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By: Dr. Nisha Rana Moderator: Dr.

Rashmi Arora

Bones are rigid organs that constitute part of the endoskeleton of vertebrates. They support and protect the various organs of the body, produce red and white blood cells and store minerals. Bone tissue is a type of dense connective tissue. Bones come in a variety of shapes and have a complex internal and external structure, are lightweight yet strong and hard, and serve multiple functions.

One of the types of tissue that makes up bone is the mineralized osseous tissue, also called bone tissue, that gives it rigidity and a coral-like three-dimensional internal structure. Other types of tissue found include marrow, endosteum, periosteum, nerves, blood vessels and cartilage.

The hard outer layer of bones is composed of compact bone tissue, so-called due to its minimal gaps and spaces. Its porosity is 5 30%.This tissue gives bones their smooth, white, and solid appearance, and accounts for 80% of the total bone mass of an adult skeleton. Compact bone may also be referred to as dense bone.

Filling the interior of the bone is the trabecular bone tissue ,which is composed of a network of rod and plate like elements that make the overall organ lighter and allow room for blood vessels and marrow. Trabecular bone accounts for the remaining 20% of total bone mass but has nearly ten times the surface area of compact bone. Its porosity is 3090%. The microscopic difference between compact and cancellous bone is that compact bone consists of haversian sites and osteons, while cancellous bones do not. Also, bone surrounds blood in the compact bone, while blood surrounds bone in the cancellous bone.

There are several types of cells constituting the bone; Osteoblasts are mononucleate bone-forming cells that descend from osteoprogenitor cells. They make a protein mixture known as osteoid, which mineralizes to become bone.Osteoid is primarily composed of Type I collagen. Osteoblsats robustly produce alkaline phosphatase, an enzyme that has a role in the mineralisation of bone, as well as many matrix proteins. Osteoblasts are the immature bone cells, and eventually become entrapped in the bone matrix to become osteocytes- the mature bone cell.

Osteocytes originate from osteoblasts that have migrated into and become trapped and surrounded by bone matrix that they themselves produce. The spaces they occupy are known as lacunae. Osteocytes have many processes that reach out to meet osteoblasts and other osteocytes probably for the purposes of communication. Their functions include, to varying degrees: formation of bone; matrix maintenance; and calcium homeostasis. They have also been shown to act as mechano-sensory receptors regulating the bone's response to stress and mechanical load. They are mature bone cells.

Osteoclasts are the cells responsible for bone resorption, thus they break down bone. New bone is then formed by the osteoblasts (remodeling of bone to reduce its volume). Osteoclasts are large, multinucleated cells located on bone surfaces in what are called Howship's lacunae or resorption pits. These lacunae, or resorption pits, are left behind after the breakdown of the bone surface. Because the osteoclasts are derived from a monocyte stemcell lineage, they are equipped with phagocyticlike mechanisms similar to circulating macrophages. Osteoclasts mature and/or migrate to discrete bone surfaces. Upon arrival, active enzymes, such as tartrate resistant acid phosphatase, are secreted against the mineral substrate.

Matrix

The majority of bone is made of the bone matrix. It has inorganic and organic parts. Bone is formed by the hardening of this matrix entrapping the cells. When these cells become entrapped from osteoblasts they become osteocytes.

The inorganic composition of bone (bone mineral) is formed from carbonated hydroxyapatite (Ca10(PO4)6(OH)2) with lower crystallinity. The matrix is initially laid down as unmineralised osteoid (manufactured by osteoblasts). Mineralisation involves osteoblasts secreting vesicles containing alkaline phosphatase. This cleaves the phosphate groups and acts as the foci for calcium and phosphate deposition. The vesicles then rupture and act as a centre for crystals to grow on. More particularly, bone mineral is formed from globular and plate structures, distributed among the collagen fibrils of bone and forming yet larger structure

The organic part of matrix is mainly composed of Type I collagen. This is synthesised intracellularly as tropocollagen and then exported, forming fibrils. The organic part is also composed of various growth factors, the functions of which are not fully known. Factors present include glycosaminoglycans, osteocalcin, osteone ctin, bone sialo protein, osteopontin and Cell Attachment Factor. One of the main things that distinguishes the matrix of a bone from that of another cell is that the matrix in bone is hard.

