02/15/2023

STUDY OF HARD TISSUES

1
 DECALCIFICATION

02/15/2023
2
CONTENTS

02/15/2023
 Introduction

 Basic structure of tooth and bone

 Definition and Ideal requisites of decalcifying agents

 Procedure for decalcification

 Different methods employed for decalcification

 Factors influencing rate of decalcification

3
 Tests for end point of decalcification

02/15/2023
 Treatment following decalcification

 Further processing

 Embedding

 Staining

 Recent advances

 Clinical implications
4
 Conclusion
 02/15/2023
INTRODUCTION

5
STRUCTURE OF TOOTH

02/15/2023
Inorganic % Organic %
Enamel 96% 4%
Dentin 65% 35%
Cementum 45-50% 50-55% 6
Bone 67% 33%
STRUCTURE OF BONE
ARTICULAR CARTILAGE

02/15/2023
CANCELLOUS TISSUE

1. Periosteum
2. Compact bone
3. Medullary canal
4. Marrow
5. Artery
TRANSVERSE
SECTION 6. Nerve
7. Vein 7
02/15/2023
8
Decalcification:

To remove calcium or calcium compounds from bones or

02/15/2023
teeth. (Merriam Webster dictionary)

Purpose : is the removal of calcium ions from the bone

through histological process thereby making the bone flexible and
easy for pathological investigation.
9
CRITERIA OF A GOOD DECALCIFYING
AGENT

02/15/2023
Complete removal of inorganic ions

Minimal damage to cells and tissues

Non-impairment of subsequent staining

Reasonable speed 10
The choice of decalcifier is influenced by four independent factors -

02/15/2023
 Urgency of case
 Degree of mineralization
 Extent of investigation
 Staining techniques required

11
 PROCEDURE

 Extracted teeth- in decalcifying solution (20:1)

02/15/2023
 Maintained at 55ºC in an incubator

 Decalcifying solutions to be changed every three hourly

 Store in 10% formalin overnight. (process continued)

 Till complete decalcification is obtained(radiographic

method)

12
  specimens - in running tap water for 24 hrs

02/15/2023
Dehydration in an increasing ethanol series, cleared with
chloroform, soaked in liquid paraffin and embedded in solid
paraffin.

 Sectioning in a microtome(4μm thickness)

 Stain with hematoxylin and eosin.

 Mount using DPX

13
METHODS OF DECALCIFICATION

02/15/2023
Acid hydrolysis

Organic chelation

Electrolysis

14
ACID DECALCIFIERS

 Acids may be used as simple solutions, in a mixture with other

02/15/2023
reagents especially fixatives, or in buffered solutions.
 Reagents like buffer salts, chromic acid, formalin or ethanol are
added to the mixtures to reduce the swelling effects on tissues
caused by acids.

Acid decalcifiers
Strong Weak
(inorganic) (organic)
15
STRONG INORGANIC ACIDS

02/15/2023
Eg NITRIC ACID 5-10%

HYDROCHLORIC ACID

Decalcifies rapidly, cause tissue swelling and damages the

tissue stainability if used longer than 24 hours.

Old nitric acid is particularly damaging and should be replaced

with fresh stock.

Strong acids tend to be more damaging to tissue antigens.

16
Mainly used for needle and small biopsy specimens
CLAYDEN SOLUTION (5-10% NITRIC
ACID)

02/15/2023
 Gives the quickest results
 Used when sections are to be made in short period
 0.5mm thickness -1/2 to 2 hrs
 Thicker sections take 6 hrs

Disadvantage:

Discolors the tissue yellow

The addition of 0.1 % urea avoids this color formation .
17
10% HYDROCHLORIC ACID

02/15/2023
 when sections are to be made quickly
 Decalcification time depends on size of the specimen

 2 to 6 hrs for complete decalcification

18
Aqueous nitric acid Perenyi’s fluid Formalin nitric acid
5 -10% (Perenyi 1882)
(Clayden 1952)

02/15/2023
Nitric acid 5-10ml •10% Nitric acid Formaldehyde 10ml
40 ml (37-40%)
Distilled water
100 ml •Absolute ethanol Distilled water 80 ml
30ml

•0.5% chromic acid Nitric acid 10 ml

30ml
To be mixed shortly
before use.

