Lecture Topic: Introduction to Biomaterials

Biomaterial is a synthetic material used to replace part of a living system or to function in intimate contact with living tissue.

Biological material is a material such as bone matrix or tooth enamel, produced by a biological system.

Artificial material is that simply are in contact with the skin, such as hearing aids and wearable artificial limbs, are not biomaterials since the skin acts as a barrier with the external world.

Involves the acceptance of an artificial implant by the surrounding tissues and by the body as a whole. Biocompatible materials do not irritate the surrounding structures, do not provoke an inflammatory response, do not incite allergic reactions and do not cause cancer.

Is defined as average number or repeating units or mers per polymer chain.

Materials obtained by combining two or more materials at a macroscale taking advantage of salient features of each material. An example is (high strength ) fiber-reinforced epoxy resin.

Stress is defined as a force per unit area, usually expressed in N/m² (Pascal).

Tensile stress is generated in response to loads that pull an object apart.

Compressive stresses: tend to squeeze it together.

Shear stress: resist loads that deform or separate by sliding layers of molecules past each other on one or more planes.

Requirements for biomaterials: can be divided into 2 categories:

	Bulk properties
	Interface or surface properties

Major requirements are:

	Functional feasibility (appropriate mechanical and physical prop): function of organ and tissue must be guaranteed, even for long term use.
	Appropriate aging property in the body (stability or degradability ): biological environment must not impair the functioning of the biomaterial.
	Biocompatibility (cell, tissues and blood): biomaterial must not disturb the biological system or give minimum histotoxicity, non-carcinogenicity).
	Sterilizability: sterilization process must not impair the function of the material
	Shelf stability.

	Replacement of diseased or damaged part artificial hip joint, kidney dialysis m/c
	Assist in healing sutures, bone plates and screws
	Improve function cardiac pace maker, contact lense
	Correct cosmetic problem chin augmentation
	Aid to treatment catheters, drains

Table1: Materials for use in the body

The success of a biomaterial in the body depends on factors:

- Materials: properties- optical prop.--> is important when used in eyes, skin, teeth

   - mechanical prop-> include strength, stiffness and                                                  fatigue.

                                                                                ---> are important for handling and functioning.

                                            - physical prop--> include density and porosity

                                            - design, and biocompatibility

- Surgeon (techniques)

- Patient (health, condition and activities)

If we can assign a numerical value ‘f to the probability of failure of and implant , then the reliability 'r' is as follows.

Reliability (r) = 1 – f

If, as is usually the case, there are multiple model of failure, the total reliability r_t is given by the product of the individual reliabilities.

r₁ = (1 – f₁), r₂ = (1 – f₂), etc

r_t = r₁r₂r₃…..r_n

Example: consider the case of a total joint replacement in which infection is most likely soon after surgery, while loosening and implant fracture become progressively more important as the time goes on.

Structure of solid properties:

The structure of solid : atomic or molecular level (0.1 – 1 nm)

Nanoscale or ultrastructural level (1nm - 1m m)

Microstructural level (1m m – 1 mm)

Macrostructural level (> 1 mm)

Atomic bonding: could be ionic or metallic or covalent bonding or hydrogen bond.

	metallic bonds (the electrons are loosely held to the ion)
	ionic bond (formed by exchanging electrons between metallic and non- metallic atoms.
	covalent bonds are formed when atoms share the valence electrons to satisfy/fit their partially filled their electronic orbitals.

Intermolecular force: dipole-dipole interactions

Van der waals forces (forces between the molecules of a non-polar compound:- such compound can solidify)

Table2: Strength of Different Chemical Bonds as Reflected in their Heat of vaporization

Covalent and ionic bonds are primary bonds which are strong bonds comparing with secondary bonds.

Secondary bonds are hydrogen bond and van der Waals bonds.

Polymers:

- synthetic polymer

biopolymer (from biological/natural products eg; chitin/chitosan, collagen, protein)

Polymers have very long-chain molecules that are formed by covalent bonding along backbone chain. The long chain are held together either by secondary bonding forces such as van der Waals and hydrogen bonds or by primary covalent bonding forces through cross-links between chains.