There

are five types of bones in the human body: long, short, flat, irregular, and sesamoid. Long bones are characterized by a shaft, the diaphysis, that is much longer than it is wide. They are made up mostly ofcompact bone, with lesser amounts of marrow, located within the medullary cavity, and spongy bone. Most bones of the limbs, including those of the fingers and toes, are long bones. The exceptions are those of the wrist, ankle and kneecap

Short bones are roughly cube-shaped, and have only a thin layer of compact bone surrounding a spongy interior. The bones of the wrist and ankle are short bones, as are the sesamoid bones. Flat bones are thin and generally curved, with two parallel layers of compact bones sandwiching a layer of spongy bone. Most of the bones of the skull are flat bones, as is the sternum. Sesamoid bones are bones embedded in tendons. Since they act to hold the tendon further away from the joint, the angle of the tendon is increased and thus the leverage of the muscle is increased. Examples of sesamoid bones are the patella and the pisiform. Irregular bones do not fit into the above categories. They consist of thin layers of compact bone surrounding a spongy interior. As implied by the name, their shapes are irregular and complicated. The bones of the spine and hips are irregular bones.

These can be: Biopsies- for diagnosis of tumor, hematopoietic disorders, infection etc. Amputation specimens: resulting from tumor, chronic osteomyelitis and gangrene. Resection specimens: for benign or low grade malignant tumors and arthritis.

Radiographs of bone slabs, blocks or fragments are useful for four main purposes: To examine the nature & extent of a lesion. To provide a diagram of lesion prior to block selection for processing. To check progress of decalcification i.e. decalcification endpoint test. To confirm the presence of large foreign materials e.g. prosthetic devices, metal or glass fragments implanted by trauma.

The

faxitron cabinet x-ray system is used. It is a tabletop unit measuring 56 cm wide, 51 cm deep & 89 m high. Energy output is 10-110 kV with 3 mA tube current. It has adjustable shelf levels for film-tosource distances of 31-61 mm. X-ray film used for specimen radiography is KODAK X-OMAT.

good band saw is an essential piece of equipment. Handymans bench band saws are inexpensive, small & light weight and these saws cut through cortical bone slowly with cuts no deeper than 7.5 cm. Wetter or meat cutters saw is used for cutting wet, fatty bone.it is heavier, rigid, has floor standing construction and strong coarse blades for deeper cuts. Electric saw is also available.

Handyman saw

Meat cutters saw

On

small saws: 0.5 cm width and 12 to 16 teeth per inch (tpi) making finer cleaner cuts. On large saws: 1.25 cm wide with 6 teeth per inch

Soft

tissues and dense connective tissue e.g. tendons should be removed before sawing the sample. The first cut is made through the midplane, then approximately 3-5 mm thick slabs are cut parallel to the first cut. A saw guide or wood plank is held against the first cut edge to ensure an even slice. After sawing, the blades are cleaned by using a slow sream of water ans a soft brush.

It

protects bone and surrounding soft tissue from damaging effects of acid decalcification. 10% natural buffered formalin is suitable for both paraffin and non-tetracycline labeled bone in MMA (methyl methacrylate). Alcoholic formalin or 70% ethanol fixation can be used to fix tetracycline labeled mineralized bone in MMA. Fixation proceeds faster by reducing the size of the bone & removing excess of soft tissue.

Techniques for demonstration of bones and its components are: For decalcified bone: frozen, paraffin, or celloidin sections and transmission electron microscopy For mineralised bone: frozen, plastic for microtomed sections, scanning and tansmission EM. The technique chosen for examination of bone is influenced by clinical diagnosis, case urgency and extent of investigation.

Fixation

99% ethyl alcohol


Tissue

1-2 mm thick bone pieces


Solutions

2% aqueous silver nitrate

Reducer : Sodium hypophosphite 0.1M sodium hydroxide Distilled water

5 gm 0.2 ml 100 ml

5% aqueous sodium thiosulphate Decalcifier :

10% aqueous formic acid Van Giesons picro-fuchsin

Wash in several changes of distilled water, 4 hrs. Place 2% silver nitrate, 48 hrs in dark. Rinse in 3 changes of distilled water, 15-20 sec each. Wash in running tap water, 4 hrs. Place in reducer, 48 hrs. Wash in running tap water, 1 hr. Place in sodium thiosulphate sol, 24 hrs. Wash in running tap water, 1 hr. Decalcify in 10% formic acid. Process to paraffin wax, cut & mount. Dewax & stain with van giesons stain, 2 min. Dehydrate, clear & mount.

