Osteoblasts

Osteoblasts are fully differentiated to carry out the primary function of bone formation by producing and laying down osteoid. They are seen on surfaces of actively forming bone as plump cells with basophilic cytoplasm and eccentric nuclei distal to the bone surface. The cytoplasm is basophilic due to ribonucleic acid, and before becoming fully differentiated frequently contains glycogen. Acid phosphatase is found in osteoblasts and the surrounding tissues, but decreases at onset of calcification. Upon completion of bone formation, most osteoblasts revert to a quiescent (small undifferentiated cell) state and reside among the heterogeneous cell population.

Osteocytes

In one respect osteoblasts do represent immature cells, as some are trapped in the osteoid matrix they lay down, and become mature osteocytes residing in tiny spaces or lacunae within the bone. Lacunae are connected to each other and to the vascular spaces by canaliculi – tiny channels into which osteocyte processes project for the purpose of passing fluids and dissolved substances necessary for cell metabolism (Fig. 16.3).

Figure 16.3 Schmorl’s picro-thionin stain showing bone canaliculi.

Celloidin section of normal femoral shaft.

Osteoclasts

These are the cells responsible for bone resorption or erosion. Osteoclasts are large multinucleated (giant) cells whose cytoplasm contains numerous mitochondria and alkaline phosphatase. They occur in small clusters or singly on bone surfaces undergoing resorption and are often seen in the depressions (Howship’s lacunae) they are actively creating by erosion. These surfaces have an irregular outline and lack osteoid (Fig. 16.4). The direction of resorption is random, with no relationship to lamellar structure. Osteoclasts respond to altered mechanical stresses on the skeleton and to growth, and their activity contributes to remodeling. They also respond to hormones that can either stimulate or inhibit their activity. When resorption halts, the process of bone formation resumes (osteoid, mineralization, etc.). Cement lines will occur at the junction between old and newly formed bone.

Figure 16.4 Osteoclasts lining trabecular bone.

Undecalcified rat knee in GMA microtomed at 2 µm. Hematoxylin and eosin stain.

Intramembranous ossification

This occurs in flat bones, e.g. skull, sternum, pelvic bones. A fibrous membrane first develops at the bone formation site in which mesenchymal cells differentiate into osteoblasts that begin the bone formation process by laying down osteoid. This starts in small islets that gradually unite to become trabeculated and finally form an external layer of compact bone.

Endochondral ossification

This occurs in long bones and major parts of the skeleton. This type of bone development begins with differentiation of mesenchymal cells at sites where bone will be formed, but this bone is laid down in a cartilage model that resembles the final shape of the bone. This cartilage model becomes covered with a connective tissue sheath or perichondrium and grows by both apposition and interstitially. Appositional growth or the laying down of more cartilage begins towards the exterior and is mainly responsible for increased diameter; interstitial growth is by cells dividing within the model and mainly occurs towards the extremities, resulting in increased length.

In the central part of the model, cells continue to differentiate, cartilage begins to calcify, blood vessels invade, and the cartilage is broken up into strands. Ossification, by differentiation of perichondrial cells into osteoblasts, begins around the exterior of the primary ossification site at the center of the model. Osteoblasts invade the strands of calcified cartilage and deposit osteoid that soon becomes calcified. This process continues and secondary ossification sites appear at each end of the model, separated from the new bony shaft by cartilage growth or epiphyseal plates capable of interstitial growth. Once bone has ossified, it can only grow by apposition. Remodeling is continuous. In some places deposition exceeds resorption, whereas in others this is reversed and results in the characteristic shape of the bone. When bone is fully grown, the three ossification sites unite and the cartilage growth plates disappear.

Area selection for embedding

In urgent cases of suspected tumor or infection, an attempt should be made to select a sample with the least mineralization in order to provide the quickest possible diagnosis. These pieces can be fixed, rapidly decalcified, and processed to meet urgent clinical requirements.

The ideal thickness of larger bone pieces is 3–5 mm. If bone slabs are too thick, both decalcification and processing are prolonged while overly thin bone slabs (less than 2 mm) tend to become brittle and bend during processing and embedding which may cause the tissue to pop out of the paraffin block during sectioning. The dense collagen matrix tends to prevent adequate paraffin wax penetration and thin pieces are not held firmly in the softer paraffin embedding media during microtomy.

An addition, an aid in area selection of a specimen is radiography when available, a diagram or ‘map’ of the lesion can be made from the X-ray to locate area of interest for processing.