19
WEAK ORGANIC ACIDS

02/15/2023
Eg. Formic acid

Acetic acid

Picric acid

Formic acid is most frequently used as a primary

decalcifier.

Acetic and picric acids cause tissue swelling and are not
used alone as decalcifiers but are found as components
20
in cornoy’s and bouin’s fixatives.
 AQUEOUS FORMIC ACID (5-10%)

90% stock formic acid 5-10 ml

02/15/2023
Distilled water 100 ml

 BUFFERED FORMIC ACID (Evans & Krajian 1930)

20% aqueous sodium citrate 65 ml

90% stock formic acid 35 ml

21
 FORMIC ACID – FORMALIN (Gooding & Stewart 1932)

90% stock formic acid 5-10 ml

02/15/2023
Formaldehyde(37- 40%) 5 ml

Distilled water to 100 ml

 Advantage: Fixes and decalcifies
 Recommended for very small bone pieces or Jamshidi
needle.

NOTE: it is recommended for complete fixation prior to any

acid decalcification.
22

Acid is gently added to water .

5% FORMIC ACID IN DISTILLED
WATER

02/15/2023
 Quicker than EDTA but results in hydrolysis of DNA
 5% acid in <24hrs-preserves DNA
 Prolonged time with 10%acid results in failure
 Formic acid is gentler and slower than HCl or HNO3 and
is suitable for surgical specimens, particularly when IHC
is needed.

23
DECALCIFYIG TISSUE COMMENT +ADVANTAGE
METHOD -
DISADVANTAGE

02/15/2023
3% HCl Routine 1.Compatible -Swells cells
decalcification of with most routine
cortical bone, and special
large thick bone, staining methods.
ossified cancellous 2.large specimens
bone

5% HNO3 Routine Compatible with -Damages nuclear

decalcification of most routine and details
cortical bone, special staining -immunohisto
large thick bone, methods but chemistry
ossified cancellous yields -Swells cells
bone substandard
results
24
DECALCIFYING TISSUE COMMENT +ADVANTAGE
METHOD -DISADVANTAGE

02/15/2023
5% FORMIC Light 1.Compatible with +immunohistochemistry
ACID decalcification most routine and +Good preservation of
of delicate special staining nuclear details
tissue, bone methods
marrow 2.Small specimens

25
CHELATING AGENTS
 Most commonly used chelating agent-EDTA

02/15/2023
 First described by Hillemen and Lee 1953

Mechanism of action:
• EDTA binds to
ionized calcium

• Outer layer depleted

• Calcium reforms
from within
• Reduction in crystal 26
size
 The efficiency of EDTA on the decalcification is
influenced by pH.

02/15/2023
 The greatest demineralizing efficiency - pH 5.0 and 6.0.
 The dissociation of the EDTA- attraction for Ca ions
 EDTA and the Ca ions form a stable complex and the
reaction proceeds until equilibrium is reached.

27
ADVANTAGES

02/15/2023
 It does not damage the tissues or their stainability.
 An excellent bone decalcifier for immunohistochemical
staining and electron microscopy.
 EDTA holds an advantage of pH adjustment for enzyme
staining.

DISADVANTAGES

Slow process

28
FORMALIN-EDTA (Hillemann & Lee)
EDTA ,disodium salt 5.5g

02/15/2023
Distilled water 90 ml
Formaldehyde(37-40%) 10 ml

EDTA(aqueous) pH 7 - 7.4
EDTA , disodium salt 250 g
Distilled water 1750 ml

29
ION EXCHANGE RESINS

02/15/2023
 Remove the Ca ions from the fluid more rapid rate
of solubility of the Ca from the tissue and in the time of
decalcification.
 NH4 form of a sulphonated polystyrene resin is used.
 Limited to those decalcifying fluids not containing
mineral acids.( formic acid is recommended).
 Should not be less than 10% the bulk of the decalcifying
30
agent.
ELECTROPHORETIC DECALCIFICATION