Edges Bone

of mineralized bone

Black.

Brown to Yellow
Red

Osteoid

In order to obtain satisfactory paraffin or celloidin sections of bone, inorganic calcium must be removed from the organic collagen matrix, calcified cartilage and surrounding tissues. This is called decalcification. It is carried out by chemical agents, either with acids to form soluble calcium salts or with chelating agents that bind to calcium ions.

It is influenced by four interdependent factors: Urgency of case degree of mineralisation Extent of the investigation Staining techniques required.

There are 2 major types of decalcifying agents: Acids Chelating agents

These can be divided into 2 groups: Strong (inorganic) acids Weak (organic) acids

These

may be used as simple aqueous solutions with recommended concentrations of 5-10%. They decalcify rapidly, cause tissue swelling can damage tissue stainability if used longer than 24-48 hours. They tend to be more damaging to tissue antigens for immunohistochemical staining and enzymes may be totally lost.

Strong

acids are used for needle and small biopsy specimens to permit rapid diagnosis within 24 hrs or less. They can be used for large or heavily mineralised cortical bone specimens with decalcification progress carefully monitored by a decalcification endpoint test. Examples include nitric acid & hydrochloric acid.

Nitric

acid 5-10 ml Distilled water to 100 ml

10%

nitric acid Absolute ethanol 0.5% chromic acid

40 ml 30 ml 30 ml

mix shortly before use.

Use

inside a fume hood Formaldehyde (37-40%) Distilled water Nitric acid

- 10 ml - 80 ml - 10 ml

Examples

include formic acid, picric acid, acetic acid. Formic acid is the only weak acid used extensively as a primary decalcifier. Formis acid solutions can be aqueous (510%), buffered or combined with formalin. The formain-10% formic acid mixture simultaneously fixes and decalcifies, & is recommended for very small bone pieces or jamshidi needle biopsies.

Formic

acid is gentler and slower than HCl or nitric acids. It is suitable for most routine surgical specimens particularly when immunohistochemical staining is needed. Also it should be endpoint tested. Decalcification is complete in 1-10 days, depending on size, type of bone & acid concentration.

90%

stock formic acid - 5-10 ml Distilled water to - 100 ml

90%

stock formic acid - 5-10 ml Formaldehyde (37-40%) - 5 ml Distilled water to - 100 ml

20%

aqueous sodium citrate - 65 ml 90% stock formic acid - 35 ml

This solution has a pH of approximately 2.3

Ethylene-diaminetetracetic

acid (EDTA) is

used for decalcification. It binds metallic ions, notably calcium and magnesium. EDTA will not bind to calcium below pH 3 and is faster at pH 7-7.4 It binds to ionised calcium on the outside of the apatite crystal and as this layer becomes depleted, more calcium ions reform from within and the crystal becomes progressively smaller.

EDTA

disodium salt - 250 gm Distilled water - 90 ml Formaldehyde (37-40%) 10 ml

pH

7.0-7.4 EDTA, disodium salt Distilled water

- 250 gm - 1750 ml

If the solution is cloudy, adjust to pH 7 with approximately 25 gm sodium hydroxide.

Concentration

and volume of the active

reagent. More concentrated acid solutions decalcify more rapidly but are harmful to tissues. Alcohol or buffers that protect tissue slow down the decalcification rate. Temperature Increased temperature accelerates the rate while decreased temperature reduces the rate of dcalcification.

Age

of the patient Type of bone Size of specimen Solution agitation

The

two most reliable tests are:


Specimen radiography using an X-ray unit Chemical method to test acids and EDTA solutions

Other method first used to test nitric acid is a weight loss, weight gain procedure that provides good results with all acids and EDTA. But it is considered inaccurate & is damaging to tissues.

