BIOE 4710 / 5710BIOE 4710 / 5710
Soft and Hard Tissue BiomechanicsSoft and Hard Tissue Biomechanics
Dr. Edward Nyman, Jr., Ph.D.Research Assistant ProfessorEngineering Center for Orthopaedic Research Excellence (ECORE)Departments of Bioengineering & Orthopaedic SurgeryThe University of Toledo, NI [email protected]
(BoneBone)HistologyHistology
Overview
I. Basic Principles of Histology
II. Histology Methods Related to Bone Imaging
III. Staining and Dying
IV. Bone Samples / Applications
V. Quantification of Bone Remodeling
Methods of Bone and Tissue Imaging
Microscopy X-Ray Ultrasound SPECT & Gamma Camera CT NMR & MRI PET
Microscopy
Processing bone tissue requires highly specialized techniques, unique equipment, and technical expertise.
Histological sections may be obtained on fresh frozen bones, decalcified and paraffin-embedded bones, or on non-decalcified, plastic-embedded bones.
Processing and Embedding
Fixation Washing Dehydration Infiltration Embedment
Sample Preparation: Fixation
Stabilize structures (micro-anatomical arrangement of tissue elements)
Retain constituents (stabilize cellular inclusions)
Negate autolysis and decay (putrefication) Improve contrast (enhance the refractive
index of tissues) Harden tissue (render cell constituents
insoluble and resistant to subsequent processes)
Types of Fixation
Mechanical (Physical) fixation Heating or freezing
Chemical fixation Immersion Perfusion Vapor Phase-partition
Processing
Biological issues have a high concentration of water
Embedding media are usually nonpolar
Water must be removed via dehydration with alcohol or acetone
Replaced with either wax or plastic (resin)
Microtomy
Microtomy (small cuts) takes practice and experience to produce quality microscope preparations.
4 critical requirements for sample success Practice and experience A sharp microtome knife A proper microtome Well prepared tissue
https://www.youtube.com/watch?v=O0D2fW1a39Y
Types of Microtomes
Handheld Cambridge rocking microtome Rotary microtomes Base-sledge and sliding microtomes Freezing microtomes Cryostats Vibrotomes
Rotary Microtomes
Most widely used type of microtome Stationary knife Sample placed on a “ball-joint” Cutting stroke simply down/up motion
Produces flat sections
Staining and Dying
Principle of creating differential contrast
Can be used to detect specific tissue constituents
Can be used to determine cell viability
Can be used to determine relative pH
Common Laboratory Stains
StainStain Common useCommon use NucleusNucleus CytoplasmCytoplasmRed Blood Red Blood CellsCells(RBC)(RBC)
Collagen Collagen FibersFibers
Specifically stainsSpecifically stains
HematoxylinHematoxylinGeneral staining General staining when paired with when paired with EosinEosin
BlueBlue N/AN/A N/AN/A N/AN/ANucleic acids - Blue Nucleic acids - Blue blue eER (ergastoplasm) - Blueblue eER (ergastoplasm) - Blue
EosinEosinGeneral staining General staining when paired with when paired with HaematoxylinHaematoxylin
N/AN/A PinkPink Orange/RedOrange/Red PinkPinkElastic fibers - pink, Elastic fibers - pink, reticular fibers - pinkreticular fibers - pink
Toluidine blue General stainingGeneral staining BlueBlue BlueBlue BlueBlue BlueBlue Mast cells granules – purpleMast cells granules – purple
Gomori's Gomori's trichrome staintrichrome stain
Connective and Connective and muscle tissuemuscle tissue
Gray/BlueGray/Blue RedRed RedRed GreenGreen Muscle Fibers – RedMuscle Fibers – Red
Masson's Masson's trichrome staintrichrome stain
Connective tissueConnective tissue BlackBlack Red/PinkRed/Pink RedRed Blue/GreenBlue/Green Cartilage - Blue/green, Muscle fibers – RedCartilage - Blue/green, Muscle fibers – Red
Mallory's trichrome stain
Connective tissueConnective tissue RedRed Pale RedPale Red OrangeOrange Deep BlueDeep BlueKeratin - Orange, Keratin - Orange, Cartilage - Blue, Bone matrix - Deep Blue, Cartilage - Blue, Bone matrix - Deep Blue, Muscle fibers - RedMuscle fibers - Red
Table sourced from Michael H. Ross, Wojciech Pawlina, (2006). Histology: A Text and Atlas. Hagerstwon, MD: Lippincott Williams & Wilkins. ISBN 0-7817-5056-3
Unstained SectionUnstained Section
Von Kossa StainVon Kossa Stain
Iron StainIron Stain TetracyclineTetracycline
Goldner’s Trichrome StainGoldner’s Trichrome Stain
Microscopes
Brightfield Light Microscopy (optical) Phase contrast Polarized Differential contrast Reflection
Fluorescence Microscopy Confocal Microscopy Multiphoton Transmission Electron Microscopy Scanning Electron Microscopy Scanning Transmission Electron Microscopy Atomic Force & Scanning Tunneling
Optical or light microscopy involves passing visible light transmitted through, or reflected from, the sample through a single or multiple lenses to allow a magnified view of the sample.
The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally.
The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage and support, makes up the basic light microscope.