02/15/2023
 This method depends upon the solution of calcium
ions in the electrolyte and their attraction to the
cathode
 Basket movement - by an electric motor
 The only advantage - speeding up of the
decalcification process due to the heat generated
during electrophoresis.
31
02/15/2023
32
 FACTORS INFLUENCING THE RATE OF
DECALCIFICATION

02/15/2023
S
C
A
o
T
u
g
en
sm
ic
p
e
p
ten
e
art
n
tatra
s
iut
iri
o
o
eo
n
n
n

33
CONCENTRATION

02/15/2023
 More concentrated –more decalcified
 Additives like alcohol/buffers- slows down the rate
 In mixture of fixative and acid- decalcification rate
should not exceed the fixation.
 Acid should be end point tested to avoid over
decalcification.

34
TEMPERATURE

02/15/2023
 A chemical reaction is accelerated two to three times for
every 10°C rise in temperature.
 Increased temperature accelerates decalcification, but
also damages the tissue.
 25°C is the ideal recommended temperature with range
of 18-30°C.

35
AGITATION
 mechanical agitation hastens the exchange of fluids with in as

02/15/2023
well as around tissues.

SUSPENSION
 Fluid in contact with all

the surfaces of the specimen.

36
DECALCIFICATION END POINT TEST

02/15/2023
 Accurate determination of the end point is necessary to
prevent the harmful effects of decalcifying fluids on
tissues.

Physical Chemical Radiological

37
Physical

palpation, needling, probing, cutting or trimming, bending

02/15/2023
and squeezing

inaccurate and damaging to tissues and can create artifacts

Chemical

Detection of Ca in decalcifying fluid.

EDTA can be chemically end point tested by acidifying used

solution which forces EDTA to release Ca for precipitation

38
by NH4 oxalate.
CA OXALATE TEST
 involves the detection of Ca in acid solutions by

precipitation of insoluble Ca (OH2) or Ca Oxalate

02/15/2023
39
 CA LCIUM OXALATE TEST

 Result: absence of turbidity after a delay of 5min

02/15/2023
indicates that the fluid is free of calcium and
decalcification is hence, complete.

 Presence of precipitate after addition of ammonium

hydroxide indicates large amount of calcium still present
in the fluid. A precipitate following addition of
ammonium oxalate suggests that decalcification is nearly
complete.

40
BUBBLE TEST

02/15/2023
 Acids react with CaCO3 CO2, seen as a layer of bubbles
on the bone surface.
 As an endpoint test, bubble test is subjective and unreliable
but can be used as a guide to check the progress of
decalcification i.e. tiny bubbles indicate less Ca present.

41
 RADIOGRAPHY
 Most sensitive test

02/15/2023
 In an appropriately decalcified specimen, the bone should have
similar radiological contrast to that of surrounding muscle.
 most reliable method, as even the smallest pocket of calcium
can be detected with modern x ray machines like FAXITRON.

 Newer methods – atomic absorption, spectrophotometry,

calorimetry.

42
 02/15/2023
 Another simple and cheap method to test end point
decalcification is the measure of weight change in the
tissue after decalcification.

Verdenius and Alma in their investigation of decalcifying

methods used weight loss as an indicator of the rate at
which calcium salts are removed.

43

. Verdinius ,L. Alma: A Quantitative study of decalcification methods in histology: J Clin Pathol 1958 ; 11 : 229
SURFACE DECALCIFICATION

 If tissue is still slightly calcified after paraffin embedment

02/15/2023
and sectioning surface decalcification can be done.
• Use of a pad of cotton or sponge soaked with decalcifying
solution (strong acids like 1% HCl in 70% ethanol or 10%
formic acid) placed over the surface of block face for 10 min.