Probing,

needling, slicing, bending or squeezing tissue can create artifacts e.g. needle tracks, disrupt small tumor from bone or cause false +ve microfractures of fine trabeculae.

It

is the most sensitive test for detecting calcium in bone. It uses FAXITRON with a manual exposure setting of approx. 1 minute, 30 kV and KODAK X-OMAT X-ray film. Acid is rinsed from sample and bone is placed on waterproof polyethylene sheet on the top of x-ray film. The bone is exposed and is left in place until film is developed. It is then examined for calcifications.

Areas

of mineralization are easily identified with tiny calcifications best viewed using a hand-held magnifier. Metal dust particles from saw blades are radio-opaque, sharply delineated fragments that never change in size.they appear as grey specs & can be easily removed. Spicules of metal, metallic paint or glass forced deep into tissue by traumatic injury are also sharply delineated but cannot be removed without damaging tissue.

They

detect the presence of calcium released from the bone. When no calcium is found or the result is negative, decalcification is said to be complete. EDTA can be chemically endpoint tested by acidifying the used solution: this forces EDTA to release calcium for precipitation by ammonium oxalate.

This

method detects the calcium in acid solutions by precipitation of insoluble calcium hydroxide or calcium oxalate.

Solutions:

Ammonium hydroxide, concentrated Saturated aqueous ammonium oxalate.

Method:

Take 5 ml of used decalcifying fluid, add a piece of litmus paper or use a pH meter. 2. Add ammonium hydroxide drop by drop, shaking after each drop, until pH is 7. 3. Add 5ml of saturated ammonium oxalate and shake well. 4. Allow solution to stand for 30 min.
1.

If

a white precipitate (calcium hydroxide) forms immediately after adding ammonium hydroxide, a large quantity of calcium is present & it is positive. If the precipitate is not formed, then ammonium oxalate is added. If precipitation occurs after adding ammonium oxalate, less calcium is present & therefore it takes longer to form a precipitate, so if the fluid remains clear after 30 min, decalcification is complete.

Acids

react with calcium carbonate in bone to produce carbon dioxide, seen as a layer of bubbles on bone surface. They disperse with agitation but reform becoming smaller as less calcium carbonate is reduced. It can be used as a guide to check the progress of decalcification but as an endpoint test it is subjective & unreliable.

Acids

are removed from tissues after decalcification is complete. Chemical neutralization is done by immersing decalcified bone into 5-10% aqueous sodium bicarbonate solution for several hours. Specimens can also be rinsed with running tap water in 30 min for small samples & 1-4 hrs for larger samples. Samples needing immediate processing e.g. needle biopsies can be blotted or quickly rinsed to remove acid from the surface.

Paraffin

waxes developed in recent years have been improved by addition of plastic polymers & other chemicals that allow better wax penetration & sectioning. Decalcified bone sectioning is made easier after infiltration & embedding in a harder paraffin wax. Small bone & needle biopsies containing little cortical bone can be processed with soft tissues.

Oversized,

thick bone slabs require an extended processing to obtain adequate dehydration, clearing & paraffin infiltration. Automatic processors are also available in which time for dehydration, clearing solvent and paraffin may vary from 2-4 hrs. Simple hand processing with a vaccum dessicator for dehydration & clearing steps & infiltration in a heated vaccum oven with 3 changes of paraffin, upto 8 hrs per change can also be done.

If

an endpoint tested decalcified bone appears chalky & crumbles out out of the block during sectioning then dehydration or paraffin infiltration maybe incomplete. For this blocks can be melted down & reinfiltrated with paraffin or by melting paraffin from bone & going back through 2 changes of xylene, 2 changes of 100 % alcohol to remove residual water.

Embedding

methods using metal molds with plastic tissue cassettes are also available. A labeled cassette contains the tissue throughout processing & after embedding, the plastic back of a block fits into a microtome cassette lamp. A large specimen is the limiting factor for embedding with cassettes.

These

are used to cut bone biopsies and smaller cancellous bone blocks. Newer microtomes are more powerful, heavier and automated, making them capable of sectioning both paraffin and plastic bone blocks. Hard, dense bone samples are sectioned on a large sledge or heavy duty motorized sliding microtome. Good microtone blades include heavy c profile steel and disposable blades.