Degree of polymerization (defined as average number of mers or repeating units per molecule/chain). The relationship between molecular weight and degree of polymerization can be expressed as:

Molecular Weight = DP X Molecular wt of repeating unit

Crystalinity:

Polymers are never perfectly crystalline, but contain disordered regions and crystallites of varying size. The process is normally incomplete because crystallization takes place when the polymer is viscous liquid. In this state, the chains are highly entangled, and as sufficient time must be allowed for the chains to diffuse into the 3D order required for crystalline formation, the crystalline perfection of the sample is affected by the thermal history. Thus rapid cooling from the melt usually prevents the development of significant crystallinity.

Factors affecting crystallinity and T_m are symmetry of polymer chain, intermolecular bonding, branching and mass of polymer.

Shows a number of important property comparisons, indicates how the branching plays a significant role in determining the properties and hence the end uses of the three polymers.

Crystallinity only affects the mechanical response in the temperature range T_g to T_m and below T_g the effect on the modulus is small.

The modulus of a semi-crystalline polymer is directly proportional to the degree of crystallinity, and remains independent of temperature if the amount of crystalline order remains unchanged.

Apparently, mechanical resistance would depend on the linking energy between the material’s structural elements. Nevertheless, the investigations performed showed that the real resistance of a crystalline material is sometimes 100 times lower than the theoretical one, calculated by means of interatomic or interionic linking energies. This is due to the structural defects of real crystals and to presence of a greater number of defects and fissures to be met in every material.

chemical structure and distribution of molecular weight

molecule flexibility

stress-strain behavior

mechanical failure

- Tg, Tm

coefficient of expansion, thermal conductivity (esp. dental materials-temp. change from foods and drinks)

Dry Heat:

Disadvantage: change shape and properties of rubber and plastic.

Autoclave-steam:

Killing effects on microorganisms by denaturing protein. Efficiency depend on moisture content, heat content, penetration. Penetration depends on the removal of air from the chamber and the load.

Chemical Sterilization:

Killing effect is by alkylation.

ETO:

Advantages: Low temperature process.

Can be used for heat sensitive materials except rubber which absorb toxic residues. ETO will not kill microbes hidden and protect in a dried organic matter.

Disadvantages: toxicity, carcinogenicity and productive hazards, require aeration time.

Efficiency: depend on concentration of gas, temperature 45-65⁰C, relative humidity, gas contact time.

Packaging: PE films, papers, cloths. But cannot use nylon film, Aluminium foil, glass etc.

Formaldehyde:

Low temperature process. It reacts with DNA during replication. Sporicidal action is accelerated by a combination of saturated steam at 70-75⁰C.

Aqueous 2% alkaline gluteraldehyde is a liquid disinfectant of choice for fiber endospores. Alkaline solution (pH 4.5 – 8.5) slowly decrease in activity as polymerization occurs. Immerse the products in gluteraldehyde for 10 hours for sterilization.

Radiation:

Gamma ( g ) ray, X ray, accelerated electron beam,

Direct action:

Initial physical chemical reaction takes place on the molecules of important cell constituents eg: DNA (rather than H₂O). Direct ionization or excitation event in the biological molecule will cause a change that leads to loss of biological activity in the molecule by bond disruption or radical formation which inactivate the target molecule.

Indirect action:

Results from the action of the radiolytic products of water on the target of biological importance.

This results in an ion pair or ionization of the target molecule.

High energy radiation may affect mechanical or biological performance of polymer and biomaterials. Generally high energy radiation of polymers leads to compatibility degradation and/or cross-linking depend on the chemical structure. Crystallinity is known to be one important factor affecting the blood compatibility of polymer.

Surface Properties:

Implants should not have any molecular contamination on the surface and several cleaning procedures can be employed.

Sterile packaging may affect surface properties of an implant by deposition of wear products. If it is allowed to rub against the packaging material. Also volatile components from packaging materials may be vaporized and deposited on the implant.

Effect of polymer structure of biodegradation:

Biopolymers such as protein, cellulose (natural macromolecules) generally degraded in biological systems by hydrolysis followed by oxidation.

Synthetic biodegradable polymers contain hydrolytic linkage along the polymers are susceptible to biodegradation by microbes and hydrolytic enzymes.

Many protelytic enzymes specially catalyze the hydrolysis of peptide linkage adjacent to substituents eg: Benzyle hydroxy, carboxy, methyle, phenyle groups have been prepared in anticipation that the introduction of substituents could increase biodegradability.

