CHAPTER 6

Bone

Part l. DecalcificationDecalcification methods using acid solutions

Formulas for acid decalcifying agentslon-exchange methods for decalcificationElectrolytic decalcif icationHistochemical methods for decalcification

Butfer mixturesChelating agentsDemonstration of specific enzymesDemonstration of glycogen in decalcified tissues

Part ll. Basic preparation and staining ofundecalcified boneMineralized bone technic

Preparation of undecalcified sections of boneSectioning machinesGround sectioning

Method of preparing ground sectionsPlastic embedding

Procedure for routine processing and melhacrylateembedding of bone tissue

Preparation of bone specimen for simultaneoustetracycline-osteoid seam assessment andhistomorphometric quantitative analysisPreparation of polymethyl methacrylate

embedding mediumProcessing and embedding procedureSectioning

Procedure for dry sectionsProcedure for wet sections

CommentsMethods of staining

Frost's basic fuchsin stainVillanueva's bone stain

Villanueva's tetrachrome bone stainModified Villanueva-Gomori trichromeModified Villanueva-Goldner trichrome

Villanueva's blood stainA modification of Movat's pentachrome stainGordon and Sweet's method for cement linesPhosphotungstic acid hematoxylinModified Von Kossa's methodSolochrome cyanin RToluidine blue stain for calcification front. cement

line, and osteoid seamsModified methyl green pyronine Y stain

Part lll. Histomorphometric quantitativeanalysis of boneMethodologyLabeling techniquesRecognition of histologic structures

Osteoid seamsTrabecular surfaceHaversian surfacePrimary osteonsSecondary osteonsOsteocyte's lacunaeBone resorption

Microscopic measurementsTrabecular surface

Circumference formulaSurface measurements of trabeculaRates involved with bone remodeling

Haversian surfaceCortical areaMeasurements of haversian surface

Part l. DecalcificationBefore bone or any calcified tissue can be

processed and sectioned with a routine micro-tome, the calcium salts must be removed by aprocess called "decalcification." Failure to de-calcify tissue with large amounts of calciumsalts will result in torn and ragged sectons anddamage to the cutting edge of the microtomeknife.

To ensure adequate fixation and decalcifica-tion, tissue selected should not exceed 4 or 5mm in thickness. A fine-toothed hacksaw or theBuehler Isomet Low Speed Saw (Fig. l-21) may

be used to obtain thin slices of bone. At the com-pletion of the decalcification process, the cutsurfaces should be trimmed to remove the areasdamaged by the saw in obtaining the specimen.When the marrow of cancellous bone is to bestudied, time is saved and acid damage to themarrow cells reduced when the cortical bone iscut of as soon as the cancellous portion is softenough.

Fixation is the next step after tissue selectionand is usually performed before the actual de-calcification process. Fixatives for bone andmarrow should be chosen with the primary ob-

89

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90 THEoRY AND PRAcTIcE oF HISToTEcHNoLoGY

Fig. 5-1. Acid decalcification method. For best results,gauze-wrapped bone should be suspended in center ofdecalcifying fluid.

jective of study in mind and should have cri-teria similar to those used for soft-tissue fixa-tives. For routine diagnostic purposes, formalinis the fixative of choice. It is used unbufferedsince the calcium phosphate present in thebone substance serves as an adequate buffer tokeep the pH level of the fixative above 6.0 with-in the tissue and prevent the formation of for-malin pigment. Nucleic acids are susceptible toribonuclease digestion or digestion by mineralacids if formalin fixation is prolonged more than2 days. Therefore formalin-fixed bone is bestdecalcified with I normal acetic or formic acidor with buffer mixtures of pH 2.0 or higher.Chromate fixatives such as Orth's, Kose's, andMtiller's potassium dichromate formalin mix-tures, and formalin-Zenker variants, are pre-ferred by many for mamow cell study. Thesefixatives, or 1 normal hydrochloric or nitric acid,render nucleic acids less susceptible to hydroly-sis either by ribonuclease or by acids and makethe tissue moderately resistant.

Routine decalciftcation methods includethose using (1) acids, (2) ion-exchange resins,and (3) electrical ionization. All these methodsare greatly accelerated by heat. Heat, however,is not recommended since at 55' to 60' C theloss of'calcium salts occurs rapidly, followed byswelling and hydrolysis of bone collagen, re-sulting in complete digestion of the tlssue. Thisdigestion will occur in about 24 hours wtth 8Vohydrochloric acid, 24 hours with formic acidmixture, or 2 to 3 days wlth 5% formic acid.Decalcification at 37" C also causes undueswelling of tissue and may also impair subse-

quent staining processes. AIum hematoxylins,Weigert's iron hematoxylin, Feulgen reaction,and azure eosin stains are all impaired by de-calcification at 37' C with formic-hydrochloricacid mixtures and by SVo formic acid. Five-per-cent formic acid may also impair Van Giesonand Masson trichrome staining results. Lilliestates that when decalcification is performed atl5o to 20o C, satisfactory H & E, Van Gieson,Masson, and azure eosin stains are obtainedwith mineral acids, formic acid. and mixturesof both acids. Feulgen staining of nuclei is wellpreserved after formic acid decalcification at24o C, but with mineral acids, even at a tem-perature of 3' C, this staining process is ino-paired.

DECALCIFICATION METHODS USINGACID SOLUTIONS

Acid solutions are most widely used for rou-tine.decalcification of large amounts of boneand calcified tissue. The principle underlyrngthe action of acid decalcifying agents involvesthe solubilities of metallic sdts. Calcium occursin bones"chiefly as the carbonate and phosphatesalts, and these salts are only slightly soluble inwater. An acid will act to release the calciumfrom its combination with the anions and effectan ion exchange to give a soluble calcium salt.For example, when hydrochloric acid is used asthe decalcifying agent, the released calciumcombines with the chloride ion to form calciumchloride, a soluble calcium salt. The calciumions released will remain in the decalcifyingsolution itself and are effectively removed fromthe bone. The general technic to be followedwhen one employs acid decalcifying agents isas follows:

1. Selection of tissue (previously discussed).2. Fixation (previously discussed). After fixa-

tion, the tissue is washed to remove excess fixa-tive.

3. Decalcification. The selected tissue shouldbe loosely wrapped in gauze and then sus-pended in the center of a large jar that is filledwith the decalcifying fluid of choice (Fig. 6-1).About 100 times the volume of the tissue is agood approximate amount, and this large vol-ume is necessary since the mineral content of agood-sized piece of bone will soon neutralize thesmall amount of acid present in the solution.Decalcifying fluids that may be used includeaqueous or alcoholic solutions of the acids listedin Table 6-1. Although some authors claim thatalcoholic solutions aid in preventing undue

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BONE 91

Table 5-1. Acid decalcifying agents

Agent Advantages Disadvantages

Formic acid

Hydrochloricacid

Sulfurousacid

Nitric acid

Trichloroace-tic acid

Chromic acid

Acetic, lactic,and picricacids

Trifluoroace-tic acid

1Vo formic acid is considered the best generaldecalcifying agent by Lillie, It is more eco-nomical than buffer mixtures and equallysatisfactory and effective. It permits satis-factory nuclear and marrow cell stainingwhen decalcification is performed at roomtemperature.

Action is rapid, even in dilute solutions. Thisacid, when used at 37" C, preserves eosinstains of cytoplasm. When used at 15' to25" C, satisfactory H & E, Van Gieson, Mas-son, and azure eosin stains may be done, ifexposure is not prolonged. To remedy swell-ing of tissues, chromic acid or alcohol maybe added to the solution. l5% NaCl mayalso be added to a 3Vo acid solution to coun-teract the swelling action.

Acts rapidly and preserves well. Best usedafter fixation in formalin.

Many writers highly recommend 1Vo, whichcauses no swelling and acts powerfully.

4Vo solution gives energetic action, good pres-ewation, and satisfactory nuclear and mar-row staining.

A strong acid with powerful decalcifying prop-erties, with a rapid action. Stains are good,

bright, and sharp with little distortion ofnuclear and cytoplasmic structure. Densebone decalcified in 2 to 3 davs in lOVo.5 to6 days in SVo.

If decalcification is done at 37o C, alum hema-toxylin staining and Weigert's iron he-

matoxylin staining of nuclei are impaired tosome extent; Feulgen staining of nuclei isunsatisfactory and azure eosin stains givepink cytoplasm and nuclei. At 37'C, it alsoimpairs Van Gieson and Masson staining ofcollagen and bone matrix.

Causes serious swelling of tissue. At 55o to60' C loss of calcium salts occurs rapidly,followed by swelling and hydrolysis of bonematrix, which soon results in complete di-gestion. This occurs in as little as 24 hr in8% hydrochloric acid. Decalcification at 37'C impairs alum hematoxylin staining andWeigert's iron hematoxylin staining of nu-clei to some extent. Feulgen staining ofnuclei is unsuccessful and azure eosinstains give pink cytoplasm and nuclei.

If action is prolonged over 48 hours, nuclearstaining is seriously impaired.

Even br[ef exposure to this acid renders un-satisfactory staining of bone marrow withGiemsa, Maximow's azure ll-eosin or Lil-lie's azure eosin formula.

Because of its high molecular weight, as com-pared with formic acid, much larger quanti-ties are needed for efficient decalcification-it takes 372 times more solution than for-mic acid.

Weak decalcifuing action and strong shrinkingaction. It should never be used in more than17o solution.

Usually unsatisfactory. Acetic and lactic acidshave considerable decalcifying power, butcause great swelling. Picric acid has a veryslow action and is suitable for very smallStructures.

swelling of the tissue, others believe that alco-holic solutions make inefficient decalcifyingagents. Acids in TOVo or 80Vo alcohol act slowly.A solution of 0.5 N hydrochioric acid in707o or8O7o alcohol decalcifies in 8 days at 25" C, com-pa.red with 4 days for the same concentrationin 4OVo alcohol, and 2 days for the aqueoussolution. Five-percent aqueous formic acid solu-tions will decalcify in 3 to 5 days; the same con-

centration in 3O% alcohol will take 4 months;but the acid will not decalcify in several monthsin SOVo alcohol. This delay occurs probably be-cause the alcohol acts to suppress ionization,which in turn slows the ion-exchange reaction,rather than being caused by the insolubility ofcertain calcium salts in alcohol. Other formulasthat may be used as acid decalcifying agentsinclude those in Table 6-1.