Decalcifying agents

As noted previously there are two major types of decalcifying agent, i.e. acids and chelating agents, although Gray (1954) lists over 50 different mixtures. Many of these mixtures were developed for special purposes with one used as a fixing and dehydrating agent. Other mixtures contain reagents, e.g. buffer salts, chromic acid, formalin, or ethanol, intended to counteract the undesirable swelling effects that acids have on tissues. Many popular mixtures used today are from the original formulae developed many years ago (Evans & Krajian 1930; Kristensen 1948; Clayden 1952). For most practical purposes, today’s laboratories seem to prefer simpler solutions for routine work. Provided the bone is totally fixed and treated with a decalcifier suitable for removal of the amount of mineral present, the simple mixtures work as well or better than more complex mixtures.

Acid decalcifiers

Acid decalcifiers can be divided into two groups: strong (inorganic) and weak (organic) acids. As Brain (1966) suggested, many laboratories keep an acid from each group available for either rapid diagnostic or slower, routine work.

Proprietary decalcifiers

The components in proprietary decalcifying solutions are often trade secrets. Manufacturers provide Material Safety Data Sheets (MSDS) that frequently indicate the type and concentration of acid. Their product data sheets usually indicate if a solution is rapid or slow, and give decalcification instructions and warnings against prolonged use. Rapid proprietary solutions usually contain hydrochloric acid (HCl), whereas slow proprietary mixtures contain buffered formic acid or formalin/formic acid. A study (Callis & Sterchi 1998) found that dilution of a proprietary HCl solution was not deleterious for effective decalcification or staining, and this is an option if a strong mixture is considered too concentrated. Chelating reagents such as EDTA mixtures are also available pre-mixed. Although proprietary mixtures have no obvious advantages over solutions prepared in laboratories, their use is increasingly popular in busy laboratories because they are reliable, time and cost-effective while addressing some safety issues by eliminating handling and storage of concentrated acids.

Strong inorganic acids, e.g. nitric, hydrochloric

These may be used as simple aqueous solutions with recommended concentrations of 5–10%. They decalcify rapidly, cause tissue swelling, and can seriously damage tissue stainability if used longer than 24–48 hours. Old nitric acid is particularly damaging, and should be replaced with fresh stock. Strong acids, however, 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 hours or less. They can be used for large or heavily mineralized cortical bone specimens with decalcification progress carefully monitored by a decalcification endpoint test (Callis & Sterchi 1998). The following is a list of strong acid decalcifying solutions. The formulae and preparations are available in Bancroft & Gamble, sixth ed. (2008).

Weak, organic acids, e.g. formic, acetic, picric

Of these, formic is the only weak acid used extensively as a primary decalcifier. Acetic and picric acids cause tissue swelling and are not used alone as decalcifiers but are found as components in Carnoy’s, Bouin’s, and Zenker’s fixatives. These fixatives will act as incidental, although weak, decalcifiers and could be used in urgent cases with only minimal calcification. Formic acid solutions can be aqueous (5–10%), buffered or combined with formalin. The formalin–10% formic acid mixture simultaneously fixes and decalcifies, and is recommended for very small bone pieces or needle biopsies. However, it is still advisable to have complete fixation before any acid decalcifier is used. The salts, sodium formate (Kristensen 1948) or sodium citrate (Evans & Krajian 1930), are added to formic acid solutions making ‘acidic’ buffers. Buffering is used to counteract the injurious effects of the acid. However, in addition to low 4–5% formic acid concentration, increased time is needed for complete decalcification. Formic acid is gentler and slower than HCl or nitric acids, and is suitable for most routine surgical specimens, particularly when immunohistochemical staining is needed. Formic acid can still damage tissue, antigens, and enzyme staining, and should be endpoint tested. Decalcification is usually complete in 1–10 days, depending on the size, type of bone, and acid concentration. Dense cortical or large bones have been effectively decalcified with 15% aqueous formic acid and a 4% hydrochloric acid–4% formic acid mixture (Callis & Sterchi 1998). The following is a list of weak acid decalcifying solutions. The formulae and preparations are available in Bancroft & Gamble, sixth ed. (2008).

Chelating agents

The chelating agent generally used for decalcification is ethylenediaminetetraacetic acid (EDTA). Although EDTA is nominally ‘acidic’, it does not act like inorganic or organic acids but binds metallic ions, notably calcium and magnesium. EDTA will not bind to calcium below pH 3 and is faster at pH 7–7.4; even though pH 8 and above gives optimal binding, the higher pH may damage alkali-sensitive protein linkages (Callis & Sterchi 1998). EDTA binds to ionized calcium on the outside of the apatite crystal and as this layer becomes depleted more calcium ions reform from within; the crystal becomes progressively smaller during decalcification. This is a very slow process that does not damage tissues or their stainability. When time permits, EDTA is an excellent bone decalcifier for enzyme staining, and electron microscopy. Enzymes require specific pH conditions in order to maintain activity, and EDTA solutions can be adjusted to a specific pH for enzyme staining. EDTA does inactivate alkaline phosphatase but activity can be restored by addition of magnesium chloride.

