dose actually absorbed by a cell and exposed dosage is the amount applied to the system. In-vitro testing observes delivered dosage and in-vivo testing, exposed dosage.
Sensitive test systems are essential where dosage amounts can be reduced to a minimum in animal models and increased to a maximum in cell culture models, thus providing two extremes.
Solubility of implanted medical devices is important in testing its compatibility. Polymers, ceramics & metals are generally water insoluble (maximum of 1 part in 10,000 parts of water). However, other components may be soluble.
Three cell culture assays are used for in-vitro biocompatibility. The result is changes in the morphology of cells. The material to be tested is exposed differently to the three assays. Standardization is carried out & interpretation of the assay result is done on the basis of number of cells. There are 4 categories: slight, moderate, severe and absence of any response (9, 10).
Direct contact test involves the preparation of a near-confluent monolayer of L-929 mammalian fibroblast cells in a culture plate. The culture medium is then replaced with a fresh one. The test sample and control specimens are placed in individual cultures and incubated for 24 hrs at 37Â±1Â°C in a humidified incubator. The culture medium and specimens are removed and cell fixing and staining with a cytochemical stain is done. Toxicity can be observed by the absence of stained and the result is that damaged cells exist at the interface between dead and live cells.
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Agar diffusion test uses a similar cell culture and incubation. The replaced culture is 2% agar. Neutral red vital stain is used in the agar mixture, allowing immediate visualization of live cells. Dead or injured cells do not absorb any colour, whereas live cells do. Here, constancy in the thickness of the agar is important.
In the elution test, by using 0.9% NaCl, the material is extracted. Specified surface area per ml of extractant is used. The extract is placed on a similar culture, but incubated for 48 hrs to evaluate its toxicity. Dead cells may be distinguished using stains.
In-vivo tests are selected to simulate end-use applications. Devices and materials can be categorized by the area, where they contact the body (skin, mucosa, bone, dentin, blood) duration of contact (limited, prolonged and permanent). The in-vivo tests (11) for biocompatibility are as follows:
Sensitization reactions are systemic immune responses & can result from exposure to even tiny amounts of potential leachable substances released from devices/biomaterials. Tests for these determine the potential for contact sensitization to irritants. Symptoms are topical.
Irritation is a local tissue inflammation response. The most irritating leachable chemicals are discovered by in-vitro cytotoxicity testing. Biomaterial extracts reveal the irritating effects of chemicals.
Intracutaneous reactivity tests determine the localised reaction of tissue to material extracts injected intracutaneously. The physiological relevance of the test-material preparation and the extract solution is important.
Systemic Toxicity testing is done to assess the harmful effects in vivo, on tissues of interest situated away from the implantation site. It evaluates the effect of released particles from medical devices & also includes pyrogenicity testing.
Acute toxicity occurs within 24 hrs of administering a single or multiple doses of a sample. Subacute toxicity is the testing of adverse effects after dose administration for duration of 14-28 days.
Subchronic toxicity is the determination of adverse effects over a period of 90 days.
Genotoxicity testing is conducted to check if the chemical composition of a material shows risk of genotoxicity such as, DNA destruction, gene mutations etc or in-vitro tests show genotoxicity. In-vivo testing includes: micronucleus test, chromosomal analysis, rodent dominant lethal test etc.
Implantation tests evaluate local pathological effects induced by a material, on tissue structure & function. Histological examination is done to observe the microscopic changes and characterize the biological response i.e. distribution of inflammatory cells, degeneration in tissue morphology, presence of necrosis etc.
Hemocompatibility testing determines the effect materials/devices have on blood components. These tests replicate in-vivo geometry, flow dynamics, contact conditions on device exposure. Specie specific differences in blood reactions may make it harder to predict the performance of the material in humans.
Carcinogenicity testing checks if the material/device is potential of causing cancer, if used over a long duration. However, tumors associated with device implantation occur rarely. Both chronic toxicity and carcinogenicity can be studied in a single experimentation. Some metal ions used in orthopaedics i.e. chromium, cobalt etc are carcinogenic.
Reproductive and developmental toxicity testing examines if devices/materials have an effect on the reproductive function of the mother, embryonic development, prenatal and postnatal development of the infant.
Biodegradation testing studies the effects that a biodegradable substance and the products of degradation can have on the tissue response. It focuses on the kinetics of biodegradation, nature & origin of degradation products and leachables present around the tissue site.
Immunological testing determines if the device/material has an adverse effect on the immune system or any other system, as a result of its dysfunction. Artificial materials are generally not immunotoxic. Adverse effects are observed when immunity needed by the host to defend against an infection or tissue damage, is compromised.
II CELLULAR RESPONSE AND SEQUENCE OF EVENTS
Any biomaterial when introduced into the body will initially be considered a threat. The body's initial reaction is to eliminate it. Biomaterials generally have three types of chemical reactivity i.e. being chemically inert and non-degradable, being biodegradable and being non-degradable but possessing some level of chemical reactivity. When any biomaterial is first introduced into the human body, there will be some inflammatory response by the tissue as it undergoes trauma.