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92 THEoRY AND PRAcTICE oF HISToTEcHNoLoGY

Formulas for acid decalcifying agentsVon Ebner's hydrochloric acid-sodium chloride mix-ture

Concentrated hydrochloric acid(specific gravity l.19)

Sodium chlorideDistilled waterDuring decalcification, add 1 ml of concentratedhydrochloric acid daily to each 200 ml of the abovemixture, until decalcification is complete.

Richman-Gelfand-Hill formic-hvdrochloric acid mix-ture

Concentrated formic acid (9OVo)Concentrated hydrochloric acid

(specific gravity 1.19)Distilled water

Kristensen's formula1 N sodium formate (6.8Vo)8 N formic acid

Evans and Krajian fluidSodium citrate crystds907o formic acidDistilled water

Krajian's variant of Evans and Krajian fluid857o formic acid95Vo ethanol (or 997o isopropanol) "Sodium citrate crystalsTrichloroacetic acidDistilled water

For the rapid decalcification of bone, strongersolutions of nitric or hydrochloric acid may beused if employed in conjunction with phloroglu-cin. The phloroglucin acts, in a way not pres-ently understood, to prevent organic constitu-ents of bone from being injured by the swell-ing and macerating action of the strong acids.Some authors state that 40Vo nitnc acid maybe used with phloroglucin. A good mixture isthe following:

5 m l70 mI

1 g m30 ml

Tissue should be removed from the decalcify-ing fluid as soon as the decalcification processis complete, otherwise the histologic and cyto-logic detail will be harmed. Testing for completedecalcification may be done by (1) flexibility oftissue, which is not recommended since it is nota completely reliable test of complete decalcifi-cation, (2) insertion of a needle to feel calciumdeposits within the tissue, which is not recom-mended since it is injurious to tissue, (3) chemi-cal testing, which is the most satisfactory testfor determining the end point of decalcificationand is performed as follows:

1. 5 ml of decalcifying fluid are nearly neu-tralized with 0.5 N NaOH (5 ml).

2. To the nearly neutralized decalcifyingfluid, I rnl of a \Vo solution of sodium orammonium oxalate is added. Turbidity inthe fluid, caused by the formation of cal-cium oxalate, indicates the presence ofcalcium ions in the fluid and in the tissue.Absence of turbidity indicates a nega-tive reaction, but several minutes shouldelapse before one should assume that thetissue is completely decalcified. If the so-lution turns turbid either immediatelyor in a few rninutes, the solutions arechanged and the test is repeated the nextday. With tissues containing only smallareas of calcium. the test should be re-peated every hour while the tissues are inthe decalcifying fluid.

3. It is important that the pH range of thenearly neutralized solution lie between 3.band 7.5. If the pH of the solution is lessthan 3.5, precipitation of the calcium ionswith the oxalate will be incomplete, result-ing in the possibility that a positive testwill be read as negative and the tissue willbe removed from the decalcifying fluid toosoon. If the pH of the nearly neutralizedsolution is greater than 7.5, magnesiumwill be precipitated as Mg(OH), andMgNHnPOo. This will also give a false endpoint, indicating that further decalcifica-tion is necessary when in fact it is nol

Most books on histologic procedures statethat when the tissue is completely decalcifiedwith mineral acid technics, the tissue should beneutralized before washing by treatment withalkali. This alkali treatment is optional, but itcan be accomplished by placing the decalcifiedtissue in a 1Vo solution of lithium or sodiumsulfate overnight. More important than a neu-tralizafon procedure, however, is a thoroughwashing of the tissue after decalcification be-fore subsequent processing technics are done.This washing is easily accomplished when thetissue is left in running tap water for 24 hours.This is a necessary step to remove all traces ofacid (or alkali if neutralization has been done).Failure to do so will impair a subsequent stain-ing procedure.

ION.EXCHANGE METHODS FORDECALCIFICATION

Before proceeding with this method for de-calcification, one must fix and wash the selected

15 ml

175 gm1000 ml

100 ml80 ml

820 ml

500 ml500 ml

l0 gm25 ml/D InI

100 ml100 ml20 gm

l g m100 ml

Nitric acidAbsolute alcoholPhloroglucinDistilled water

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Fig. 6-2. Ion-exchange methd. Gauze-wrapped boneis placed on top of resin.

tissue. Following these steps, the bone is de-calcified with a mixture of formic acid and acommercially available ion-exchange resin. Thecalcium is rapidly removed from the solution offormic acid into the resin, and this eliminatessolution changes that must be carried out toeffect proper decalcification with the acid de-calcification methods.

Tissue is placed (Fig. 6-2) in a bottle in a mix-ture of lOVo or 20Vo resin and formic acid. Can-cellous bone (2 to 3 mm in thickness) will de-calcify in 2 to 3 hours in the solution and thickerpieces (5 to 6 mm in thickness) will take 4 to8 hours to decalcify. A 40% resin and formicacid solution may be employed where speed isessential, but for good tissue preservation, thebone should not be left in this strength solu-tion any longer than necessary (up to 8 days).If speed is not essential, tissue may be left in

Thermometer.

BONE 93

the following solution up to 20 days without dis-tortion of tissue:

WIN-3000 (ion-exchange resin)olOVo formic acid (aqueous)

100 gm800 ml

The advantages of using the ion-exchangemethod for bone decalcification include well-preserved cellular detail, superior to that ob-tained with the acid decalcification methods;faster decalcification; and elimination of thedaily solution change. In addition, the resin,once used, may be reclaimed for further use bywashing to remove excess acid. The washing isfollowed by a l%a ammonia water wash, over-night treatment with saturated ammonium oxa-late, and final water wash the next day.

ELECTROLYTIC DECALCIFICATION

This method employs electrolysis to shortenthe time required for decalcification of bone sec-tions. Materials used in the technic include adurable glass jar containing the acid decalcify-ing solution in which is immersed the electrodeassembly and bone specimen, as shown in Fig.6-3. The bone specimen is suspended by a plati-num wire anode in the jar, and the insolublecalcium salts are changed to ionizable salts bythe action of the acid in the solution. A recom-mended electrolytic decalcifuing solution is asfollows:

887o formic acidHydrochloric acidDistilled water

100 ml80 ml

820 ml

*Winthrop-Sterns, Inc., New York, N.Y.

- - ' = -( _ )

------'-/Formic ocid solution

Bone (

Corbon

Fig. 5-3. Electrolytic decalcification apparatus.

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94 THEoRY AND PRAcTICE OF HISTOTECHNOLOGY

Current, which is supplied by a power unit,causes an electric field between the electrodes,and this enables the calcium ions to migraterapidly from the specimen (anode) to the carbonelectrode (cathode). The acid radicals will mi-grate from the cathode to the anode. The tem-perature of the reaction is regulated between30" to 45" C. Temperatures exceeding the upperlimit of 45' C will cause the disintegration of thespecimen. Solutions should be changed after 8hours of use to ensure maximum speed of de-calcification. Prolonged washing of the speci-

men after electrolysis is unnecessary; the tis-sues are rinsed well in alkaline water and thesections immersed in lithium carbonate beforestaining. The lithium carbonate treatment of acut section will neutralize any remaining acidin the tissue so that the acid cannot interferewith any staining procedure. The chief advan-tage to the electrolytic method lies in the short-ened time required for complete decalcifica-tion. This faster time will speed diagnosis andgive better preservation of soft-tissue patterns.

Staining reactions are usually better, since themethod acts fast enough and the tissues havea relatively short time in the acid bath. Gen-erally, cancellous bone 3 to 5 mm in thicknesswill decalcify in 45 minutes or less. More com-pact bone will take 16 hours or longer. A dis-advantage to this method is that only a limitednumber of specimens may be processed at anyone time. Maintaining contact between tissueand electrode may also create problems.

HISTOCHEMICAL METHODS FORDECALCIFICATION

Standard methods of decalcification using ni-tric, hydrochloric, and other acids are unsatis-factory if histochemical technics are to be doneon the tissue, since acid treatment will destroythe enzyme activity. Analytical methods fornucleic acids and polysaccharides should alsobe preceded by a histochemical, rather than

routine, method for decalcification since thesesubstances are largely destroyed by the acidsemployed in the routine technics. Histochemi-cal decalcification methods include the use ofchelating agents and buffer mixtures.

Buffer mixtures

Calcium salts may be removed from bonewhen it is placed into a buffered solution ofcitrate, pH 4.5. Calcium salts are soluble at thispH, and the zinc present in many buffered solu-tions of citrate will produce a reversible in-

activation of the alkaline phosphatases. (Sub-sequent reactivation will allow for their dem-onstration.) Daily changes of the buffer arenecessary and the decalcification progress maybe checked by use of the chemical oxalate testdescribed in the section on acid decalcificationmethods.

Tissue should first be fixed in cold 80Vo alco-hol for 24 to 48 hours. It is then placed in thebuffer solution at refrigerator temperature (4oC) until decalcification is complete. Tissue isthen washed in tap water, followed by distilledwater, and then placed in a sodium barbitalsolution at 37o C for 6 hours to neutralize thetissue from the effects of acid citrate and to re-activate the enzyme activity. After the barbitaltreatment, the fissue is washed for 3 hours inrunning tap water, and processed for subse-quent enzyme analysis. Solutions used are asfollows:

Citric acid-cirate buffer (pH 4.5)1 N citric acid (monohydrate, TVo)I N ammonium citrate

(anhydrous, 7.54Vo)l7o zinc suHateChloroform

Sodium barbital solutionSodium barbitalGlycine

Other buffer mixtures

50 ml950 ml

2 m l0.1 ml

100 ml75 mg

L Molar hydrochloric acid-cirate buffer at pH 4.5I N hydrochloric acidI M sodium citrate solution(Use 29.4Vo of the dihydrate or 35.7% of

the following compound: Na(CO,)3C3H4OH'iY2HIO.)

2. Lorch's citrate hydrochloric acid buffer at pH 4.4Citric acid crystals0.2 N sodium hydroxide0.1 N hydrochloric acid7Vo zinc sulfateChloroform

3. Acetate buffer at pH 4.51 N acetic acid1 N sodium acetate (8.27o

anhydrous or 13.6% crystelline)l%a zinc sulfateChloroform

Chelating agents

General considerations. Chelating agents areorganic compounds that have the power ofbinding certain metals. For decalcification,Sequestrene, or Versene (ethylenediaminetet-

raacetic acid, or EDTA), has the power of bind-ing calcium ions according to the equation inFig. 6-4.