Hydrophilic and hydrophobic characters.

Since most of the enzymes-catalyzed reactions occur eg: media, synthetic polymers quantity affect their biodegradability. Eg: A polymer containing both hydrophilic and hydrophobic segments seems to have higher biodegradability than those polymers containing either hydrophilic or hydrophobic structure only.

Eg: Polymer derived from C₆ and C₈ alkaline diols were more biodegradable than C₂ and C₄ alkaline diols and C₁₀ and C₁₂ diols.

For a synthetic polymer to be degraded by enzyme catalysis the polymer chain must be flexible enough to fit into the active site of the enzyme.

Ex: the flexible aliphatic polyesters are readily degraded by biological systems, the more rigid aromatic polyester is generally considered to be bioinert.

Morphology:

Protein-irregularity results in protein chain is likely to crystalize. (more biodegradable)

Synthetic polymers contain short chain repeating units. Regularity enhances crystallization making the hydrolyzable groups inaccessible to enzymes.

Microorganisms produce extracellular enzymes responsible for the biodegradation of the amorphous area prior to degradation of the crystalline spherelittes. Size shape and no of crystalline have pronounced effect on the polymer chain mobility in the amorphous region. Thus control the degradation rate.

Ex: polymer with hydrolysable backbone.

Polyesters: aliphatic polyesters are perhaps be most readily biodegraded synthetis polymer known.

Simplest polymers(a -hydroxy acid), poly(glycolic acid) has been successfully used as a biodegradable structure.

Degradation rate at temperature above Tg is faster than that temperature below Tg.

The poly(hydroxy acid) polymers derived from d-hydroxy acidsare degraded more easily than those polymers of g , d , e hydroxy analogues.

Polyamide (Nylone)

Again the incorporation of methyle, hydroxy and benzyle groups into the polyamide chains increase biodegradability of the polyamides.

Ex: of polymer with C backbones.

Vinyle polymers: Generally are not susceptible to hydrolysis. If degrade, it require oxidation.

Poly (vinyle alcohol) and Poly (vinyle acetate): PVA is the most readily biodegradable of vinyle polymers.

Polyacrylate:

Ex: Poly (2-hydroxy ethyle methacrylate) P(HEMA) is generally crosslinked with a small amont of ethylenedimethacrylate. It swells in water to from a hydrogel and has been widely used in biomedical areas because of its good biocompatibility.

Lecture Topic: Biological Responses

Standards for Biomaterials:

ISO (International Organization for Standardization)

ISO 10993-1 (1992): Biological evaluation of medical device,

Part 1: Guidance on selection of test

ISO 10993-5 (1992): Part 5: Test for cytotoxicity of biocompatibility of medical devices used in dentistry.

ISO 7405 (1997): Dentistry- Preclinical evaluation of biocompatibility of medical devices used in dentistry.

ASTM (American Society for Testing and Materials)

ASTM: F748-91 (1991): Standard practice for selecting generic biological test methods and devices.

ASTM: F619-79 (1991): Standard practice for extraction of medical plastics

ASTM: F895-84 : Standard test method for agar diffusion cell culture screening for cytotoxicity

ASTM: 813-83 : Standard practice for direct contact cell culture evaluationof materials for medical devices.

BSI (British Standard Institution)

BS 5736: part10(1998): Evaluation of medical devices for biological hazards.

Part 10: Method of test for cytotoxicity to cell in culture of extract from medical devices.

USP (United State Pharmacopeia)

USP 23 (1995): [ 87] : Biological reactivity test, in vitro, pp 1967

(includes: agar diffusion test, direct contact test and elution test)

From all standards we can conclude that in vitro biological test for biomedical materials are:

	direct contact test
	indirect contact test: - agar diffusion (solid sample) or filter diffusion (liquid sample)
	extract / elution test

Evaluation can be end point measurement or kinetic study deopending on the required information. Toxicity and biocompatibility of materials to cells can be determined the effects on cell growth/death and on the differentiation of cells.

	cell growth/death: - morphology
	number of cells --> - cell count
	biomechanical assay

         (protein content, dye staining, Cr release, DNA synthesis, LDH release)

	metabolism of cells-> MTT assay
	cell differentiation - keratinocytes-> keratins
	cell function - osteoblasts -> alkaline phosphatase(ALP) synthesis