Disposable

knives are convenient, extremely sharp, single used blades capable of sectioning properly decalcified and processed paraffin-embedded bones. Newer microtones come equipped with disposable blade holders. Heavy steel knives range in size from 16 to 18 cm for small microtomes and for 200-300 cms for base sledge microtomes with specially designed blades for the polycuts.

An

optimal thickness for bone sections is same as that for soft tissues, 4 to 5 m. Small bone samples and biopsies are sectioned with knife angles set for routine soft tissue microtomy. When sectioning, a sharp knife is necessary to get flat, uncompressed, wrinkle-free sections. Hard tissues cut more easily if cooled by a melting ice block to allow water penetration into the tissue surface.

While

floating on water, cartilage and bone sections can expand more than paraffin and small folds may form as sections dry. To prevent this, water bath temperature should be lowered to 10-15C below the paraffin melting point. If cartilage curling is a problem, drying sections flat at 37C overnight may solve the problem.

Bone

sections adhere to slides better when glass surfaces are coated with adhesive. For this, commercially silanized Plus Charge or poly-l-lysine coated slides are available. Coating can also be done by dipping the slides in a gelatin and potassium dichromate subbing solution. If some sections are persistently non adherent, a solution containing amylopectin, or HMW bloom gelatin in the chrome subbing mixture may be used.

Celloidin

or nitrocellulose embedding method is useful for large, decalcified bone. But it has been replaced with MMAembedded undecalcifed bone section methods. It doesnt harden cortical bone, is pliable and more elastic, binding tissues with different consistencies together and preventing separation during sectioning. Double embedding method combines celloidin and paraffin, uses harder paraffin and has extended processing.

The

disadvantages of celloidin are

Expense. Reduced availability of nitro cellulose. Chemical safety issues, i.e. toxic chloroform. Extended preparation time. Sections too thick for good cellular detail. Need for a sledge microtome with a knife suitable for sectioning celloidin.

Diagnostic

immunohistochemical staining is frequently done on decalcified bone sections embedded in paraffin e.g. bone marrow biopsies, tumors, cartilage. Care must be taken to fix bone specimens properly and decalcify with the least damaging agent in shortest time possible to protect antigens from damaging effects of acids. 2 m thick MMA sections after complete removal of plastic with warm xylene are taken .

Glycol

methacrylate cannot be removed & may inhibit adequate antibody preparation to the antigenic sites.

Mineralized

bone must be cut with tungsten carbide-tipped knives and need special, hard support to avoid cracked tissue sections. Paraffin and celloidin are too soft and fail to match the harness of bone, Acrylic resins and plastics are now widely used and are preferred embedding media for undecalcified bone. Frozen sections provide some support of cancellous bone but the bone itself tends to look damaged.

They

have been used to maintain the intact sections of undecalcified double embedded bone sections during microtomy. 2 methods, 1 for undecalcified bone embedded in MMA and other for decalcified paraffin embedded bone are used. Clear adhesive packaging tape is rolled on to the trimmed block face and the cut section sticks to the tape during and after sectioning. The tape section combination is then attached to an adhesive coated slide.

During

the staining process, the tape releases xylene, leaving the section transferred.

It

can be sectioned by microtomy, sawing & grinding. Thick ground sections are necessary for microradiography. Ultra-miller on a large sliding microtome can mill a flat 15-20 m sections, but it wastes bone & must be used carefully.

These

sections provide rapid diagnosis. They are made by rapid freezing of bone samples in liquid nitrogen-cooled 2methylbutane (-120 C). It must be used carefully as some bones may shatter in extremely cold temperatures. A dry ice/isopentane bath snap freezes bone coated with 4% aq polyvinyl alcohol gently without shattering (-70 C).

1.
2. 3.

4.

5.

6.
7. 8.

Mount bone on cork. Snap freeze in isopentane cooled by liquid nitrogen or with dry ice. Place bone in cryostat at -30 to -35 C. Cut sections at 5-10 m, pickup section on slide & fix with fixative. Fixation in 95% alcohol for 5 min removes fat. Stain in harris Gill II or Gill III for 1 min. Rinse with water. 1% alcoholic eosin for 10-30 sec. Dehydrate & mount in permanent mounting medium.

