BME385 Study Guide
Lab 2: Fabrication of Materials
- Biomaterial: material intended to interface with biological systems to evaluate, treat,
and augment or replace any tissue, organ or function of the body
o Biomaterial considerations
 Biocompatibility: Ability to perform with an appropriate host response in
a specific application
 Biodegradable/remodeling
 Architecture
 Mechanical properties
 Cell phenotype/function
 Fabrication/processing
 Cost & Manufacturing
- Synthetic materials
o Biodegradable polymers: poly(a-hydroxy)esters, PGA, PLA, PLGA
o Polycaprolactones, polycarbonates, polyanhydrides, polyfumarates,
polyorthoesters
o Ceramics/glasses: HA, B-TCP, bioactive glass
o Advantages: Biodegradable, processing, pore architecture, mechanical
properties
o Disadvantages: Inflammation, degradation rates, loss of cell function
- Natural materials
o Proteins: collagen, fibrin, elastin
o Polysaccharides: alginate, chitosan, GAGs
o Advantages: Cell attachment, natural function, remodeling, less inflammation
o Disadvantages: Mechanical properties, stability, processing
- Natural biopolymers
o Collagen (Type I)
 Accounts for 30% of all protein in the body
 Found in every major tissue requiring strength and flexibility
 Collagen fibers: E=500 MPA, Yield Stress=50 MPA, Max
Strain=10%
 15 Types of Collagen – Type I is most commonly used
 Abundant (>90% of all fibrous protein)
 Unique physical and biological properties
 Convenient and abundant sources in tendon, skin, bone, fascia makes it
easy to isolate
 Structure
o Triple helix: 3 left stranded helixes wound into a righthanded helix
o Individual chains contain 3 repeating peptides in subunit
 Gly – X – Y where Glycine, Proline, Hydroxyproline
o ~1050 amino acid residues, -300nm and d=1.5nm
o Collagen: Advantages
 Mild immunoreactivity due to primary amino acid sequence and helical
structure
 Individual collagen molecules will spontaneously polymerize in vitro into
strong fibers – forming large organized structures
 Can be processed to increase cross-linkage (form covalent bonds
between polymer chains)
 Increase biodegradation time
 Decrease collagen capacity to absorb water
 Increase tensile strength
 Free amines on collagen can also be used to link active agents
 Good cell attachment
 Collagen has adhesive peptide domains (DGEA, asp-gly-glu-ala)
 Integrin binding domains
 Cell attach via a1b1, a2b1, a3b1 integrins
 Degrades by specific enzymes known as matrix metalloproteinases
 Collagens are easily processed into porous sponges by freeze drying or
hydrated gels
 Cross-linking (Glutaraldehyde, EDC, etc)
 Material somewhat stiffer
 Tensile strength increased
 Degradation time increased
 Reduce the rate of tissue ingrowth
 Change biological activity?
 Co-polymers
 Collagen-chitosan co-polymers
 Collagen-HA co-polymers
o Collagen: Disadvantages
 Poor mechanical properties
o Gelatin
 Prepared by thermal denaturation of collagen
 Biodegradable, biocompatible, and non-immunogenic
 Enhance cellular adhesion, migration, proliferation, and differentiation
 Chitosan-gelatin scaffolds have been used for cartilage, bone, and skin
due to their biocompatibility and good cell adhesion
 When heated, gelatin changes to solution form, when cooled, solidifies
into gel form
 Transformation is reversible
o Chitosan
 Polysaccharide, 2nd most abundant natural polymer
 N-deacetylated derivative of chitin
 Abundant in natural resources: shells from insects and crustacea
 Polysaccharides having structural similarity to cellulose and GAGs
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Cationic in solution
Biocompatible, biodegradable, hydrophilic
100,000 < MW < 1,000,000
Insoluble above pH 7, soluble below pH 6.3
Used to immobilize GAGs
Chitosan: Advantages
 Breakdown in body by lysosome activity
 Can easily be modified to adjust mechanical properties
o Films and fibers
o Not antigenic and well-tolerated
 Applications: cell encapsulation, cell culture, cartilage
regeneration
How to fabricate Chitosan Film Membrane?
o Make 1% chitosan solution + 1% acetic acid
o Coat petri dishes with sigmacote
o Pour into petri dish mold
o Allow to dry
o Peel the film
How to fabricate porous chitosan scaffolds?