OR
• Immersing the block in decalcifying solution for 30-60 min

44
02/15/2023
45
TREATMENT AFTER
DECALCIFICATION
 Acids can be chemically neutralized after decalcification by

02/15/2023
immersing bone into either lithium carbonate or 10% aqueous
sodium bicarbonate solution for several hours

 Rinsing the specimen with running tap water. ( smaller- 30 mins

and for larger 1 to 4 hours)

 Culling (1974) washing in two changes of 70% alcohol for 12 to

18 hours before dehydration to prevent contamination of the
dehydrating solvents by acid

 Tissue decalcified with EDTA should not be placed directly in 46

70% alcohol as this causes EDTA to precipitate in the alcohol.

 PROCESSING
Automated tissue processors with vacuum and pressure
options have an improved efficiency and quality of tissue

02/15/2023
processing.

47
 SECTIONING
 Blocks are routinely sectioned on a hand controlled rotary
microtome. Base sledge and motorized microtome can also be

02/15/2023
used.

 Routine sections are cut at a thickness of 3µm, while those for

immunohistochemistry are cut at 2µm.

48
EMBEDDING

02/15/2023
49
 STAINING
STAIN APPLICATION

Hematoxylin and eosin General or diagnostic work

02/15/2023
Trichrome Collagen fibers in periodontal ligament

Van Gieson’s Collagen fibers in periodontal ligament

Picrothionin Dental tubules

Air injection Dental tubules

Silver impregnation Nerve fibers in dental pulp

Sudan black Lipid and myelin

Microradiography Mineral density

50
 02/15/2023
RECENT
ADVANCES

51
ULTRASONIC DECALCIFICATION

02/15/2023
 Rapid dissolution of ions
 up to 75% time saving
 100% preservation of morphology and antigenity.

52
02/15/2023
53
MICROWAVE DECALCIFICATION

02/15/2023
Equipment:
 Laboratory microwave equipped with a temperature probe accurate to +/- 1ºC

 Microwave-safe container holding a minimum of 250ml 54

 5% Formic Acid Solution

 Procedure
 Keep bone biopsies as thin as possible.

02/15/2023
 Place the bone biopsies in a microwave-safe container
with at least 10x volume of 5% Formic Acid Solution to
volume of tissue
 Microwave for 10 minutes at 55ºC
 Repeat this procedure until desired softness is achieved
 Rinse in running tap water for at least 10 minutes
 Proceed with either conventional or microwave
55
processing
 02/15/2023
CLINICAL IMPLICATIONS

56
COMPOUND ODONTOME

02/15/2023
57
COMPLEX ODONTOME

02/15/2023
Section from a panoramic radiograph
showing a complex odontome
associated with unerupted and
displaced teeth in the lower right
quadrant.

Photomicrograph from a
decalcified section of a complex
odontome. Present in this field
are dentine, odontoblasts and a
small amount of enamel matrix.

58
GHOST TEETH

02/15/2023
 Photomicrograph of a decalcified
section of a partially erupted ‘tooth’
with regional odontodysplasia. The
dentine is very thin with a very large
pulp chamber.

59
HYPERCEMENTOSIS

02/15/2023
60
 HYPOPHOSPHOTAEMIA

02/15/2023
Photograph demonstrating prominent interglobular
dentine pattern throughout the dentine.

61
 PAGET’S DISEASE

02/15/2023
Histology of the decalcified surgical specimens showing
irregular, thickened trabeculae with cement lines in a
‘‘mosaic’’Pattern.

62
 SUMMARY

02/15/2023
ADVANTAGES DISADVANTAGES
NITRIC ACID Fast Too much RNA lost for
diagnosis
FORMIC ACID Faster than EDTA .Less RNA Slower than nitric acid.
lost than with nitric acid. Some loss of RNA

EDTA Minimal loss of RNA Slow

63
REFERENCES
1.Cullings: Cellular Histological Techniques

02/15/2023
2.John D. Bancroft, Marilyn Gamble: Theory and Practice of Histological
Techniques ; sixth edition pg 338-345

