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 5040edward.nyman@utoledo.edu

(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