PART IV

Biocompatibility of Biomaterials
Protein structure, interaction of proteins with synthetic
material and characterization of cell material
interactions

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 Biocompatibility Assessment
• In vitro Assays
• Cell morphology and circularity
• Cell density
• Cell migration
• MTT Assay
• Dye inclusion assay
• DNA laddering
• 3D-spheroids
• Ex-vivo Assays
• In vivo Assays- animal models

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 BIOCOMPATIBILITY ASSESSMENT
Reference: Jacqueline M. Morais,1 Fotios Papadimitrakopoulos,2 and Diane J. Burgess1,3, Biomaterials/Tissue Interactions: Possible Solutions to Overcome Foreign Body Response, The AAPS Journal, Vol. 12, No. 2, June 2010

• Biocompatibility studies on an implantable device require complex in vitro and in vivo experiments to test the
local and systemic effects of the material on the host

• Evaluation of biocompatibility and biofunctionality of materials is performed mainly by methods based on the
assessment of cytotoxicity, mutagenesis and/or carcinogenesis, and cell function

• In fact, the extent of nonspecific protein absorption (biofouling) can be used to evaluate the degree of
biocompatibility of the implant

• In vitro cell culture tests are often used to screen the tissue compatibility of implantable devices.

• The objective of cell culture techniques in biocompatibility assessments is (a) to simulate the biological response
of the body environment on which the biomaterial is placed and (b) predict its functional performance.

• Three primary cell culture assays are used to evaluate biocompatibility: (a) direct contact, (b) agar diffusion, and
(c) elution (also known as extract dilution). (US Pharmacopeia and American Society for Testing and Materials,
the British Standards Institute, and the International Standards Organization (ISO)).

• These are morphological assays, meaning that the outcome is measured by observation of changes in cell3
morphology.
• To standardize the methods and compare the results of these assays, it is important to carefully control: (a) the
number of cells, (b) the growth phase of the cells (period of frequent cell replication), (c) the cell type, (d) the
duration of exposure, (e) the test sample size (e.g., geometry, density, shape, thickness), and (f) the surface area
of test sample.

• It is worth mentioning that cell lines that have been developed for growth in vitro have been preferred to
primary cells that are freshly harvest from live organisms because these cell lines have improved reproducibility
and reduced variability among laboratories

• L-929 mouse fibroblast cell line has been extensively used for testing biomaterials. Initially, L-929 cells were
selected because they are easy to maintain in culture and produce results that have a high correlation with
specific animal bioassays.

• In addition, fibroblasts are appropriate for these assays because they are one of the early cells to populate a
healing wound and are often the major cell in the tissues that adhere to implanted devices.

• Cell lines from other tissues or species may also be used.

• Ultimately, the selection of a cell line should be based upon the type of assay, the investigator’s experience, and
measurement endpoints (viability, enzymatic activity, species receptors, etc.).

• These in vitro tests include positive and negative control materials, extraction conditions, and choice of cell lines
and cell media.
• Important aspects of the test procedures include tests on extracts and on direct and indirect contents.

• Such tests are a sensitive, reliable, convenient, and reproducible screening method

• Relevant to the ove

• rall in vivo assessment of tissue compatibility of a biomaterial or device is knowledge of the chemical composition
of the materials and the conditions of tissue exposure (including nature, degree, frequency, and duration of
exposure).
 Proteins: Why It is Important to Study Surface Interaction with Biomaterials
• In as short a time as can be measured after implantation in a living system (<1 sec), proteins are already deposited on
biomaterial surfaces.

• In seconds to minutes, a monolayer of as a tightly bound adsorbate, adsorbs to most surfaces.

• The protein adsorption events complete before cellular interactions at the surface.

• Therefore, cells see primarily a protein layer, rather than the actual surface of the biomaterial.

• Since cells respond specifically to proteins, this interfacial protein film may be the event that controls subsequent
bioreaction to implants.

• Protein adsorption is also of concern for biosensors, immunoassays, marine fouling, a host of other phenomena.

• After proteins adsorb, cells arrive at an implant surface propelled by diffusive, convective, or active (locomotion) mech-
layer on a solid substrate.

