ch.1 lec 4

• Types of biocompatibility tests

  • Cytotoxicity
  • Systemic Toxicity (acute Toxicity)
  • Sub-acute Toxicity (Sub-chronic Toxicity)
  • Chronic Toxicity
  • Sensitization
  • Irritation
  • Mutagenicity
  • Genotoxicity
  • Carcinogenicity
  • Implantation
  • Hemocompatibility
  • Reproductive & Developmental toxicity
  • Intracutaneous reactivity
  • Pyrogenicity
  • Biodegradation
• Cytotoxicity tests


  • Performed in vitro on isolated cells
  • Measure cell death or damage, cell number or growth before and after exposure to material
  • Measure material's effect on cell growth, cell depth, and other effects
  • If material is solid, measure density of cells at different distances and describe zone of inhibited cell growth

  
• Cytotoxicity test procedure


  1. Cells plated in well of cell culture dish

  2. Material placed in test system

  3. If material not cytotoxic, cells remain attached and grow

  4. If material cytotoxic, cells may stop growing, exhibit cytopathic features, or detach
• Ways to extract biomaterials for cytotoxicity testing

  • Direct contact with surface
  • Indirect contact via diffusion layer like agar
  • Elution or extract test
• Direct contact with surface

  • Rapidity, high sensitivity
  • Simple technique and less experience
  • Exposing cells to extract of biomaterials
  • Difficulty obtaining reproducible number of cells on test material
• Indirect contact via diffusion layer like agar

  • Components from test material allowed to diffuse through agar or agarose to cells
  • Cytotoxicity of diffusible components determined by staining cells with viability dye and measuring zone of dead cells
• Elution or extract test

  • Determine toxic doses and changes in cell growth or proliferation compared to non-treated cells over 24-78 hours
  • Quantitative evaluations preferred over qualitative
• MTT assay


  • Preferred quantitative cytotoxicity test
  • Limitations: unable to detect cellular damage in early stages, relies on detecting cell death only
• Membrane permeability cytotoxicity test


  • Identifies cells that are alive or dead under microscope
  • Based on loss of membrane permeability being equivalent to cell death
• Systemic toxicity


  Potential toxicity in living body caused by leachables (chemicals) released from implant materials or devices at sites distant from implant site
• Categories of systemic toxicity

  • Acute Systemic Toxicity
  • Sub-Acute Toxicity (Sub-Chronic Toxicity)
  • Chronic Toxicity
• Mutagenicity


  Increase in rate of mutation of individual genes or chromosomal mutation
• Epigenetic mutagens


  Do not alter DNA themselves, but support tumor growth by altering cell's biochemistry, immune system, acting as hormones, or other mechanisms
• Genotoxicity


  Determines whether material induces gene mutations, changes in chromosome structure or number, or other DNA or gene toxicities
• Types of genotoxicity tests

  • In vitro gene mutation (AMES test)
  • In vivo chromosomal damage (Micronucleus test)
• Carcinogenicity


  Measures tumorigenic potential during significant portion of test animal's life cycle
• Carcinogenic compounds can cause malignant tumors, increase frequency or severity of tumor occurrence, or speed up onset of tumor manifestation
• Carcinogenicity potential evaluated through implantation on rodents using non-carcinogenic polyethylene as control
• Reproductive and developmental toxicity

  Evaluates genotoxicity, gene mutations, chromosomal abnormalities, DNA effects, endocrine toxicity, and effects on reproductive function, embryonic development, and fertility
• Carcinogenic material


  Polyethylene used as control
• Carcinogenicity test


  1. Extended time period of 1 year to complete

  2. If test is negative, may still induce carcinogenic response once induced

  3. Occurs over multiple stages and is characterized by complex biological interactions influenced by factors such as genetics, age, dietary habits, environmental exposures, and hormonal imbalances
• Carcinogenicity test


  • Breast Prosthesis
• Powder produces almost no tumours
• Reproductive and developmental Toxicity Test

  Evaluates genotoxicity, gene mutations, chromosomal abnormalities, DNA effects, endocrine toxicity, which can affect reproductive functions and embryonic development (teratogenicity), and fertility
• Reproductive and developmental Toxicity Test


  • Intrauterine devices, energy depositing devices and resorbable devices
• New experiments have been done using transgenic animals whose DNA is replaced by human DNA
• Reproductive and developmental Toxicity Test is similar to carcinogenicity and mutagenicity test
• Hemocompatibility


  Compatibility of implanted materials with mixing blood while a medical device can maintain contact with blood without any adverse reactions
• Hemocompatibility Test


  Measures the potential for hemolysis, thrombogenicity, and the effect of materials and their surfaces on blood clotting

  
• Thrombosis, coagulation, platelets, hematology, and immunology tests are recommended to be performed for any form of interaction

  
• Hemocompatibility Test Procedure


  Materials or their extracts are incubated with red blood cells, isolated from (rabbits, mice, or rats) for three hours with alternating shaking to keep samples mixed and in contact with blood. The amount of hemoglobin released into the supernatant from the cells is determined spectrophotometrically and reported as percent hemolysis with respect to negative controls. The number of adherent platelets may be determined per unit area after exposure to whole blood.
• ISO 10993-4 specifies specific examinations based on the blood contact group of the device to achieve hemocompatibility

  
• All materials are incompatible with blood, as they can cause hemolysis by disrupting blood cells; activating coagulation pathways, resulting in thrombogenicity; or triggering the complement system
• Intracutaneous Reactivity Test


  Measures or assesses the localized reaction of tissue to medical device / material extracts. It is applicable to devices that can break the skin and contact circulating blood and other tissues.
• Intracutaneous Reactivity Test


  • Devices having access to the blood path or where material extractables are hydrophobic
• Pyrogenicity Test


  Assesses a material's potential to induce fever-producing reactions in test animals. Fever-inducing is limited to devices causing biological effects. Pyrogenicity tests are also conducted to ensure the safe level of endotoxins in the finished / sterilized product.
• Biodegradation Test


  Determines the process of degradation, biotransformation, and elimination of degradation products. It is essential when the device contains a biodegradable component, is intended for implantation exceeding 30 days, or when a complete analysis of the material composition indicates the possibility of releasing toxic substances upon contact with the body.
• Replicating biodegradation mechanisms in vitro is recommended to determine degradation rates and the release of potentially harmful materials for the performance evaluation. In some cases, in vivo assessments may be necessary to evaluate the biodegradation of a material.
• The need for biodegradation tests may be avoided if the potential sources of degradation are present in expected quantities and their generation rate is similar to what has been demonstrated to be sustainable in previous clinical applications.
• ISO 10993-9 provides a widely used framework for conducting biodegradation assessments, while ISO 10993-13, ISO 10993-14, and ISO 10993-15 offer specific in vitro procedures for evaluating biodegradation in polymers, ceramics, and metals, respectively.