EDTA and EDTA disodium salt (10%) or EDTA tetrasodium salt (14%) are approaching saturation and can be simple aqueous or buffered solutions at a neutral pH of 7–7.4, or added to formalin. EDTA tetrasodium solution is alkaline, and the pH should be adjusted to 7.4 using concentrated acetic acid. The time required to totally decalcify dense cortical bone may be 6–8 weeks or longer, although small bone spicules may be decalcified in less than a week. Formulae and preparations are available in Bancroft & Gamble, sixth ed. (2008).

Factors influencing the rate of decalcification

Several factors influence the rate of decalcification, and there are ways to speed up or slow down this process. The concentration and volume of the active reagent, including the temperature at which the reaction takes place, are important at all times. Other factors that contribute to how fast bone decalcifies are the age of the patient, type of bone, size of specimen, and solution agitation. Mature cortical bone decalcifies slower than immature, developing cortical or trabecular bone. Another factor of mature bone is that the marrow may contain more adipose cells than a young bone. This requires diligent attention to make sure specimens stay immersed in decalcification solution. Of all the above factors, the effectiveness of agitation is still open to debate.

Completion of decalcification

Ideally, bone should be taken from the acid solution as soon as all calcium has been removed from it. It is still possible that the outer parts of a sample will be overexposed to the acid, although these parts usually stain no differently from inner portions, which are the last to be decalcified. Tissues decalcified in acids for long time periods or in high acid concentrations are more likely to show the effects of over-decalcification, whether or not all mineral has been removed.

Consequently, it is important for a laboratory to control a decalcification procedure by using a decalcification endpoint test to know when calcium removal is complete and, if incomplete, renew the decalcifying agent. If laboratories do not perform endpoint testing, it is recommended they should do so. When using formic, HCl or nitric acids, daily testing is recommended unless near the endpoint; then test every 3–5 hours when possible. With EDTA, weekly tests are sufficient unless solution changes are more frequent. It is recommended that EDTA be changed often at first, since the reaction with calcium is initially quicker and then slows down as the calcium in the tissue is depleted. Minimally calcified tissues and needle biopsies decalcified by a strong acid may be tested only once. It is wise practice for a laboratory that performs a high number of bone biopsy specimens to establish a time range (that depends on the amount of cortical bone present) for ideal decalcification time for bone biopsies. Once the process is established it would eliminate guessing, testing, and handling of those delicate samples. Biopsy decalcification is fairly consistent on multiple-sized biopsies within an hour or two of acid exposure. Wrapping a bone needle biopsy in tissue paper and leaving it wrapped until embedding prevents any loss of cells or tissue during the washing or decalcification process. Sponges sometimes pull cells from a sample when washing and decalcifying. Often these may be urgent cases where a shorter decalcification time is allowed, but the sample must be carefully treated and incomplete decalcification is still possible. If tissue is still slightly under-decalcified after paraffin embedment and sectioning, surface decalcification can be done. Problem blocks should be identified as such so that proper treatment is given should further microtomy be requested.

Decalcification endpoint test

There are several methods for testing the completion of decalcification, with two considered to be the most reliable. These are specimen radiography, using an X-ray unit and the chemical method to test acids and EDTA solutions. Another method first used to test nitric acid is a weight loss, weight gain procedure that provides relatively good, quick results with all acids and EDTA (Mawhinney et al. 1984; Sanderson et al. 1995). Although still used, ‘physical’ tests are considered inaccurate and damaging to tissues. Probing, ‘needling’, slicing, bending, or squeezing tissue can create artifacts, e.g. needle tracks, disrupt soft tumor from bone, or cause false-positive microfractures of fine trabeculae, a potential misdiagnosis. The ‘bubble’ test is subjective and dependent on worker interpretation.

Methods for chemical testing of acid decalcifying fluids detect the presence of calcium released from bone. When no calcium is found or the result is negative, decalcification is said to be complete and may entail using one extra change of decalcifier after actual completion. EDTA can be chemically endpoint tested by acidifying the used solution; this forces EDTA to release calcium for precipitation by ammonium oxalate (Rosen 1981).

Calcium oxalate test (Clayden 1952)

This method involves the detection of calcium in acid solutions by precipitation of insoluble calcium hydroxide or calcium oxalate, but is unsuitable for solutions containing over 10% acid even though these could be diluted and result in a less sensitive test.