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Cellular host response to injury is generally expressed in the form of inflammation, wound healing and foreign-body reactions (6, 11). The sequence of reactions that occur when a foreign material is introduced into the body can be given as follows:
Implantation of any material in-vivo causes injury (11). Changes in blood components & cellular events characterize the inflammatory response. Due to the effect of injury/biomaterial, cells produce chemical factors that mediate vascular and cellular responses of inflammation. The injury response depends upon the degree of injury, blood-material interactions, tissue necrosis etc.
Blood material interaction is linked to the inflammatory response (11). When a biomaterial is implanted, initial inflammatory response happens due to the injury to the vasculature. Blood clotting, thrombosis can occur as blood is involved. Here, fluid, proteins and blood cells move into the injured area (exudation process).
As vascularised tissue undergoes injury, immediately provisional matrix formation (11) is observed at the implantation site (blood-protein deposition on the surface of the biomaterial).
This matrix consists of inflammatory products, activated platelets, inflammatory cells and endothelial cells. Components released from this matrix system initiate resolution, reorganization and repair processes. This matrix provides structural and biochemical components to the wound healing process.
The acute inflammatory response (11, 12) is marked by a stereotypical chain of events. Immediate body responses to any unidentified material are swelling, redness and pain. Since tissues undergo trauma, mediators of inflammation (histamines) are released from cells within the tissue and nerve endings release neuropeptides which aim to stimulate the smooth muscle cells (in vessel wall), thus narrowing the blood vessel. Change of flow direction of blood cells i.e. from an axial, centralized flow to turbulent flow occurs. They collide with each other, forming aggregates. Gaps present between endothelial cells (vessel wall) increase permitting the seepage of plasma from the blood vessel into surrounding tissue. New mediators against inflammation (kinins) are released by the plasma proteins. Complement proteins present in the plasma get activated on contact with foreign substances and produce smaller activation products (complement activation products).
Neutrophils (phagocytes) are attracted by these products. Blood neutrophils are attracted into the tissue from blood vessels. After squeezing through the vessel wall, they reach the site where the concentration of complement activation products is maximum. If a foreign body is present in the vicinity, the neutrophils adhere to it and get activated. But, they would be unable to destroy it. They release toxic oxygen radicals, which cause their death. While dying, they continue to release other mediators of inflammation that attract mononuclear phagocytes (leukotrines, prostaglandins).
Monocytes are attracted from the vessel into the tissue, and gather where these attractants are being produced. In tissues, the monocytes get converted into bigger, more active macrophages, and ingest the dead neutrophils. When macrophages adhere to the foreign body, it produces pro-inflammatory cytokines (IL-1, IL-6, TNF-Î±), and employ more macrophages to the tissue site.
The healing response (11, 13) is initiated by monocytes and macrophages. If the biomaterial is inert, the reaction is dependent upon its size. If the size is within phagocytosible range, then the macrophages will take it to the phagosomes of the cell, but will be unable to consume it. Thus, by releasing cytokines, more macrophages are attracted as a distress event. Granulation tissue is thus formed, as a result of the proliferation of macrophages at the implant site. This tissue is specialized and marks healing.
Foreign Body Reaction stage (11, 13) is comprised of foreign body giant cells (FBGC's) and granulation tissue components i.e. capillaries, macrophages etc. It is seen that macrophages adhere and form a wall around the material, fusing their cell membranes to form FBGC's. These giant-cells are multi-nucleated and the biomaterial is located within its vacuole. Scar formation occurs in healing when the original architecture cannot be entirely reconstructed by the parenchymal cells. If the implant surface is smooth, the reaction is observed to be a layer of macrophages, 1-2 cells thick and if the surface is rough, the reaction is composed of FBGC's & macrophages at the surface. If the material is biocompatible with the body, the reaction composition is controlled by its surface properties. Reaction may persist for as long as the implant exists within the body. Fibrous encapsulation (11, 13) is the body's most typical response to a biomaterial. New extracellular matrix is laid down by fibroblasts, which is not as organized as normal tissue. A layer of fibrous tissue forms a capsule around the implant. As this tissue stabilizes, the macrophages move out of it. The vascular capsule blocks the implant from interacting with the surrounding environment. Capsule thickness depends upon the reactivity of the material. If the material is inert and motion-less, the capsule is thin. If the implant releases chemicals or ions, some macrophages stay within the capsule to keep the situation under control, and the capsule is thicker.
DISCUSSION & CONCLUSION:
Material biocompatibility basically depends upon the end-application of the device. New biomaterials will require new testing standards and new procedures for evaluation. In the previous years, tissue engineering has been given more importance in the development of candidate biomaterials, which has encouraged active approach rather than passive. In-vivo assessment plays an important role in R&D of any device and ensuring safety of its usage. Development of in-vivo tests (for biocompatibility) will be significantly contributed by the usage of tissue-engineered devices. Also, it must be remembered that extrinsic materials may cause various effects on the cells, when placed in-vivo. Thus, inappropriate, inadequate or adverse reactions may occur in the cell in the presence of any external factor. This should be taken into perspective when designing biocompatibility programs. Lastly, animal tissue usage, during the device development, poses a major challenge to the in-vivo assessment. An alternative must be developed to address this issue urgently (11).