o Make 1.5% chitosan solution + 1% or 0.2M acetic acid
o Coat petri dishes with sigmacote
o Pour into petri dish molds
o Lyophilize (freeze drying technique)
Polydimethylsiloxane (PDMS) Elastomer
o Most widely used silicon based organic polymer
o Inert, non-toxic, non-flammable
o Applications: contact lenses and medical devices to elastomers, caulking,
lubricating oils, heat resistant tiles, and biomedical applications
 Patterning of cells and proteins, cell-cell or cell-ECM interactions
o Fabrication
 Two parts: silicone elastomer base and curing agent
 10:1 ratio base: curing agent
Lab 4: FT-IR Characterization of Biomaterials
- FT-IR: Fourier Transform Infrared Spectroscopy
o Chemically specific analysis technique
o Identify chemical compounds and substituent groups
o (1) Can identify unknown materials (2) quality or consistency of a sample (3)
number of components in mixture
- Most common spectrophotometers are used in UV and visible spectrum
o Infrared light occurs between 0.7 and 500um between visible and microwave
regions
- How does a spectrometer work?
o IR spectroscopy is an absorption technique where IR radiation is passed through
a sample
 Some IR radiation is absorbed by the sample and some is passed through
(transmitted), and is then detected
 Resulting spectrum represents molecular absorption and
transmission
o Relies on Michelson Interferometer
 Relies on interference of infrared waves
 At the interferometer, light strikes a beam splitter which passes
50% of light to one mirror and 50% of light to the second mirror
o Mirror 1 is oscillating and mirror 2 is stationary
o The beam splitter recombines the light which is guided
toward the sample
o Light is absorbed by the samples and passes to the
detector
o Interpretation of IR spectrum provides information about functional groups
present in a molecule
 How does IR work?
 Molecules are always in motion, as the organic compounds
absorbed infrared radiation, different types of bonds absorbed
infrared radiation at different frequencies cause an increase in
amplitude of bond vibration
 Plot of wavelength (frequency) versus absorption
o IR bands
 Described by location, intensity, and shape
 Location – reported as a wavenumber value of the absorption minimum
 Intensity – describes the % transmittance, the size of the peak is relative
to other peaks
 Shape – describes width of the band, broad, sharp, narrow etc.
 PDMS
 Si-O-Si: 1015
 Si-(CH3)2: 790
 Si-C: 851
 CH3 symmetric: 1259
 CH3 asymmetric: 1408
 Methyl CH: 2959
Lab 3: Protein Assay
- Dye-binding assay in which a color change of a dye occurs in response to various
concentrations of protein
o Used for total protein quantification
o Protein dyes to Coomassie dye (binding dye)
 Shift in absorbance maximum from 465 nm to 595 nm
 Color change from brown to blue
o 1. Protein Albumin has NH3+ groups in side chains of amino acids of proteins
o 2. Binding Dye Coomassie Blue is negatively charged and reddish/brown
o 3. Creates a Protein Dye Complex that is blue
o 4. Read by spectrophotometer
- Albumin: body’s predominant serum-binding protein
o Transports a variety of substances including fatty acids, bilirubin, metals, ions,
hormones, and exogenous drugs => molecular “taxi”
o Albumin: 75-80% of normal plasma colloid osmotic pressure and 50% protein
content
o Serum albumin in mostly abundant blood plasma protein produced in the liver,
hepatic cells
- Binding dye: Coomassie
- Spectrophotometer: instrument for measuring the absorbance or transmittance of a
sample as a function of the wavelength of electromagnetic radiation
o Consists of two instruments (Spectrometer + Photometer)
 Spectrometer: producing light of any selected color (wavelength)
 Photometer: for measuring the intensity of light
o 1. Light source shines through sample
o 2. Sample absorbs light
o 3. Detector detects how much light the sample has absorbed
o 4. Detector then converts how much light the sample absorbed into a number
o 5. Numbers are plotted or transmitted to computer
- Wavelength ranges
o Most common spectrophotometers are used in the UV and visible regions of the
spectrum
 Visible: 400-700nm
 FT-IR: used in characterization and identification of organic compounds
- Beer’s law: when light of a specific wavelength passes through a solution there is usually
a quantitative relationship between the solute concentration and intensity of the light
transmitted
o A=abc
o A: Absorbance, a= molar absorptivity (L/cm mole) b=pathlength (1/cm)
c=concentration (mol/L)
o Amount of light is directly related to concentration of chemical in solution
- Comparison to a standard curve provides a relative measurement of protein
concentration
Lab 5: Flow Cytometry
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Flow cytometry: measuring properties of cells while in fluid stream (Flow – cells in
motion, cyto – cells, metry – measure)
o Cytometry: localization of antigen possible, poor enumeration of cell subtypes,
limiting number of simultaneous measurements
o Flow cytometry: cannot tell where antigen is, can analyze cells in a short time
frame, can look at numerous parameters at once
Uses of flow cytometry
o Immunophenotyping, DNA cell cycle, membrane potential, ion flux, cell viability,
intracellular protein staining, ph changes, cell tracking and proliferation,
chromatin structure, total protein
o Analyze many properties of cells in short amount of time
How does it work?