3. Whinney W M, Richardson E.: Technical methods Control of rapid nitric

acid decalcification: J Clin Pathol 1984; 37: 1409-1415

4. Reineke, Tanja et al: Ultrasonic Decalcification Offers New Perspectives

for Rapid FISH, DNA, and RT-PCR Analysis in Bone Marrow Trephines:
The American Journal of Surgical Pathology: July 2006:30:7

5. Verdinius ,L. Alma: A Quantitativestudy of decalcification

methods in histology: J Clin Pathol 1958 ; 11 : 229
64
 02/15/2023
GOOD

MORNING

65
GROUND SECTIONS

02/15/2023
Dr.A.Saileela
I MDS
MSRDC 66
CONTENTS
Introduction

02/15/2023
History

Procedure for preparation

Microscopic appearance of normal teeth

Clinical implications

67
INTRODUCTION
 The visualization of the architecture of an
undecalcified section
 Ground sections are the sections prepared without
using any chemical and thus
maintaining normal anatomy and constituent.

02/15/2023 68
APPLICATIONS

 Diagnostic tool for forensic anthropologist’s,

paleopathologist’s
 Disasters such as earthquakes, airplane crash
investigation
 Age estimation.

An Alternative Efficient Technique For Thin Tooth Sectioning,IeJSME 2011: 5 (1): 27-30
69
HISTORY

 1958- Frost recommended preparation of thin

undecalcified bone sections

Manual Preparation of Ground Sections for the Microscopy of Natural Bone Tissue:
Update and Modification of Frost’s ‘Rapid Manual Method’,International Journal of Osteoarchaeology, 11:
366–374 (2001 70
PROCEDURE
Frost’s method:

With just the help of very basic

instrumentation an intact

10–15 micron thin section.

Manual Preparation of Ground Sections for the Microscopy of Natural Bone Tissue:
Update and Modification of Frost’s ‘Rapid Manual Method’,International Journal of Osteoarchaeology, 11:
366–374 (2001 71
ADVANTAGES:

1. Simple

2. Extremely cheap

3. Reliable

72
 Modification of frost’s method

Rapid manual method

73
PROCEDURE

74
75
76
77
78
79
USES:

 Microscopical investigation of historical human

bones

 Observation of bones which are damaged by fire

and/or by biotic and abiotic environmental factors.

80
TROUBLE SHOOTERS

 Air bubbles
 A section that is too thick
 Section of uneven thickness
 Broken section
 Dirty specimen

81
A SIMPLE METHOD ADOPTED FOR
TOOTH SECTIONING

 A simplified cost effective technique is given which was

used before but usually not carried out nowadays as
newer gadgets (expensive) have replaced the older
simpler methods.

Journal of the university Dow university of health sciences 2010;4(2):84-6 82

ARMAMENTARIUM
Plaster

Rubber bowl

Plaster spatula

Plaster cutter

Glass slab

Model trimmer(carborundum stone)

Arkansas stone

02/15/2023 83
Pumice powder

Glass slide

Cover slip

Microscope

02/15/2023 84
 Preparation of plaster slab

A slab of 4x3” was made and teeth to be embedded

(mesioistally and labiolingually) in such a way that only
half of the tooth is embedded.

02/15/2023 85
PREPARATION OF GROUND SECTION

Model trimmer for initial preparation until 4 to 5mm

02/15/2023 86
Section from trimmer replaced by bench grinder upto 1mm.
Coarse abrasive replaced by fine abrasive wheel

02/15/2023 87
FINAL FINISHING AND POLISHING
•Slurry of pumice and water rubbed with section on arkansas stone

•Until 0.25microns

02/15/2023 88
New tooth sectioning techniques continue to be
introduced

Research into the hard tissues of enamel and dentine

requires a histological technique which preserves
this mineralized tissue and takes into account the
hardness and brittleness of the tissues

89
.An alternative efficient technique for thin ground
sections

 A simple method is described by which thin sections

(approximately 100 ± 20 mm) from fresh specimens
of teeth and bones can be obtained.