540 ml460 ml

14.7 gm700 ml300 ml

2 m l0.1 ml

520 ml480 ml

2 m l0.1 ml

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Thin sections of bone are fixed for 24 hours incold 80Vo alcohol, washed in running tap water,and transferred to a solution of EDTA, adjustedwith sodium hydroxide to a pH of 6.0 to 6.5. Theexact pH is not critical since satisfactory resultscan be obtained at values ranging from 5.0 to7.2. A slightly acid pH is preferable, however,since alkaline phosphatase is more stable in anacid rather than alkaline solution. About 50 mlof a 5Vo solution is usually adequate, but theconcentration is unimportant provided that asufficient excess of the chelating agent is avail-able to bind all the calcium and magnesiumpresent in the bone. Decalcification by thismethod is slow and requires three changes ofsolution lasting 4 to 5 days each. The tlssuesalso tend to harden, but tissues decalcified bythis method also show a minimum of artifactand may be stained with most staining tech-niques with good results. The actual rate of de-calcification varies with the size and density ofthe bone sample and can be hastened by oc-casional agitation or addition of fresh solution.Cancellous bone will usually be completely de-calcified in about 3 days, whereas dense corticalbone requires as much as 2 weeks. Tissues thathave been exposed to the chelating agent for aslong as 6 weeks have yielded satisfactory prep-arations. When decalcification is complete, thetissue is washed thoroughly in running waterand processed. Since the EDTA inactivates thealkaline phosphatase system in the process ofdecalcification, sections should be reactivatedwith a suitable activating ion, preferably mag-nesium. Placing the sections in a 1Vo solutionof magnesium chloride for 2 to 6 hours or longeris usually sufficient to return them to maximalactivity. During staining technics, a controlsection omitting the substrate is essential, asincomplete decalcification can produce areas offalse reaction.

Demonstration of specific enzymes

1. For the demonstration of succinic dehydro-genase and NAD- and NADP-linked dehy-

otl

NaO-C-CH,\ . /

N - C H r - C H 2 - N

oll

cHr-c -oNa

BONE 95

drogenases and diaphorases, bone can bede-calcified by a 3- to S-day immersion at 4"to 10' c in 10vo EDTA in a 0,1 M phosphatebuffer of pH 7.0. The pH should be read-justed to 7.0 when both the buffer and EDTAare in the solution.

2. For the demonstration of acid phosphatase,tissues should be fixed for a maximum af 24hours i.n a l07o to 20Vo solution of neutralformalin at 4" C. Since the use of chelatingagents such as EDTA causes loss of acidphosphatase, a decalcifying solution com-posed of citrate and formic acid pH 4.2 isrecommended. A pH below 3.8 will destroythe enzyme activity, The following solutionmay be used:

2OVo sodium citrateFormic acidDistilled waterpH to 4.2

50 ml2.5 ml

47.5 ml

After decalcification, frozen sections are pre-pared. Paraffin embedding will destroy theenzyme activity.

3. For the demonstration of alkaline phospha-tase, tissues should be fixed in 7O% ethanolor isopropanol at 20" to 25' C. Decalcificationis carried out at 0o to 5o C in a buffer mix-ture of pH 4.5 or higher. Ammonium citrateand citric acid is effective. as well as normalacetate buffer. Both these mixtures will de-calcify more rapidly than will Lorch's mix-ture or the 0.1 M citrate and citrate-hy-drochloric acid mixtures. EDTA may alsobe used to decalcifi'. Solutions should bechanged daily until the oxalate test is nega-tive. If it becomes difficult to continue dailychanges for decalcification, one may inter-rupt the process, washing briefly in water forl0 to 15 minutes, transfening to 80Vo alco-hol, and storing at -2O" to -25'C in a deep-freeze compartment until daily changes canbe resumed. Tissues should be washed for10 to 15 minutes in running tap water be-fore being returned to the decalcifying solu-

o o

N a O - C - C H , . r C H 2 - C - O N a

N - C H " - C H . - N

, / . - - - - - - - - ' - \ + 2 N a *H r C

- C a a * C H ,

\ , / \ /c-o o-c

/ / \o o

EDTA-colcium complex

, / \NaO-C-CH2 CHg-C-ONa

t lo o

EDTA

+ [ C a * + ] +

Fig.5-a. Calcium chelation by EDTA.

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96 THEoRY AND PRAcTIcE oF HISToTEcHNoLoGY

tion. When the decalcification process iscompleted, tissues should be washed over-night in running water, incubated 6 hoursin a 7Vo sodium barbital solution (containing75 mg/dl glycine) to reactivate the enzyme,washed 2 to 4 hours to remove the glycine,dehydrated, cleared in gasoline or petroleumether, and infiltrated 10 to 15 minutes inparaffin at 58' C in a vacuum.

Demonstration of glycogen indecalcified tissues

Total or partial losses of glycogen result whentissues are decalcified with acids or EDTA. Gly-cogen in muscle and marrow cells will with-stand decalcification best when fixed in aque-ous formalin acidified with acetic or formic acid.Thorough fixation of protein surrounding gly-cogen and embedding in celloidin before paraf-fin will create semipermeable membranes sur-rounding the glycogen and thus hinder diffu-sion and re-solution. Lillie uses the followingtechnics for decalcification to subsequentlydemonstrate glycogen:

Technics1. Celloidin

a. Tissue should be fixed in aceric alcohol for-malin 24 hours at room temperature (25" C)or 3 to 4 days at 5' C. Dehydrate with alco-hols and infiltrate for 3 days trurth l%o celloi-din in equal volumes of alcohol and ether.

b. Transfer to SOVo alcohol to harden celloidin.c. Decalcify with 1Vo formic acid (aqueous);

solution should be changed daily until a neg-ative chemical test is obtained.

d. Wash 6 to 8 hours in running water, dehy-drate, clear, and embed in paraffin.

2. Hard protein fixativea. Fix tissues as described above.b. Transfer to Bouin's fluid for 3 days at2tr C.c. Decalcift in daily changes of l}Vo formalin

with 1Vo formic acid.d. Wash 8 hours in running water; dehydrate,

clear, and embed in paraffin.

REFERENCES

Dotti, L., Paparo, G. P., and Clarke, B. E.: The use ofion exchange resin in decalcification of bone, Am. J.Clin. Pathol. 212475-479, 1951.

Lillie, R. D.: Histopathologic technic and practicalhistochemistry, ed. 3, New York, 1965, McGraw-Hill Book Co.

Pearse, A. G. E.: Histochemistry, theoretical and ap-plied, ed. 2, Boston, 1960, Little, Brown & Co.

Preece, A.: A manual for histologic technicians, ed. 2,Boston, 1965, Little, Brown & Co.

Richman, I., Gelfand, M., and HilI, J. M.: A methodfor decalcifying bone for histologic section, Arch.Pathol. 44:92-95. 7947.

Sobel, A. E., and Hanok, A.: Rapid method for deter-mination of ultramicro quantities of calcium andrnagnesium, Proc. Soc. Exp. Biol. Med. 77:737-740,1951 .

Part ll. Basic preparation andstaining of undecalcified boneAntonio R. Villanueva

The primary objective of using undecalcifiedbone in histopathology is to gain insight into thehistologic diagnosis and investigation of certainmetabolic bone diseases, particularly the osteo-malacias. r,6'rr'1e It is also an essential prerequi-site for distinguishing mineralized from non-mineralized areas, for contact microradiogra-phy, and for histomorphometric analysis of boneremodeling. Mineralized sections of bone suit-able for microscopy have been used by severalinvestigatofs,2'3'ro'ra,r5'rz but such sections wererarely employed in the routine histopathologylaboratory.

During the last few years, many biopsies fromsuspected cases of metabolic bone diseases havebeen reported routinely from decalcified sec-tions. The use of decalcified sections, however,

have not proved as reliable as undecalcified sec-tions in distinguishing between mineralizedbone and osteoid. Therefore, for routine histo-logic and pathologic study of certain metabolicbone diseases, the ideal histologic technic isthe use of undecalcified bone, a condition wherethe tissue deviates minimally from the livingstate thus permitting maximum resolution ofthe components.

MINERALIZED BONE TECHNIC

(In this text, "mineralized" is interchangeablewith "undecalcified.")

Preparation of undecalcified sectionsof bone

There are four methods of preparing and sec-tioning mineralized bone. First, there is the useof electrically powered machines that have adiamond cutting wheel and can do thin section-ing; second, there is the use of plastic embed-

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ding procedures, followed by cutting with one ofthe thin-sectioning machines, Jung "K" micro-tome or other heavy duty microtomes; third,there is the use of double embedding tech-niques with low-viscosity nitrocellulose or cel-loidin in combination with paraffin wax afterimpregnation with the celloidin, followed bycutting in a heavy-duty base sledge microtome;and fourth, there is the method of Frost's forpreparing mineralized, fresh, ground, thin sec-tions of bone.8'e

In general, procedures of preparing undecal-cified bone for histopathology have very muchin common with routine paraffin sections. Ex-cept for a little modification, the sequence isthe following: (1) fixation, (2) dehydration, (3)clearing, (4) infiltration, (5) embedding (plas-tic), (6) sectioning (grinding), (7) staining.

Sectioning machines

The increasing use of mineralized bone workresulted in the development of various thin-sectioning machines. Currently there are sev-

BONE 97

eral on the market, but the most common onesused today are the following: Gillings-HamcoThin Sectioning Machine, Buehler Isomet, TheH/I Bright 5030 Universal Rotary RetractingMicrotome, Sorval Porter Blum JB 4 Microtome,Jung-K Microtome, and Leltz Sledge Micro-tome.o The first two are excellent for cuttingbone sections at 50 to 100 p.m thick, and theother four are for celloidin or plastic embeddedsections at 5 to l0 p.m thick.

Ground sectioning

There is another method developed as a tech-nic for manual grinding of fresh, unembed-ded bone specimens. It is a very simple methodfor preparing sections of undecalcified bone

*Comparries making thin-sectioning machines: Gillings-Hamco, Bronwill Scientific, Rochester, N.Y.; Buehler Isomet,Buehler Ltd., Evanston, Ill.; H/I Bright 5030, Hacker Instru-ments, Inc., Fairfield, N.J.; JB-4, DuPont Insruments-Sor-vall, Newtown, Conn.; Jung-K, American Optical Corp., Buf-falo, N.Y.; Leitz Slcdge, Scientiffc Products Div., AmericanHospital Supply Corp., McGaw Park, Ill.