- hepatocytes -> cytochrome P450, urea synthesis

Cell cycle:

	M phase = mitosis (the contents of the nucleus condense to from visible chromosomes, which through an elaborately orchestrated series of movements are pulled apart into 2 equal sets) + Cytokines ( the cell itself splits into 2 daughter cells, each receiveing one of the 2 sets of chromosomes).
	G1 phase = the biosynthetic activities of the cells resume at a high rate. (proceed very slowly during mitosis)
	S phase = begins when DNA synthesis starts and ends when DNA contents of nucleus has doubled and the chromosomes have replicated.
	G2 phase = separates the end of DNA synthesis from the beginning of the next M phase.

Note:

Aneupolid: Describe a cell having a chromosome number slightly different from a multiple of the haploid number. One or more chromosomes may present in a greater or lesser number that the rest. The chromosomes may or may not show rearrangements.

Diploid: The state of a cell in which chromosome (except sex chromosome) are paired and structurally identical with those of the pieces from which they are derived. Each species has a characteristic number of chromosome in the diploid state e.g. human = 46, mouse = 40, cat = 38, monkey = 42 etc.)

Haploid: Describes the state of germ cells after meiosis when each chromosome is presented once.

Cells used: - primary cells (have a diploid chromosome pattern )

- establish cell lines (have aneuploid chromosome pattern)

Summary: There is no universally right answer to what cell types should be used for an in vitro experiment. More than one cell types should be used. It must be remembered that biomaterial evaluation in vitro always is a relative measurement which in itself has little relevance to direct clinical interpretations.

Evaluation Assays:

qualitative assays ------> - microscopic assay (need experience/training) looking cell morphology and other in details using light microscope, SEM, TEM, etc.

quantitative assay: ------> number of cells

    chromium release

   determination of protein  content

   determination of DNA content

   fluorimetric assay

   leakage of cytosolic enzyme (LDH                   leakage)

   metabolism assay (MTT assay)

DNA synthesis Rate:

Determined by incubating the cell with radio-isotopically labeled nucleotide eg: ³H - thymidine and measuring the amount of radioactivity incorporated per 10⁶ cells or per milligram protein over a set period of time.

Protein Synthesis Rate:

Incubate the cells with radioisotopically labeled amino acids eg: ³H - leusine or ³⁵S - methionine measuring the radioactivity incorporated per 10⁶ cells or per milligram proteins over a period of time.

Plating Efficiency (PE);

PE = no of colonies formed X 100

            No of cells seeded

Cells are plated out as single cell suspension at low cell density (d - 50 cells/cm²), they will grow as discrete colonies. It should be measured at a time where the maximum no of cells has attached but before mitosis starts.

Growth Fraction:

Useful in accessing the growth stimulatory effect of an exogenous agent (eg: growth factor) on a quiescent cell population, that is in determining the conditions that cause cells to re-enter the cell cycle.

Method: Exposure of the culture to ³H - thymine for a length of the entire cell cycle (usually 24 hours) and then determine the percentage labeled cells.

Labeling Index (LI):

The percentage of labeled cells, determined by autoradiography is known as the LI. LI is commonly used to quantify the response of a cell population to a mitotic stimulus.

Log phase stage cells more actively absorb the labeled chemicals.

Mitotic Index:

Is the fraction or percentage of cells in mitosis determined by counting mitoses in stained cultures a proportion of the whole population.

Protein Content:

Lowry Assay: depend on the presence of aromatic amino acids (tyrosine and phenyle alanine ) residues and will under estimates if the frequency of these amino acids is low as in nuclear histone proteins.

Bradford Assay: Give more sensitive assay than Lowry.

Principal: Coomasie blue undergoes a spectral change on binding to protein in acetic acid.

References:

	Culture of Animal Cell: A manual of basic technique, Freshney, R. Iam, 3rd Ed, Wiley-Wiss Inc. Ny-1994.
	Molecular Biology of the Cell 2nd Ed. Bruce Alberts, Tannis Bray, Julian Levis, Martin Ralf, Keith, Garland Publishing Inc. Ny 1989
	Cell Biology, Organelle structure and Function, David E Gadava, Jhon and Barlet Publishers London 1993.

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