An Alternative Efficient Technique For Thin Tooth Sectioning,IeJSME 2011: 5 (1): 27-30
90
Microslice 2 Precision Slicing Machine (Malvern Instruments, UK)

02/15/2023 91
Grinding Plate, StruersRotopol - 1, Copenhagen, Denmark

02/15/2023 92
02/15/2023 93
INTERPRETATION

02/15/2023 94
NORMAL TOOTH STRUCTURE
 Enamel:
 Incremental Lines of Retzius
 Enamel Tufts

 Enamel Lamellae

 Enamel Spindles

 Neonatal Line

02/15/2023 95
STRIAE OF RETZIUS

02/15/2023 96
 ENAMEL TUFTS, LAMELLAE AND
SPINDLES

Enamel spindles represent odontoblast

Enamel tufts resemble tufts of grass in processes trapped in enamel.
ground section

Transmitted light image of cross-sectional

ground section of a tooth showing a lamella
and concentric lines/bands representing the
striae of Retzius.

02/15/2023 97
NEONATAL LINE

02/15/2023 98
DENTIN

 Primary Dentin
 Secondary Dentin
 Interglobular Dentin
 Dentinal Tubules
 Dead Tract
 DEJ

02/15/2023 99
PRIMARY DENTIN

02/15/2023 100
SECONDARY DENTIN

02/15/2023 101
INTERGLOBULAR DENTIN

02/15/2023 102
DENTINAL TUBULES

Permanent tooth dentinal tubules Primary tooth dentinal tubules

following an s-shaped curve following a straight path

02/15/2023 103
DEAD TRACTS

02/15/2023 104
CEMENTUM

 Cellular Cementum
 Acellular cementum
 Tome's Granular Layer
 Cementoenamel Junction (Edge to edge type)
 Cementoenamel Junction (Overlap type)
 Cementoenamel Junction (Gap type)

02/15/2023 105
CELLULAR CEMENTUM

02/15/2023 106
CEMENTOENAMEL JUNCTION

(a) Gap type CEJ (b) edge type CEJ (c) Overlap type CEJ

02/15/2023 107
PATHOLOGY

 Caries

 Amelogenesis imperfecta
 Dentinogenesis imperfecta
 Invaginated odontome
 Tetracycline stains

02/15/2023 108
CARIES

02/15/2023 109
AMELOGENESIS IMPERFECTA

02/15/2023 110
DENTINOGENESIS IMPERFECTA

02/15/2023 111
INVAGINATED ODONTOME

Sectioned upper lateral incisor with an invagination evident within the

crown of the tooth.

02/15/2023 112
GROUNDSECTION OF TETRACYCLINE
STAINED TEETH

 ground section from a permanent molar tooth examined by incident ultraviolet

light and showing brilliantly fluorescing bands attributable to tetracycline.

02/15/2023 113
CONCLUSION

 The knowledge of the normal histology of oral

tissues is the basis for being able to understand
and differentiate from pathology.
 Ground sections do play an important role.

02/15/2023 114
REFERENCES
1.Manual Preparation of Ground Sections for the Microscopy
of Natural Bone Tissue: Update and Modification of Frost’s
‘Rapid Manual Method’,International Journal of
Osteoarchaeology, 11: 366–374 (2001)

2.An Alternative Efficient Technique For Thin Tooth

Sectioning, IeJSME 2011: 5 (1): 27-30

3.Manual Preparation of Ground Sections for the Microscopy

of Natural Bone Tissue: Update and Modification of Frost’s
‘Rapid Manual Method’,International Journal of
Osteoarchaeology, 11: 366–374 (2001)

02/15/2023 115
4.Serial sectioning of teeth and microscopy in cariology
research, Microscopy: Science, Technology, Applications
and Education
Tencate oral histology- 7 th edn,

5.Scientific journal 2007;vol-1

6.Cementum analysis in cleidocranial dysostosis,Indian

journal of Dental Research, 19(3),2008

02/15/2023 116
7.Dentin comparison in primary and permanent molars
under transmitted and polarised light microscopy: An in
vitro study2010;28(3):167-72

02/15/2023 117