Fig. 6-5. Abrasive paper is wrapped around a microslide and held as indicated in this figure. Bonesection will adhere to this strip of paper when placed upon it.

Copyright © 1987 by Battelle Memorial Institute 

98 THEoRY AND PRAcTIcE oF HISToTEcHNoLoGY

Fig. 6-6. Completed ground section approximately 75 s.cm in thickness.

with use of abrasive sandpaper of various grades(wet or dry waterproof adhesive) on a ffat, plas-tic or glass sheet where the sections are groundby hand under gently running water.

Method of preparing ground sections1. Bone specimens are sawed to 2 or 3 mm thick

with a very fine toothed jewelers' saw or copingsaw. The material may be held in a small vise,or in a conventional wooden V-block duringthe sawing process. While cutting, moisten thespecimen with water intermittently in order tominimize friction. Finally bone slabs are placedin a beaker of saline or alcohol (or acetone) de-pending on the substance under study.

2. The slab of bony tissue is then ground to ap-proximately 75 to 100 g,m thick. Abrasive warer-proof'Carborundum sandpaper is employed forthis purpose. This abrasive material is a sand-paper fbund in most paint and hardware storesand known as "wet-or-dry" sandpaper becausethe adhesive is waterproof.

Section grinding is simple. The sandpapershould be placed on a flat, smooth surface of

aciylic plastic (Plexiglas) while rhe sections areground by hand in a circular motion undergently running tap water. The trick to the pro-cedure is illustrated in Figs. 6-5 and 6-6. A stripof the same abrasive paper wrapped around amicroslide as indicated in Fig. 6-5 is used to at-tach the section intack during grinding. Thespecimen will adhere to this strip when placedupon it. Should the sections fail to adhere to thestrip of paper, a fresh one must be substituted.Accordingly, when the hand ground section isfinished, as in Fig. 6-6, the section of bone ad-heres to the paper beneath.

Each finished ground section is placed into abeaker of distilled water until ready for washingin a detergent solution (A.}lVo mild detergent orO.lVo Zephiran Chloride solution). The proces'sremoves grinding debris and bone dust and pre-pa.res the bone section for staining. The averagetime required to grind a section such as a rib,is approximately 2 minutes, whereas larger onessuch as the tibia and femur would take at least15 minutes or longer.

Copyright © 1987 by Battelle Memorial Institute 

PLASTIC EMBEDDING

Several types of plastic materials used differchiefly in the hardness of the final product andthe ease of impregnation and polymerization.Some of these plastics used today are the fol-lowing: methyl (or butyl) methacrylate embed-ding, Araldite embedding, Bio-Plastic, and Poly/Bed 812 embedding media. In our laboratorypolymethyl methacrylate is used because it isrelatively simple and can be used either forJung K microtome sectioning or can be sawn forground preparations.

The number of methods for plastic embed-ding are legion, but all of them have the samething in common, which ls they all go throughthe process of fixation, dehydration, infilnationin the plastic monomer, embedding with poly-mer, and polymerization or hardening of theplastic.

Procedure for routine processing andmethacrylate embedding of bone tissue

When bone specimens are received, such asby iliac bone trephination or by needle or ribbiopsies, they are placed immediately in one ofthe fixatives conducive to such study. Exam-ples of these fixatives are alcohol,l}Vo bufferedformalin, and formol-calcium. Heaay-metal

fixatiaes, dichromate, regular 10Vo formalin,and fixatiaes containing acid interfere uith thestaining and should not be employed.

If tetracycline has been given, or if bone-seeking radionuclides such as plutonium areused and radioautography is the main objective,alcohol fixations should be employed to mini-mize bleaching of the tetracycline or isotope.

Preparation of bone specimen forsimultaneous tetracycline-osteoid seamassessment and histomorphometricquantitative analysisl 3

Preparation of polymethyl methacrylateembedding medium

Add the following ingredients (do not makemore than 250 ml at a time): 250 ml of methylmethacrylate monomer;2.5 gm of benzoyl per-oxide. Stir and dissolve thoroughly for 2 min-utes; then add 100 gm of polymethyl methacry-late beads. Stir the mixture in a closed containeron a magnetic stiner. Frequently, at hourly in-tervals, mix beads manually into the mixture.Total stirring time may require up to 2 fullworking days. Note: Do not let mixture bestirred oaernight When all the beads are dis-solved, the medium is ready for embedding.

BONE 99

Stor" in the refrigerator in a tightly closed con-tainer. Note: If catalyzed monomer has beenstored in the refrigerator, it shou.ld be alloutedto warm gradually to room tempera.ture heforeuse. This is necessary to preuent condensationof water, which utill interfere with polymeriza-tion.

Processrn g and embedding procedure

1. Fresh or fixed bone specimens are stainedin Villanueva's bone stain for 72 hours.

2. Dehydrate in vacuum desiccator accordingto the following:a. I change of707o ethanol for t hourb. 2 changes of 95Vo ethanol, lYz hours eachc. 2 changes of l00Vo ethanol, 11lz hours

eachd. Acetone, lYz hours

3. InfiItrate in the following:a. Equal parts of acetone and methyl meth-

acrylate monomer, for overnightb. Methyl methacrylate monomer, for 24

hoursc. Embedding medium for 24 hours; place

in the refrigerator. Nofe: These are thestandard times used for iliac bone speci-mens. For larger or denser boneE, doublyincrease sta.ining time and dehydrationand infi.ltration times to those giuenaboae.

4. Using Peel-A-Way embedding molds,oembed specimens in the embedding me-dium.

5. Polymerize in a vacuum oven at 37" to 40' Cat a pressure of 15 to 20 pounds of mercury.Note: If there is a shrinhage of plastic uhilepolymerizing, add more embedding mediumto compensate for this phenomenon. Repeatas necessa.ra.

6. Polymerization takes approximately 3 to 5days. Plastic must be completely hardenedbefore cutting.

Sectioning

There are two methods used for cutting sec-tions: (1) dry method and (2) wet method. Inthe dry method, sections are cut without appli-cation of alcohol on the block and may be usedfor qualitative as well as quantitative analysis.They are most suitable for observation of tetra-cycline labels. The wet method involves theapplication of alcohol on the block during cut-

*Peel-A-Way Scientific, 1800 Floradale Ave., South El Monte,cA 91733.

Copyright © 1987 by Battelle Memorial Institute 

lOO THEoRY AND PHAoTIcE oF HISToTEcHNoLoGY

ting and are used for future staining to suitindividual requirements.

Procedure for dry sections1. Cut sections at 5 or l0 g.m thick.2. Do not use ethanol in cutting these sections.3. Trim away excess plastic from each section,

handling as carefully as possible to minimizeshattering.

4. Mount with Eukitt's mounring medium.

Procedure for wet sections1. Apply 35Vo ethaxrol to the cutting surface of

the specimen block.2. Cut sections 5 to 10 g,m thick.3. Trim excess plastic from each section, han-

dling as carefully as possible to minimize shat-tering.

4. Place a liberal amount of Haupt's gelatin af-fixative on a slide.

5. Place bone section on top of affixative andcover with bibulous paper.

6. Flatten gently with a roller and allow to ser forseveral minutes.

7. Lfit bibulous paper and place several drops of27o formalur on the section and blot again withfresh bibulous paper.

8. After 5 minutes remove bibulous paper andplace slides on a slide-warming plate set at b0'to 60' C for t hour.

9. Dissolve plastic off these sections by immers-ing slides in fresh xylene and then place in rheoven at 50' to 60' C for 3 to 5 hours.

10. Remove slides from xylene and allow to air-dryfor at least I hour. Stain as desired.

CommentsIn our laboratory most of the bone specimens

received are prestained with the Villanuevabone stain for 72 hours before dehydration andembedding because it gives a better differentia-tion of osteoid seams and tetracycline fluores-cence when studied with the light and fluores-cence microscope respectively. Fixation ofbone specimen prior to staining with the bonestain is not necessary; the bone stain itself actsas a fixative. since it contains TAVo alcohol.

METHODS OF STAINING

Methods of staining mineralized, thin sec-tions of bone have changed little in recent years.The many attempts to stain fresh, hydrated,unembedded, and plastic-embedded mineral-ized bone sections have resulted in few suc-cesses.

The following staining methods have provedto be excellent in the approach to mineralizedor undecalcified bone staining.

Frost's basic fuchsin stains (for stainingJresh or fixed, unembedded sections of bo.ne)

Fixation. 70Vo alcohol.

SolutionBasic fuchsin solution I gmEthanol,807a 100 mlDissolve the basic fuchsin thoroughly in the aI-cohol and then filter.Nofe: For the bone of infants, young children, orvery immature animals, reduce the stain concen-tration to O.3Vo, since such bones contain largevolumes of incompletely mineralized matrix,which will be deeply, often opaquely stained.

Procedure1. Grind sections to approximately 50 to 100 pm

thick.2. Rinse in distilled water.3. Stain in the basic fuchsin solution for 48 hours.4. After staining, place sections in tap water then

lightly grind surface stains.5. Wash sections in O.lVo Zephiran Chloride or in

0.1Vo wnld household detergent, such as Lux orIvory.

6. Again wash sections in tap water and then rinsein distilled water.

7. Pldce sections between two slides with one slidesurface covered with filter paper trimmed to thelength and width of the slide. Clamp the slidesgently with a Hoffrnan clamp and allow to dryin a 45'C oven overnight.

B. Mount in Eukitt's mounting medium.

ResultsFully mineralized bone-unstainedEarly stages of mineralization and new bone forma-tion-fuchsin stainHalo volumes (perilacunar red density of appre-ciable numbers around the osteocytes)?

Villanueva's bone stain20 (for staining fresh,or fixed, unembedded, or plastic-embeddedsections of bone)

Fixation. TOVo ethanol.

SolutionsVillanueva's bone stainoA.OlVo glacial acetic acid in TOVo methanolO.lvo Zephiran Chloride (or O.OlVo mild house-

hold detergent)

Procedurel. Cut bone into slabs to approximately 2 to 3 mm

thick.2. Grind sections to 50 to 100 pm thick under

gently running water. (This method may be

'Either from Histo-Cyto-Prep Inc., Box 524, Bloomfield Hills,MI 48013, or from Polysciences, Inc., Paul Valley IndustrialPark, Warrington, PA 18976.