o 1. Fluidics: Cells in suspension flow single file past
o 2. Interrogation: A focused laser where they scatter light and emit fluorescence
that is filtered and collected
o 3. Electronics: Converted to a digitized value that are stored in a file
o 4. Interpretation: which can then be read by a specialized software
o When light from a laser interrogates a cell, that cell scatters light in all different
directions
o The scattered light can travel from the interrogation point down a path to the
detector
Light scattering
o Light that is scattered in the forward direction (along the same axis the laser is
traveling) is detected in the FSC (Forward Scatter Channels) = FALS=LALS
 Intensity of this signal has been attributed to cell size, refractive index
(membrane permeability)
o Laster light that is scattered at 90 degrees to the axis of the laser path is
detected in the Side Scatter Channels (SSC=RALS)
 Intensity of this signal is proportional to the amount of cytosolic structure
in the cell (granules, cell inclusions, etc.)
o Since FSC ~ size and SSC ~ internal structure, a measurement between them can
allow for differentiation of cell types in a heterogeneous cell population
Fluorescence channels
o As the laser interrogates the cell, fluorochromes on/in the cell (intrinsic or
extrinsic) may absorb some of the light and become excited
o As those fluorochromes leave their excited state, they released energy in the
form of a photon with a specific wavelength, longer than the excitation
wavelength
 Those photons pass through the collection lens and are split and steered
down specific channels with the use of filters
Spectra of common fluorochromes
o PE – Texas Red
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o PI – Propidium iodide
o Ethidium Homodimer
o PE- Phycoerythrin
o FITC – Fluorescein isothiocyanate) or GFP – Green fluorescent protein
o Cis Parinaric acid
Detectors
o Photodiodes: used for strong signals, when saturation is a potential problem (FSC
detector)
o Photomultiplier tubes (PMT): more sensitive than a photodiode, a PMT is used
for detecting small amounts of fluorescence emitted from fluorochromes
Interpretation
o Once the values for each parameter are in a list mode file, specialized software
can graphically represent
 Either in 1,2, or 3-dimensional format
 Common include CellQuest, Flowjo,
WinMDI, FCS express, flowing
software
Types of plots
o Single color histogram: Fluorescence
intensity (FI) vs count
o Two color dot plot: FI of parameter 1 vs FI of
parameter 2
o Two color contour plot: FI or P1 vs FI of P2.
Concentric rings form around populations.
The denser the population, the closer the
rings are together
o Two color density plot: FI of P1 vs FI of P2.
Areas of higher density will have different
color than other areas
Gating: used to isolate a subset of cells on a plot,
allows the ability to look at parameters specific to
only that subset
Embryonic stem cells
o Pluripotent, self-renewal capability, unlimited proliferation, capable of different
germ lineages, reliable cells source
o Ectoderm: neural cells, glial cells
o Mesoderm: cardiac muscle, endothelium, hematopoietic
o Endoderm: liver, pancreas, lung
o Oct4: embryonic stem cell marker
Hematopoietic stem cell – a cell isolated from blood or bone marrow that can renew
itself
o Differentiate into all the blood cell types
 Myeloid: monocytes and macrophages, neutrophils, basophils,
eosinophils, dendritic cells, platelets, erythrocytes
 Lymphoid: T cells and B cells
o Can mobilize out of bone marrow into circulating blood
o Found in bone marrow and placental tissue/umbilical cord blood