• After cells arrive and attach at surfaces, they may multiply to tissues. Synthetic materials can interact with or disrupt
living tissues. The organization of tissues must be understood to appreciate the response to synthetic materials implanted
in those tissues.
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Cell interaction with an adsorbed protein layer on a solid substrate
FIG. 1. The cell is shown as a circular space with a
bilayer membrane in which the adhesion receptor protein
molecules (the slingshot-shaped objects) are partly
embedded. The proteins in the extracellular fluid are
represented by squares and triangles. The receptor
proteins recognize and cause the cell to adhere to only the
surface-bound form of one protein, the one represented by
a solid circle. The bulk phase of this same adhesion
protein is represented by a triangle, indicating that the
solution and solid-phase forms of this same protein have a
different biological activity. The figure is schematic and
not to scale.

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 Amino Acids: Structures in Side Chain
In addition, phenomena at the air-water interface (e.g., interfacial coagulation and foaming), at the oil-water
interface (e.g., the "receptor" proteins located in cell mem branes that serve many important signalling functions), and at the
solid—liquid interface in nonbiomaterial settings (e.g., marine fouling, bacterial adhesion, and cell growth on surfaces in
culture), are all strongly influenced by the behavior of proteins at interfaces,
 Amino Acids: Nature of Side Chain

http://www.cryst.bbk.ac.uk/PPS2/course/section2/SideChains/primary1.html 11
pKa is the negative base-10 logarithm of
the acid dissociation constant (Ka) of
a solution.
pKa = -log10Ka
The lower the pKa value, the stronger
the acid. For example, the pKa of acetic acid
is 4.8, while the pKa of lactic acid is 3.8.
Using the pKa values, one can see lactic acid
is a stronger acid than acetic acid.

HA ⇆ A- + H+

Ka = [A-][H+]/[HA]

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Secondary Structure of Protein

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15
ADSORPTION BEHAVIOR Of PROTEINS AT SOLID—LIQUID INTERFACES

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 Density of Adsorbed State
The folded protein structures have densities of about 1.4 g/cm3. In comparison with water's density of 1.0, or the density of most
synthetic polymers of about 1.1, this basic fact about proteins reflects their tightly folded structure.

It is therefore convenient to think of proteins in a physicochemical sense as quite compact, externally charged wax droplets in
water, in which the interior hydrophobic core is analogous to wax and the surface amino acids are the charged species.

Thus, for example, typical values for adsorption of proteins are in the range of 1 microgram/cm2, a plateau or monolayer
value typically reached at higher bulk concentrations. To convert this two dimensional value into an equivalent volumetric
concentration unit, we can assume a monolayer of a typical protein for which a 100-A (or 10-6 cm) diameter is a good
approximation. Then, a 1-cm2 area containing 1 ug corresponds to a local protein concentration of 1/10-6 = 106 ug/cm3 or 1
g/cm3.

Given that the density of a pure protein is 1.4 g/cm3, this layer is indeed tightly packed.

Furthermore, the 1 g/cm3 is equivalent to 1000 mg/cm3, which is far higher than the bulk protein concentration
of solutions from which such adsorbates form (typically 1 mg/cm3).

Thus, the surface phase is often 1000 times more concentrated than the bulk phase, corresponding to high local
concentration indicative of the existence of a very different state of matter and thus truly deserving of a special term,
namely, the adsorbed state.
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Figure 2.1.2.12 The Vroman effect: (A) fibrinogen adsorption to Biomer and glass from various concentrations of blood
plasma; (B) time course of fibrinogen adsorption to glass and poly(ethyl methacrylate) (PEMA) from undiluted plasma.
(Reprinted with permission from Slack, S.M., Horbett, T.A., 1995. The Vroman effect: a critical review. In: T.A. Horbett,
J. Brash (Eds.), Proteins at Interfaces II: Fundamentals and Applications. ACS Symposium Series (vol. 602, pp. 112–
128). Washington, DC: American Chemical Society. Copyright © 1995 American Chemical Society.)
 Competitive Surface Binding

Mixture of Protein

1. Individual concentration in bulk

2. Surface activity

e.g. Albumin and Fibrinogen may

differ on a single surface
Protein adsorption on solid surface is
largely irreversible
Which makes it different from gas
molecule adsorption

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