Copyright © 1987 by Battelle Memorial Institute 

Orange GDistilled water

BONE 101

2 g m100 ml

omitted if thin-sectioning machine is used tocut sections at desired thickness prior tostaining,)

3. Rinse sections in distilled water.4. Stain in the Villanueva bone stain:

a. 1 or 2 sections for 90 minutes.b. 2 to 4 sections for 48 hours.

Note: The 9O-minute staining yields faststains of diagnostic usefulness but with in-complete permeation of the section by thestain; the 48-hour staining is for completepermeation of bone and tissue elements.

5. Transfer sections into tap water and then grindsurface stains. (Optional if sections are cut todesired thickness with thin-sectioning ma-chine.)

6. Wash sections with 0.lVo Zephiran Chloride orin O.OlVo mild household detergent.

7. Again wash sections with tap water and thenrinse with distilled water.

8. Differentiate in O.Ol7o glacial acetic acid in957o methanol:a. For the 90-minute staining, differentiate

fo r3 toSminu tes .b. For the 24-hour staining, differentiate for

l0 minutes.c. For the 48-hour staining, differentiate for

20 to 25 minutes.9. Dehydrate in the following:

a. 957o alcoholb. l3o7odcohol

10. Clear in the following:a. Equal patrs of alcohol, 10O7o, l0 min

plus xyleneb. 1 part of lO}Vo alcohol and

3 parts of xylenec. 1 part of lODVo alcohol and

9 parts of xylened. Xylenee. Xylenef. Xylene

11. Mount in Eukitt's mounting medium.

Resultsosteoid seams-transparent green to iade grcen

or homogeneous redZone of demarcation-orange-redLow-density bone-redModerately permeable bone-orangeOsteocytes, canaliculi, halo volumes-redNuclei of osteoclasts, osteoblasts-greenish blue to

dark purpleCytoplasm-green or light grcen

V ittanueva's tetrach rome bone staln le

(for staining fresh or fixed unembeddedmineralized sections of bone)

Fixation. 70% alcohol

SolutionsA. Fast green FCF 0.1 gm

Note; The mixture is adjusted between pH 6.5and 6.8 with 3 N acetic acid. Add a few crystalsof thymol.Azure II 0.25 gmEthanol, 50% 100 mlBasic fuchsin 1 gmEthanol, SOVo 100 mlFor staining, mix 1 ml of solution B and 9 mlof solution C.

Procedure1. Prepare sections about 75 to 100 p,m thick by

Frost'sr method. Before making sections, re-hydrate any alcoholated material by soaking itin a large volume of distilled (or tap) water foran hour to decrease its brittleness. Even 5%alcohol may make the bone unworkably brittle.

2. By manual or mechanical agitation wash in0.1% invert soap (Zephiran Chloride) in tapwater and then rinse in distilled water for 1minute each.

3. Stain in solution A for 12 to 15 hours. (In biop-sy material, t hour yields fast stains of diagnos-tic usefulness but with incomplete permeationof the section.)

4. Remove surface stain by regrinding lightlyunder tap water. Rinse in distilled water.

5. Counterstain in solution B for I to 2 hours.(In biopsy material, 15 to 30 minutes will per-mit quick reading but incomplete permeationof the section.)

6. Repeat step 4.7, Repeat step 2.8. Differentiate in O.01Vo acetic acid in 957o

methanol, 3 to 5 minutes. At l-minute inter-vals, examine the section under a microscopeto determine the staining of osteoid seams,taking care that the sections are not allowed todry. Differentiate until the osteoid seams haveacquired the maximum green tint.

9. Dehydrate in:957o alcohol

1007a alcohol10, Clear in the following:

Equal parts of 1007o alcoholplus xylene

1 part of l1OVa alcohol and3 parts of xylene

1 part of l1OVo alcohol andI parts of xylene

Xylene, 2 changes

B.

C.

D.

15 min15 min

5 min

5 min

10 min5 min5 min

9 min8 min

7 min

6 min

5 min

4 and 3 minrespectively

11. Mount in Eukitt's mounting medium,

ResultsSee results in the bone stain method.

Moditied V illanuev a-Gamori trichrome2l(for plastic-embedded sections of bone)

Copyright © 1987 by Battelle Memorial Institute 

1O2 THEoRY AND PRACTICE OF HISTOTECHNOLOGY

Fixation. 70Vo ethanol 10Vo buffered forma-lin.

SolutionsWeigert's hematoxylin (see solution C in preced-

ing column)Masson's Ponceau-fuchsin stock solution

Ponceau de xylidine, C.l. 16150, 3 partslVc aqueous solution

Acid fuchsin C.L 42685,7Vo I partaqueous solution

Dilute 1 :10 uith 0.2Va acetic acid,for staining.Phosphotun gstic-phosphomolybdic solu tion

SolutionsWei gert's hematoxylin-modifi ed

Solution AHematoxylin crystalsEthanol,95Vo

Solution BFenic chloride, FeCl3 ' HzO.62VcDistilled waterHydrochloric acid

2gm200 ml

8 m l190 ml

2 m ISolution C

Equal parts of solutions A and B. (Make upfresh working solution for each new batch ofslides, and prepare stain immediately beforeuse. )

Trichrome stain mixture-modifi edChromotrope 2R C.l. 16570 0.2 gmLight green C.I. 42095 0.1 gmGlacial acetic acid 1.0 mlPhosphotungstic acid 0.8 gmDistilled water 100 mlFilter solution before using.

Procedure1. Deplasticize to distilled water.2. Stain in Weigert's hematoxylin solution for

5 minutes.3. Rinse under running water, differentiate in l7o

aqueous hydrochloric acid, rinse in water, bluewith saturated aqueous solution of lithium car-bonate, and wash under running water for 3to 5 minutes.

4. Transfer to the modified trichrome mixture for5 minutes.

5. Differentiate in aqueous O.1Vo or l.OVo acettcacid for 1 minute.

6. Rinse in distilled water, Check microscopicallyand repeat steps 4 and 5 until proper differ-entiation of bone matrix is reached.

7. Dehydrate:95Vo alcohol l0 dipslOOTo alcohol 15 dipsIOOVo alcohol 20 dips

8. Clear in:Xylene 25 dipsXylene I minXylene 5 min

9. Mount in Eukitt's mounting medium.

ResultsBone matrix and collagen-bluish greenNuclei-dark purple to blackOsteoid seam-bright redMuscle-red

M od it ied V i lla n uev a -G old ner tric h romezl(for plastic-embedded secfions of bone)

Fixation. 70% ethanol. lOVo buffered forma-lin.

Phosphotungstic acidPhosphomolybdic acidDistilled waterDissolve thoroughly.

Naphthol green B solution, 17oNaphthol green B, C.I. 10200Distilled waterAcetic acid

Alcohol saffron solutionSa{?an du Gitinais Chroma" 54-394 1 gmAbsolute ethanol 100 mlPlace in stoppered flask in 55o to 60'C ovenfor 2 days. Agitate from time to time. Cool andfilter.

Procedure1. Deplasticize to distilled water.2. Stain in modified Weigert's iron hematoxylin

for 20 minutes. Rinse under running water,differentiate with 17o aqueous hydrochloric acid,rinse with water, blue with saturated aqueoussolution of lithium carbonate, and wash underrunning water for 3 to 5 minutes.

3. Transfer to Masson's Ponceau-fuchsin (diluteformula) for t hour. Rinse in lVo acetic acid.

4. Place in phosphotungstic-phosphomolybdic acidfor 5 minutes. Rinse with l% acetic acid, andplace in fresh lvo acetic acid for 5 minutes.

5. Transfer to naphthol green B solution for 15minutes.

6, Rinse in the following:Distilled water95% alcoholTOOVo alcohollO}Vo alcohol

7. Place in the alcoholic saffronminutes.

8. Transfer toIOOVo alcoholIOOVo alcoholXyleneXyleneXylene

8 dips20 dips20 dips20 dips

solution for 15

2.5 gm2.5 gm100 ml

1 g m100 ml

l m l

l5 dips15 dips15 dips15 dipsI min

9. Mount in Eukitt's mounting medium,

ResultsNuclei-dark purple to blackCytoplasm-bright pink

'Roboz Surgical Instrument Co., Inc., Washington, DC20006.

Copyright © 1987 by Battelle Memorial Institute 

Bone matrix and collagen-bright greenish yellowMuscle-scarletOsteoid seams--bright red

Villanueva's blood stain22(for plastic-embedded sections of bone)

Fixation. 70Vo ethanol. lOVo buffered forma-lin, B-5.

SolutionsStock Villanueva's blood stain I*Stock Villanueva's blood stain II*Working phosphate buffer

Solution CSolution DDistilled water

Working staining mixture ISolution ADistilled water (or solution E)

Working staining mixture IISolution BDistilled water (or solution E)

O.SVo glacial acetic acid, aqueous

Procedurel. Cut sections to approximately 5 pm.2. Decalciff plastic embedded section of bone in

one of the decalcifying solutions for 2 to 5 min-utes.

3. Wash in water for 5 minutes and then rinse indistilled water.

4. Stain in working staining mixture I for 10 min-utes. Discard after use.

5. Transfer directly to working staining mixtureII for 45 minutes. (It may be left in this solutionIonger to suit individual requirements.) Nofe:Carry one slide at a time in the nuct remainingsfeps. Discard mixture after each use.

6. Differentiate in O.1Vo glacial acetic acid solu-tion, 5 to 10 dips. Immerse shde in distilledwater and then check differentiation under amicroscope to ascertain the depth of stainingbut take care that the preparation is not allowedto dry. If the desired degree of staining has notbeen attained, retum the slide to the differentiat-ing solution and rinse again with distilled waterbefore further examination. This process may berepeated several times until differentiation iscomplete. The Inndmarhs are pinh eosinophilgranulcs and sharp, brilliant purple nuclei.

7. Dehydrate and clear in the following:95Vo ethanol

700Vo ethanol5 to l0 dips5 to 10 dips

Equal parts:Xylene plus 1007o ethanol 10 to 20 dips

BONE 103

ResultsNuclei of bone marrow cells-dark bluish purple

' Cytoplasm-Iight blue to grayNuclei of osteocytes, osteoblasts, osteoclasts-dark

purpleCytoplasm-Iight blue to grayEosinophil granules-bright pinkMast cell and basophil granules-dark purple

A moditication of Movat's pentachrome stain 16

(for plastic-embedded secfions of bone)

Fixation. SVo phosphate-buffered formalin,4OVo ethanol, Carnoy's fluid.