Fresh

frozen sections can be fixed, rinsed then decalcified in 10% EDTA before immunostainig. For cryoprotection, formalin fixed biopsies are immersed in 15-20% sucrose for 1-8 hrs to replace water before freezing.

FIXATION
1.

The specimen is allowed sufficient time for it to be fully fixed with 10% formol calcium (at least 24 hours after removal from the body) before processing is commenced. Specimens which are labelled with tetracycline (noted on request form) for subsequent demonstration with fluorescence, must be fixed only in 70% alcohol and not formol calcium, for at least 24 hours after removal from the body.

2.

If the specimen is too large, it should be trimmed/cut so that adequate size block(s) are taken from the part(s) required for examination. Surplus tissue not required for examination should be cut off. Should cutting/trimming be necessary, then advice must be sought from an experienced MLSO. 3. Large specimens especially those which require cutting/trimming may require longer fixation than 24 hours before processing is commenced.

The

following processing schedule is suitable for biopsy size specimens which are processed in a 10ml glass vial. 1. 70% Alcohol 2 hours 2. 90% Alcohol 2 hours 3. 100% Ethanol x 2 2 hours each 4. Ethanol/methyl methacrylate monomer (1:1) overnight 5. Infiltrating solution A x 2 4 hours each 6. Infiltrating/embedding solution B 23 hours

1.

Specimens must only be processed under an operational fume hood. 2. Specimens are agitated continuously on a roller mixer for all steps. 3. Waste solutions containing alcohol can be discarded down the sink. 4. Any waste solution containing methyl methacrylate must be discarded after being allowed to evaporate off according to wastedosposal policies.

STORE

AT 4C. Methyl methacrylate monomer (stabilised) 70ml Dibutyl phthalate 30ml Benzoyl peroxide (previously dried) 2g. Make up solution under a fume hood in a darkened bottle. Benzoyl peroxide must be dissolved before use by agitating on a roller mixer.

STORE

AT 4C. Infiltrating solution A 100ml Poly methyl methacrylate low mol wt beads 30g Make up mixture under a fume hood in a darkened bottle, mixing on a roller mixer until beads have fully dissolved (approximately 4 days)

Benzoyl peroxide is supplied damped with water as it is potentially explosive in its dry form. However, it is essential that only dried benzoyl peroxide is used in the solutions A and B. It is dried as follows: 1. Spoon out approximately 10g of benzoyl peroxide into a filter paper bent in the shape of a cone on a beaker. 2. Dry in a 37C oven overnight. Use only the oven in the laboratory, and place a sign to warn staff. 3. Excess dried benzoyl peroxide is stored on the shelf in the container provided away from direct heat.

Allow

specimen in resin embedding solution B to stand for 1 hour. Orientate the label using a wooden cocktail stick, so that it is bent at right angles across the specimen. Secure lid on vial/container. Partially immerse in pre-heated 60C water inside a staining dish. Ensure lid is replaced on dish. Polymerise resin overnight at 60C in the oven. Use only the 60C oven in the resin laboratory.

Place

glass container (minus lid if possible) in freezing compartment of the refrigerator for 15 minutes. Wrapping the container well in paper towels, break the container by hitting gently with the hammer provided. Wear eye protection. Discard both waste paper and broken glass in the glass bin, and ensure no glass is lying on the bench. Carefully wash block under a running tap.

Clamp block in specially designed holder on Reichert-Jung Autocut so that the bone is vertical to the knife edge. Using the trimming tungsten carbide knife, trim into block until a representative area is reached, lubricating both knife and block with 30% alcohol with the aid of an artist's brush. Remove the trimming knife and replace it with the cutting tungsten carbide knife and cut sections at 6, lubricating both block and knife as previously described. Sections that require fluorescence to examine tetracycline labelling should be cut at 12 and kept separate from other sections. Sections can be picked up using a pair of fine forceps.

Cut

at least 6 sections per routine case so that spares are available should they be required. Store sections in 30% alcohol (or 70% alcohol for tetracycline-labelled tissue). Use a separate labelled small specimen pot for sections from each block.