Solutions

Ammonium hydroxide, concentrated.

Saturated aqueous ammonium oxalate.

Method

1. Take 5 ml of used decalcifying fluid, add a piece of litmus paper or use a pH meter with magnetic stirrer.

2. Add ammonium hydroxide drop by drop, shaking after each drop, until litmus indicates solution is neutral (pH 7).

3. Add 5 ml of saturated ammonium oxalate and shake well.

4. Allow solution to stand for 30 minutes.

Result

If a white precipitate (calcium hydroxide) forms immediately after adding the ammonium hydroxide, a large quantity of calcium is present, making it unnecessary to proceed further to step 3 which would also be positive. Testing can be stopped and a change to fresh decalcifying solution made at this point. If step 2 is negative or clear after adding ammonium hydroxide, then proceed to step 3 to add ammonium oxalate. If precipitation occurs after adding the ammonium oxalate, less calcium is present. When a smaller amount of calcium is present, it takes longer to form a precipitate in the fluid, so, if the fluid remains clear after 30 minutes, it is safe to assume decalcification is complete.

‘Bubble’ test. Acids react with calcium carbonate in bone to produce carbon dioxide, seen as a layer of bubbles on the bone surface. The bubbles disperse with agitation or shaking but reform, becoming smaller as less calcium carbonate is reduced. As an endpoint test, a 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 calcium present.

Radiography. This is the most sensitive test for detecting calcium in bone or tissue calcification. The method is the same as specimen radiography using a FAXITRON with a manual exposure setting of approximately 1 minute, 30 kV, and Kodak X-OMAT X-ray film on the bottom shelf. It is possible to expose several specimens at the same time. The method is to rinse acid from the sample, carefully place identified bones on waterproof polyethylene sheet on top of the X-ray film, expose according to directions, and leave bones in place until the film is developed and examined for calcifications. Bones with irregular shapes and variable thickness can occasionally mislead workers on interpretation of results. This problem is resolved by comparing the test radiograph to the pre-decalcification specimen radiograph and correlate suspected calcified areas with specimen variations. Areas of mineralization are easily identified, although tiny calcifications are best viewed using a hand-held magnifier. Metal dust particles from saw blades are radio-opaque, sharply delineated fragments that never change in size. These are unaffected by decalcification, appearing as gray specks on the bone surface and can be easily removed. Spicules of metal, metallic paint, or glass forced deep into tissue by a traumatic injury are also sharply delineated but cannot be removed without damaging the tissue. Radiography only indicates the presence of deeper foreign objects, and care must be taken during microtomy to not damage the knife (Fig. 16.5).

Figure 16.5 An example of a FAXITRON X-ray (a) before decalcification (b) after decalcification is completed. The bright white in the sample is calcium. As the calcium is removed, the bone becomes opaque.

Treatment following decalcification

Acids can be removed from tissues or neutralized chemically after decalcification is complete. Chemical neutralization is accomplished by immersing decalcified bone into either saturated lithium carbonate solution or 5–10% aqueous sodium bicarbonate solution for several hours. Many laboratories simply rinse the specimens with running tap water for a period of time. Culling (1974) recommended washing in two changes of 70% alcohol for 12–18 hours before continuing with dehydration in processing, a way to avoid contamination of dehydration solvents even though the dehydration process would remove the acid along with the water.

Adequate water rinsing can generally be done in 30 minutes for small samples and larger bones in 1–4 hours in order to not delay processing. Samples needing immediate processing, e.g. needle biopsies, can be blotted or quickly rinsed to remove acid from surfaces before proceeding to the first dehydrating fluid. It is important to avoid contaminating the first dehydrating fluid with acids, and washing bones even for a short time is good practice particularly with large bone slabs.

Acid-decalcified tissues for cryomicrotomy must be thoroughly washed in water or stored in formal saline containing minimal amounts of sucrose (3–10%), or PBS with 3–10% sucrose, at 4°C before freezing. This helps avoid any residual acid in the tissue from corroding the metal knife. Caution: higher percentages of sucrose may prevent the tissue from fully freezing or freezing unevenly, causing soft spots in the tissue which will create thick-thin appearing sections or the tissue will fall out due to improper freezing.

Tissues decalcified in EDTA solutions should not be placed directly into 70% alcohol, as this causes residual EDTA to precipitate in the alcohol and within the tissue. The precipitate does not appear to affect tissue staining since EDTA is washed out during these procedures, but may be noticeable during microtomy or storage when a crystalline crust forms on the block surface. A water rinse after decalcification or overnight storage in formal saline, NBF, or PBS should prevent this.