SolutionsWeigert's iron hematoxylin

Solution AHematoxylinEthanol,95Vo

Solution BFerric chloride (FeCl, .

Distilled waterHydrochloric acid, 25Va

Worhing solutionMix equal parts of solution A and solufionB. The mixture is stable for approximately 8days.

Brilliant crocein-acid fuchsinSolution A

Brilliant crocein (C.1. 27 2gO)Distilled waterGlacial acetic acid

Solution BAcid fuchsin (C.I. 42685)Distilled waterGlacial acetic acid

Worhing solutionMix 8 parts of solution A with 2 parts of so-lution B. The mixture is stable for severalmonths, but it should be replaced when itbegins to stain weakly.

Alcoholic saffron

2mL2 rnl

96 ml

10 rnt30 rrit

5 rnl45 ml

10 to 20 dipsI to 5min

Safran du Gitinais (C.I. 75100)Ethanol. ljo%o

l g m100 ml

THrO) 1.19 gm98 ml

l m l

0 . 1 g m99.5 ml0.5 rnl

0.1 gm99.5 ml

0.5 rnl

3 g m100 ml

8. Mount in Eukitt's mounting medium.

*Polysciences, Inc., Paul Valley Industrial Park, Warrington,PA 18976.

Place the solution in a tightly plugged bottlein an incubator at 50" C for 48 hours beforeuse. This is done to extract the saffron. Keepthe solution in a fully filled air-tight dark bottleto prevent hydration, which will make thesolution useless. The stability of the solutionis largely dependent on the adherence to theseprecautions.

Alcian blueAlcian blue 8 GS (C.L 74240) I gmDistilled water 100 mlGlacial acetic acid 1 mlThe solution is stable for 2 to 4 weeks.

Procedurel. Remove methyl methacrylate with 2 changes

of methyl Cellosolve acetate followed by 2

XyleneXylene

Copyright © 1987 by Battelle Memorial Institute 

104 THEoRY AND PRAcTIcE oF HISToTEcHNoLoGY

changes of methyl Cellosolve acetate contain-ing lVo celloidin, 15 minutes each.Hydrate to water through TOVo and 4OVo etha-nol, 5 minutes each.Rinse in distilled water.Wash in running tap water for 6 minutes.Transfer to alkaline ethanol (pH over 8) for Ihour. Prepare by adding l0 ml of concen-trated ammonium hydroxide to 90 ml of 95Voethanol. This converts the alcian blue intothe insoluble nonfading pigment monastralfast blue.

6. Wash in running tap water for 10 minutes.7. Immerse sections in Weigert's iron hematoxy-

8.L

10.1 1 .

72.13.

lin for 10 minutes.Rinse in distilled water.Wash in running tap water for l0 minutes.Rinse again in distilled water.Immerse sections in brilliant crocein-acidfuchsin for 8 minutes.Rinse in 0.5Vo acetic acid.Differentiate in 5Vo aqueous phosphotungsticacid until mineralized bone matrix is merelypale pink; this usually requires 15 to 20 min-utes.

14. Wash in O.1Vo acetic acid under continuousagitation for 2 minutes.

15. Rinse thoroughly in 3 changes of 1007a ethanolfor 5 minutes each. (It is essentid not to useless concentrated ethanol, which will dissolveaway the cytoplasmic stain and prevent thetissue from taking the collagen stain.)

16. Immerse sections in alcoholic saffron solutionfor 30 to 40 minutes.

17. Rinse thoroughly again in 3 changes of 100%ethanol for 3 minutes each.

18. Transfer into methyl Cellosolve acetate for 2minutes.

19. Clear in 2 changes of xylene and mount inEukitt's mounting medium.

ResultsNuclei-black to bluish grayCytoplasm-redElastic fi bers-yellow (coarse fi bers-red)Cartilage-red or yellowCalcifi ed cartilage-sea greenOsteoid-redMineralized bone-yellow

Gord on and Swee t's method fo r ce menl lines s

(for plastic-embedded sections of bone)

Fixation. 70Vo ethanol, lj%o buffered forma-lin.

SolutionsAcidifi ed perrnanganate solution

Potassium permangan ate, O.1VoSulfuric acid,3.AVoThis solution is stable for 48 hours.

Oxa]ic acid. IVo

48 rnl2ml

Iron alum (ferric ammonium sulfate),2.57oDiamine silver hydroxide solution

To l0 mI of lOVo solution of silver nitrate, add287o ammonium hydtoxide drop by drop. A pre-cipitate will form. Continue to add ammoniumhydroxide, shaking vigorously until the precipi-tate disappears. Then add l0 ml of 8% sodiumhydroxide. Again add ammonium hydroxide anddissolve the precipitate, which forms in the samemanner. Add distilled water to make 100 mL Fil-ter into brown bottle. Solution remains stablewhen refrigerated.Formalin, l0%Gold chloride, O.2Vo (Solution is very stable, may

be used repeatedly; keep refrigerated.)Sodium thiosulfate, 5%Mayer's hematoxylin or Gill II hematoxylin

Procedurel. Deplasticize for 4 hours in 55" C xylene.2. Bring to IOOVo ethanol.3. Dip in 7Vo celloidin and then air-dry for 5 to

10 seconds.4. Hydrate with:

957o ethanol7Mo ethanolDistilled water

2 changesI changeI change

5. Oxidize in acidified permanganate solution forI minute.

6. Wash in distilled water for 2 minutes.7. Bleach in l%o oxalic acid for I minute.8. Wash in tap water for 2 minutes.9. Wash in 2 changes of distilled warer, I min-

ute each.10. Mordantin2.SVo iron alum for I minute.11. Wash in tap waterfor 2 minutes, followed with

with 2 changes of distilled water for 30 sec-onds each.

12. Permeate with the diamino silver solution Iminute.

13. Rinse in distilled water for 20 seconds.14. Reduce in lOVo formalin for 3 minutes.15. Wash in water for 3 minutes.16. Tone in O.2Vo gold chloride for l0 minutes.17. Wash in tap water and then for 5 minutes in

57o Na2S2O5 (sodium thiosulfate), and againwash in tap water for 3 minutes.

18. Stain in Mayer's hematoxylin for I minute;wash in water and then rinse in disblled wa-ter.

19. Dehydrate:957o ethanol

IOOVo ethanol2 changesI change

20. Rinse in IOOVo ethanol.21. Clear in 2 changes of xylene.22. Mount in Eukitt's mounting medium.

ResultsBone-bluish gray with well-defined unstained, ce.

ment linesReticulum fibers-black

2.

3.4.5.

Copyright © 1987 by Battelle Memorial Institute 

Phosphotungslic acid hematoxy li ns(for plastic-embedded sections of bone)

Fixation. 7 OVo alcohol, 70Vo buffered formalin.

SolutionsPhosphotungstic acid hematoxylin (pTAH)

BONE 105

Mayer's hematoxylinBasic fuchsin in 70Vo ethanol, O.lVo

Procedurel. Deplasticize in xylene for 4 hours at 55' C.2. Transfer to

IOOVo ethanollO0To ethanol957o ethanol957o ethanolTOVo ethanol

Distilled water3. Incubate in silver nitrate solution at room tem-

perature under ultraviolet light for a minimumof 6 hours.

4. Wash thoroughly in distilled water for 3 ro 5minutes.

5. Rinse in the sodium thiosulfate solution for 2 to3 minutes.

6. Wash thoroughly in distilled warer for 3 to 5minutes.

7. Stain in Mayer's hematoxylin for 15 minutes.8. Wash in distilled water for 5 minutes.9. Transfer to TOVo dcohol for I minute.

10. Stain in O.lVo basic fuchsin for 15 to 30 sec-onds.

11. Dehydrate and clear in the following:95Vo alcohol

IOAVo alcohollOWo alcoholXyleneXyleneXylene

12. Mount in Eukitt's mounting medium.

ResultCalcium deposits-dark brown to blackOsteoid seams-bright pink

l0 dips15 dips15 dips20 dips20 dips

1 min

Nuclei-purpleCytoplasm-pink

Solochrome cyanin R12 (for plasfic-embe ddedsectbns of bone)

Fixation. 7 0To alcohol. lOVo b uffere d formalin.

SolutionsSolochrome cyanin R solution

SolochromecyaninR lgm2Vo acettc acid, aqueous 100 mlNote; A pH of approximately 2 is obtained bythe acetic acid.

Procedurel. Deplasticize slide for 4 hours in 55' C xylene.2. Transfer to

7OO7o ethanol70OVo ethanol95Vo ethanol95% ethanolTOVo ethanol

Distilled water3. Stain for l0 minutes in the l7o Solochrome

cyanin R solution.

HematoxylinPhosphotungstic acidDistilled warerPotassium permanganate

0.5 gm10 gm

500 ml0.09 gm

0.5 gm50 ml1 drop

10 dipsl0 dips

Dissolve the hemaroxylin with hdf of the warerby heating it moderately. In the remaining wa-ter, dissolve the phosphotungstic acid. Cooland then combine the solutions. Next add thepotassium permanganate. When combined,store in the refrigerator. The solution remainsstable for about 4 weeks.

Eosin YEosin YEthanol, gSVoGlacial acetic acid.This solution is very stable.

Procedure1. Deplasticize slides in 55' C xylene for 4 hours.2. Air-dry completely then transfer to

TOOVo ethanol1007o ethanol957o ethanol957o ethanolTOVo ethanol

Distilled water3. Incubate in PTAH either ovemight or 2 hours

at 60" C. Although the use of heat intensifiesthe colors, it also loosens the sections from theslides.

4. Differentiate in 95Vo ethanol (3 quick dips isusually optimum). Check with microscope. Os-teoid seams should be clearly differentiated, be-ing stained a medium purple.Counterstain with the eosin Y for I minure.Dehydrate rapidly:1007o ethanolTAQVo ethanol

5.6.

7. Clear in 2 changes of xylene and mount in Eu-kitt's mounting medium.

ResultsOsteoid-light to dark purpleNuclei, bone matrix-reddish pinkCytoplasm-light pink

Modified Von Kossa's method2s (for calciumdeposifs modified for plastic-embeddedsectrbns of bone)

Fixation. TOVz alcohol, 10Vo buffered forma-lin.

SolutionsAqueous silver nifiate, 1VoAqueous sodium thiosulfate, 57o

Copyright © 1987 by Battelle Memorial Institute 

106 THEoRY AND PRAcTIcE oF HISToTEcHNoLoGY

4. Wash in warm water approximately 30" C untilthe sections become bluish and the water is nolonger colored red. (Check the differentiationunder the rnicroscope; calcified bone should beblue and osteoid seams, red.)