Optical Microscopy
Resolution versus Magnification
Fluorescence Microscopy
Fluorescent molecule = fluorochrome absorbs light of specific wavelength
When excited by absorption, fluorochrome emits light of longer wavelength.
Every fluorochrome has an absorption and emission spectra.
Electron Microscopy
Developed in 1930s Uses electron beams instead of visible light. Because of the much shorter wavelength of the electron beam than
of light, resolution is far greater.
TYPES: Transmission electron microscopy (TEM) is principally quite similar
to the compound light microscope, by sending an electron beam through a very thin slice of the specimen.
The resolution limit is ~0.03 nanometer (~2.5 nm practically for biological samples)
Scanning electron microscopy (SEM) visualizes details on the surfaces of cells and particles and gives a very nice 3D view.
The magnification is in the lower range than that of the transmission electron microscope (~ 30 nm for bio samples).
Transmission Electron Transmission Electron Microscopy (TEM)Microscopy (TEM)
• Very thin sections
• Beam of electrons • (=0.05)
• Electromagnetic lenses
• Stain with metals• Stain: electron dense = dark• Unstained: light
Scanning Electron Microscopy (SEM)Scanning Electron Microscopy (SEM)
• Surface structure
• Sectioning not required
• Metal coating of specimen• Electron scattering• Primary electrons• Secondary electrons• Detector
http://www.chm.bris.ac.uk/pt/diamond/jamespthesis/chapter2_files/image002.gif
Scanning Electron Microscopy (SEM) Black Ant
Human red blood cells
Human stem cells
Neurons CNS
Neuron growing on astroglia
House Fly
Scanning Probe Microscopes
Employ a sharp tip that is scanned over a sample surface to measure some property
Two Types: Scanning Tunneling Microscope (STM)
Atomic Force Microscope (AFM)
Scanning Probe Microscopes
STM (1980s): can image individual atoms very sharp metallic wire brought to within a few
angstroms of a surface and scanned over that surface tunneling current is distance dependent - can actually
be used to manipulate atoms “one at a time”
AFM (1985): uses tiny diamond-tip glued onto a thin piece of gold
that acts like a spring actually in contact with the sample - can manipulate a
sample
Bone Histology Samples
optical SEM
Lamellar Bone
Picture courtesy Gwen Childs, PhD.
Haversian System
Osteon with central Haversian canal containing
Cells Vessels Nerves
Volkmann’s canal Connects osteons
osteon
Haversian canal
osteocyte
Volkmann’s canal
Picture courtesy Gwen Childs, PhD.
Osteoblasts
Picture courtesy Gwen Childs, Ph.D.
Osteocytes
Picture courtesy Gwen Childs, Ph.D.
Osteoclasts
Picture courtesy Gwen Childs, Ph.D.
Cartilage
HyalineFibrocartilage
Elastic
Bone Remodeling: Quantification
Histomorphometry: measurement and analysis of bone structure and bone remodeling. Usually performed on cancellous bone from transiliac biopsies.
Isotropic (randomly oriented) nature of trabeculae in iliac bone is assumed.
2D measurements (of area) converted to 3D (volume) measurements. This is a fundamental stereologic principle used in histomorphometry.
Using computer graphics, multiple fields of known medullary area/volume are analyzed. Bone tissue volume (TV) is the sum of field volumes
Trabeculae within each field are graphically outlined and trabecular bone volume (Tb.V), and total trabecular bone surface (Tb.S) are calculated.
Bone Remodeling: Quantification
Trabecular bone volume, (Tb.V) = relative volume of total cancellous bone measured (TV) (expressed as %) that is occupied by trabeculae.
Tb.V is about 20% in women and 22% in men.
Tb.V is related to cancellous bone mass and declines with age and with bone loss
Note: Tb.V is also commonly referred to as Bone Volume / Total Volume (BV/TV)
Bone Remodeling: Quantification
Bone Remodeling: Quantification
Bone Formation Rates (BFR/BV and BFR/BS): calculated rates at which cancellous bone surface and bone volume are being replaced annually.
Derived from estimates of: Mineral Appositional Rate (MAR), (interlabel distance (4/π)
(labeling interval) in µm/Day x 365. Relative Mineralizing Surface (MS), Bone Surfaces (BS) or
Bone Volume (BV)
BFR = MAR(MS/BS)
BFR= MAR(MS/BV)
Bone Remodeling: Quantification
Bone formation rates are expressed as:
BFR/BV in (mm³/mm³/yr)
BFR/BS in (mm³/mm²/yr)
NORMAL MEAN VALUESNORMAL MEAN VALUES
Parameter Female mean Male meanWall Thickness (W.Th) 49.8 µm
Mineral Apposition Rate (MAR) 0.88 µm/d 0.89 µm/d
Bone formation RateSurface (BFR/BS) (mm³/mm²/yr) 0.019 0.009
Volume (BFR/BV) (mm³/mm³/yr) 0.250 0.131
Mineralization Lag Time (M.Lt) 21.1 d 27.6 d
Activation Frequency (Ac.f) 0.42 y
Bone Remodeling: Quantification
Review
I. Basic Principles of Histology
II. Histology Methods Related to Bone Imaging
III. Staining and Dying
IV. Bone Samples / Applications
V. Quantification of Bone Remodeling