5. Dehydrate and clear:957a ethanol95Vo ethanol

1007a ethanol1007a ethanolXyleneXylene

6. Mount with Eukitt's mounting medium.

ResultsOsteoid-bright orangeNew bone-light purpleOld bone-dark purple

Toluidine blue stain tor calcification tront,sement line, and osteoid seaf?s24(for plastic-embedded secfions of bone)

Fixation. TOTo ethanol. 10Vo buffered forma-lin.

SolutionSioclc solution

Toluidine blue O staining solution, 17o aeueousWorking solution

Toluidine blue O stock solutionDistilled waterCertified buffer tablet" (pH 8.0)

0.75 ml100 ml1 tablet

Dissolve the buffer tablet in distilled water. Addtoluidine blue O and dissolve thoroughly. Note:The staining solution must be prepared fresh foreach batch of slides. Da not prepare stain untilready to use.

Procedurel. Deplasticize sections in xylene at approximately

6 to 12 hours.2. Allow sections to dry.3. Stain sections in working solution for 24 hours.4. Dehydrate sections in the following way:

a. 2 changes of 95Vo ethanol 20 dips eachb. 2 changes of IOOVo ethanol 20 dips eachc. Clear in 2 changes of xylene: 20 dips for the

first and I minute or longer in the second.5. Mount in Eukitt's mounting medium.

ResultsOsteoid seams-light blueMineralized bone-dark purpleCement line-scalloped. bright purpleCalcification front-dark, granular, bluish-purpleNuclei of osteoclasts, osteoblasts, osteocytes-dark

blue or dark purple

*Lot no. 13, Formula no. 30, Catalog no. 001-0060, The Per-kin-Elmer Corp., Coleman Instruments Division, Oak Brook,IL 60521.

Cytoplasm of osteoclasts, osteoblasts, osteocytes-light blue or light purple

Modified methyl green pyronine Y stain4 (forplastic-embedded secfions of bone)

Fixation. Absolute alcohol. cold acetone.

SolutionsStoch solutionMake 2Vo aeueous solutions of each dye and thenextract each solution in a separatory funnel sep-arately with equal volume of chloroform. Methylgreen should be extracted at least six times. Afterthis.washing, check for contaminant methyl violet by exposing filter paper with a drop of methylgreen on it to ammonia fumes. After a few sec-onds, the blue color will fade away. If any violetat all is apparent, repeat the washing process un-til the paper comes white with the test. PyronineY should be washed at least l0 times. Both solu-tions will remain quite stable if stored over chlo-roform. The chloroform wash can be cleanedand reused for washing by being filtered throughactivated charcoal.Worlting stoining solution

Prepare just before use:Aqueous methyl green, 2VoAqueous pyronine Y, ZVoDistilled water

Procedurel. Deplasticize for 4 hours in xylene at2. Transfer to:

a. ljjVo ethanolb. lO}Vo ethanolc. 95Vo ethanold. 95Vo ethanole. TOVo ethanolf. Distilled water

9 m l5 m l

20 ml

55" C.

3. Incubate 12 to 24 hours in refrigerator in meth-yl green-pyronine Y solution.

4. Blot carefully with paper towel or filter paper.5. Dehydrate rapidlg (3 dips) in each of the solu-

tions below:a. SOVo acetone in distilled waterb. 95Vo acetone in distilled waterc. IOOVo acetoned. Equal parts ofacetone and xylenee. 70OVa xylenef. IAOVo xylene

6. Mount in Eukitt's mountins medium.

ResultsDNA-greenRNA-redNuclei-blue-greenBacteria, osteoblasts, basophilic cytoplasm-redBackground-colorless to pale pink

Control for RNA in methyl green pyronineY. Because pyronine Y is not specific for ribo-

Copyright © 1987 by Battelle Memorial Institute 

nucleic acid, it is important to run controls. Itis best to use two control systems. Firsf, witheach run, include a section known to containsignificant amounts of RNA. (ln our laboratory,we use a section from a patient with Paget's dis-ease because of its significant amounts of RNAin the osteoblast's cytoplasm,) Second, sincepyronine Y is not specific for RNA, run a secondset of slides from which the RNA has been re-moved. Although other methods which showedpromise were less expensive, we received bestresults u sing ribonuclease.

lncubation medium. Weigh 0.5 to 1 mg of r i-bonuclease dissolved in I ml of O.21% sodiumbicarbonate (M/40). (According to Chayen,loptimal pH is 7.7. Others use various buffersand distilled water, but the Ml40 sodium bicar-bonate at pH 8.2 works well.)

Method. Incubate one slide at 37" C in ribo-nuclease solution for 2 hours. Incubate a secondslide just like the first, but omit the ribonu-clease. Just use sodium bicarbonate solution.

REFERENCES (Basic preparation and stainingof undecalcified bone)

l. Arnstein, R. R., Frame,8., and Frost, H. M.: Re-cent progress in osteomalacia and rickets, Ann,Intern. Med. 57:1296-1330, 1967.

2. Baylink, D. J., Stauffer, M., Wergedal, J., andRich, C.: Formation, mineralization, and resorp-tion of bone in vitamin D-deficient rats, J. Clin.Invest. 4921122-1134, 1970,

3. Bordier, P. J,, and Tun-Chot, S.: Quantitative his-tology of metabolic bone disease, J. Clin. Endocri-nol. Metab. l:197-275, 1972.

4. Chayen, H.: The methyl green pyronine method,Exp. Cell Res. 3:652, 1952.

5. Clayden, F. C.: Practical section cutting andstaining, ed. 5, Edinburgh, 1971, ChurchillLivingston.

6. Frame, B., Arnstein, A. R., Frost, H, M., andSmith, R. W., Jr.: Osteomalacia: studies withtetracycline bone labeling and metabolic balance,Am. J. Med. 38:134-744,1965.

7. Frost, H. M.: Some observations on bone mineralin a case of vitamin-D resistant rickets, HenryFord Hosp. Med. Bull. 6(4):300-310, 1958.

8. Frost, H. M.: Preparation of thin, undecalcifiedbone sections by rapid manual method, StainTechnol. 332273-277, 1958.

9. Frost, H. M.: Stainingof fresh, undecalcified thinbone sections, Stain Technol. 34:135-146, 1959.

10. Frost, H. M., Villanueva, A. R., and Roth, H.: Tet-racycline staining of newly forming bone andmineralizing cartilage "in vivo," Stain Technol.35:135-138. 1960.

BONE 1O7

11. Frost, H. M,, Frame, B., Ormond, R. S., andHunter, R. B.: Atypical axial osteomalacia: a re-port of three cases: Clin. Orthop. 23:285-295,1962.

12. Martajt, H., and Hioco, D.: Solochrome cyanin Ras an indicator dye of morphology, Stain Tech-nol. 41:97-99, 1966.

13. Mathews, C. H. E., and Mehr, L.: Staining andprocessing bone specimens for simultaneous tet-racycline-osteoid seam assessment and histo-morphometric quantitative analysis, J. Histo-technol. 2:23-24, 1979.

14. Meunier, P., and Edward C.: Quantification ofosteoid tissue in trabecular bone methodologyand results in normal iliac bone. In Jaworski, Z.F. G., editor: Proceedings of the First Internation-al Workshop on Bone Morphometry, Ottawa,1976, Ottawa University Press.

15. Melseen, F.: Histomorphometric and dynamicstudies of osteoporosis, Univ. of Utah Workshopon Osteoporosis, Sun Valley, Utah, Aug. 1977.

16. Olah, A. J., Simon, A., Gaudy, M., Herrmann, W.,and Schenk, R. K.: Differential staining of cal-cified tissue in plastic embedded microtome sec-tions by modification of Movat's pentachromestain, Stain Technol. 52:331-337, 1977.

17. Schenk, R. K., Merz, W. A., and Muller, J.' Aquantitative histological study on bone resorptionin human cancellous bone, Acts Anat. 74:44'53, 1969.

18. Villanueva, A. R., Hattner, R. S., and Frost,H. M.: A tetrachrome stain for fresh mineralizedbone sections: useful in the diagnosis of bone dis-eases, Stain Technol. 39:87-94, 7964.

19. Villanueva, A. R., Ilnicki, L., Frost, H. M., andArnstein. A. R.: Measurement of the bone forma-tion rate in a case of familial hypophosphatemicvitamin-D resistant rickets, J. La. Clin. Med, 57:973-982, 1966.

20. Villanueva, A. R.: A bone stain for osteoid seamsin fresh unembedded, mineralized bone, StainTechnol.4g:l-8, 1974.

21. Villanueva, A. R., and Mehr, L.: Modifications ofthe Goldner and Gomori one-step trichromestains for plastic-embedded thin sections of bone,Am. J. Med. Technol. 43:536-538, 1977.

22. Villanueva, A. R.: A modified blood stain useful inthe diagnosis and evaluation of hematologic ele-ments, J. Histotechnol. 1:19-22, 1977.

23. Villanueva, A. R.: A Von Kossa variant modifica-tion. (Unpublished data, 1977.)

24. Villanueva, A. R.: A toluidine blue stain for cal-cification front, cement line and osteoid seams inplastic embedded undecalcified sections of bone,J. Histotechnol. 2:139, 1979.

Copyright © 1987 by Battelle Memorial Institute 

108 THEoRY AND PRACTICE OF HISToTEcHNoLoGY

Part lll. Histomorphometricquantitative analysis of boneAntonio R. Villanueva

Microscopic histomorphometric analysis ofmineralized bone is rarely applied in a pathologylaboratory. Most of the work on morphometryis done in specialized research laborato-ries4'r6'18'24'28-3o where analytic techniques areavailable for quantitating dynamics of tetracy-cline-labeled bone. But the methods of demon-strating and measuring bone kinetics histo-morphometrically have never attracted manypathologists. Because of this oversight undecal-cified bone is one of the most neglected typesof tissue in the pathology laboratory.

Today undecalcified bone has assumed a firmrole in pathology and in medicine-a situationthat was not true 20 years ago. Probably thefirst investigator who contributed to the im-provement of quantitative bone histomorphom-etry is Frost.ro'rr'r3 He developed and pioneeredone of the most important areas of potentialstudy of patients-the quantitative histologicmethods for the study of bone dynamics.s-8 Hispioneering techniques yielded large amounts ofvalid information essential to the pathologic in-terpretation of bone biopsies, particularly in thefield of metabolic bone diseases.3'e'12'r4'2r'2s'3r

The following section is devoted to the quanti-tative measurements of various structures andtheir estimation. For the purpose of those inter-ested in the histomorp-hometric quantitativeanalysis, only two kinds of bone surfaces will bediscussed: (l) cortical and (2) trabecular sur-faces.

METHODOLOGY

Bone specimens suitable for this kind of mea-surement are obtained from the iliac crest bytrephine biopsyr'r7 or from the eleventh rib.27

Complete measurement of at least one iliaccrest (or rib) section is adequate if the measuredvalues must be significant. The iliac crest mustbe a complete section cut transversely from cor-tex to cortex, or a complete cross section of arib.

For microscopic identification of cells andother related structures, Villanueva's bonestainsz is most satisfactory, particularly if simul-taneous assessment of osteoid seams and tetra-cycline is determined.

LABELING TECHNICS

The labeling technic is basically Frost'sconceptlo but modified by Dr. A. M. Parfitt (Di

rector of the Bone Mineral Research Labora-'tory, Henry Ford Hospital, Detroit, Michigan).

The following instructions for bone labelingand biopsy protocol are described below:

l. schedule biopsy first and then initiate pro-tocol exactly 2l days earlier.

2. First label-oxytetracycline, 250 mg every8 hours for 3 days..Second label-after an interval of l1 days,demeclocycline HCI (Declomycin), 300mg every B hours for 3 days.

3. Do not vary the labeling schedule even ifosteomalacia is suspected.

4. The following precautions should be ad-hered to as closely as possible:a. The time of administration should be 7

AM, 3 plt, and 11 prvr or as close to thisas possible with the patient's schedule.

b. No milk or dairy products should beconsumed by the patient from I hour

' before to 2 hours after medication,which should be taken with wateronly.Nondairy foods do not affect absorption.

c. No alurninum-containing antacids' should be taken from t hour before to

2 hours after medication.With the above schedule of time, the periods

available for ingestion of dairy products and alu-minum-containing antacids are 9 AM to 2 ptvtand 5 PM to 10 plt.

Basically the labeling protocol calls for oxy-tetracycline,2SO mg every 8 hours for 3 days,followed by an ll-day gap, followed by Declo-mycin, 250 mg every 8 hours for 3 days. Thebiopsy will be performed on the fourth day afterthe completion of the second label. If the firstday of the label is counted as day 1, the biopsyshould be performed on day 21.

RECOGNITION OF HISTOLOGICSTRUCTURESOsteoid seams

Bone is a highly specialized tissue consistingof specialized cells and organic and inorganicsubstances. One of these specialized cells iscalled an "osteoblast." These cells are oftencalled "bone builders." They are responsible inlayrng down new unmineralized organic matrix,commonly known as osteoid seams. Some timeafter 14 days this matrix begins to mineralizeand the mineralized matrix is now called"bone." An osteoid seam therefore constitutes alayer of unmineralized organic matrix of bone.After mineralization, the convention is to termthe bone matrix simply the matrix-a highlycalcified tissue. See Figs. 6-7 and 6-8.

Copyright © 1987 by Battelle Memorial Institute 

BONE 109

Eig. 6-7. Photomicrograph of osteoid seam, gray (ongsnally green), taken from a mineralized un€m-bedded, ground cross section of bone. At periphery of seam is a dark (originally reddish orange)staining area of incompletely mineralized bone. Dark-staining cells in bone matrix are osteocytes.(Villanueva bone stain, 250x.;

Fig. 5-8. Same case as in Fig, 6-7 but this figure shows histologic feature of a red osteoid seam. It isindicated by dark circular band lining the intima of haversian canal. Just around seam is a highlymineralized bone matrix, and osteoid itself is densely stained. (Villanueva bone stain, 250x.;

.W

Copyright © 1987 by Battelle Memorial Institute 

110 THEoRY AND PRACTICE oF HISToTECHNoLoGY

Trobeculor surfqce

. . ' + . " . - . . . " . . - i . . + . " . . " . - . " i . ;| **r-*^ **r'

If a stained, undecalcified section of bone isexamined with a high dry objective of the mi-croscope and moved about on the stage, a fieldsomewhat similar to that illustrated in Figs.6-9 and 6-10 should be found easily. Osteoidseams appear in two major colors, red andgreen, lining the haversian or trabecular spacesin fresh unembedded ground sections stainedwith the Villanueva bone stain but red whenembedded in plastic. On the average, the thick-ness measured on over 5000 osteoid seamsin normal individuals is ll.G + l.2S ss11.z;'zaSeams are hyaline and clear and contain cana-liculi and lacunae, which in turn contain osteo-cytes. Special stains or phase-contrast micros-copy will reveal these features. At the junctionbetween osteoid and bone lies a zone of demar-cation measuring about 2.5 p.m in width.33 Un-der high power, the zone of demarcation revealsa highly granular structure advancing upon theosteoid seam.

Osteoid seams possess the following proper-

2 - - " . " , , , - " " - . l - ' " . . i " - . . - ? : - - t . - - - - . ! .

3 4 5 6 7 8 9 l 0

Squore-grid Integrotionsplotte l l , Zeiss

Fig. 6-9. Diagram of trabecular surface. Area and perimeter measurements by point (hits) and line(intersects). Square grid is comprised of 100 possible hits, l0 series of parallel-horizontal, and l0 seriesof superimposed parallel-vertical test lines. Each squa.re represents an area equal to l%o as indicatedabove, and a represents spacing between parallel lines. Dark bands lining the upper and lower rabec-ular surfaces are osteoid seams. Two lighter bands, peripheral to upper osteoid seam, are two tetracy-cline labels. Lining the intima of these seams are the four types of osteoblasts (I to IV). Note also twoosteoclasts-one mononucleated and the other multinucleated.

r 0

ties, which one may need to measure: (1) aperimeter, (2) athickness, (3) the rate of apposi-tion and of mineralization, (4) the number in aunit amount of bone tissue, and (5) the loca-tion or distribution throughout a given bone inthe whole skeleton.

Osteoid seams normally occur in the follow-ing sites: (l) actively forming haversian sys-tems, (2) actively forming endosteal new bone,(3) actively forming trabeculae or portions oftrabeculae in cancellous bone.

Frost's basic fuchsin, Goldner's trichrome,Villanueva's bone stain, and Von Kossa's readilydifferentiate such osteoid seams.

Trabecular surface (Fig. O-9)

The trabeculae are thin and average approxi-mately 725 p.m in thickness.rT The spaces be-tween the trabeculae are filled with marrow.The trabeculae also has the properties of shape,width, volume, and length. Similarly, they un-dergo a progressive, net loss in thickness and

Copyright © 1987 by Battelle Memorial Institute 

BONE 111

Hoversion surfqce

l.!.u^."*t

5 **T-

1 2 3 4 5 6 7 I 9 l 0Squore-grid Integrotionsploite l l , Zeiss

Fig. 6-10. Diagram of cortical surface. Areas, porosity, and circumference measurements by point(hits) and line (intersects). Square grid is comprised of 100 possible hits and 10 series of superim-posed parallel-vertical test lines. Each square represents an area equal to lVo as indicated above, and arepresents spacing between parallel lines. Dark bands lining the intima of three haversian canals areosteoid seams. Smaller seam is unlabeled but larger ones are labeled, as indicated by double lighterbands peripheral to osteoid seams. Note the three types of osteoblasts (II to /V) without type I. Asingle resorption surface is also observed at upper left. Note also two multinucleated and one mononu-cleated osteoclasts enclosed in Howship's lacunae.

6 ****-*--\-

tt

r 0

number with age. This loss in trabecular bonemass begins soon after longitudinal growthstops and continues for the remainder of life.zsThe amount of surface trabecular bone exposedto histologic remodeling activity (that is, the sur-face-to-volume ratio for trabeculae) varies de-pending on age and sampling site in comparisonto that exposed by equivalent amounts of com-pact bone.20

Haversian surface (Fig. 6-10)

The space enclosed by the walls of all thehaversian canals in bone constitutes the haver-sian surface. Its changes with normal aging arerelatively small compared with those of the oth-er surfaces, and in normal adults its volume maybe considered as approximately constant.2s In-cluded in the haversian surface are cylindricstructures, or canals, running longitudinallywith the axes of long bones. Communicatingwith the haversian canals are narrow channels.

Volkmann's canals, originating from the perios-teal and endosteal surfaces, which make a waythrough the bone obliquely or at right anglesthrough the bone's long axis. These canals con-tain blood vessels and nerves. In cross sectionsof bone, haversian canals are seen to be sur-rounded by concentric larnellae and sometimesby osteoid seams or by resorption spaces, Theinner surface of the normal haversian canal isIined with endothelium-like cells. Haversian ca-nals in stained sections are easv to identifv andcount.

Primary osteonsPrimary osteons are produced by enfolding of

the periosteal circumference around longitu-dinally coursing vessels during growth. Suchosteons are not considered in Frost's methodshut are included in Parfitt's fractional corticalcalculations.22

Copyright © 1987 by Battelle Memorial Institute 

112 THEoRY AND PRAcTIcE oF HISToTEcHNoLoGY

Secondary osteons

Secondary osteons are haversian systems thathave been fbrmed after formation of the cortex.Most of Frost's data are measured from thesesystems.

Osteocytic lacunae

Osteocytic lacunae are small holes in bone.Branching out from these lacunar walls are sev-eral canaliculi. These are small tubular holes inbone, anastomosing with one another and thenconnecting to a vascular channel.

Bone resorption

In eroding a bone surface, osteoclasts produceshallow indentations called "Howship's lacu-nae." Osteoclasts are acfive, motile cells, andonce having performed their function, they dis-appear. When osteoclasts resorb bone, theyremove both mineral and organic phases simul-taneously. Bone resorption in the whole skele-ton therefore constitutes all the destructive ac-tivity of osteoclasts. The evidence of the previ-ous existence of Howship's lacunae after theyhave been covered by bone matrix remains asscalloped irregularities in the cement lines of os-teons or trabeculae. At the light-microscope res-olution these shallow, scalloped excavations onthe surface of the bone, which are Howship's la-cunae, can be seen prominently. A good exam-ple of this type of cement line is those seen inPaget's disease.

MtcRoscoptc MEASUREMENTS2,15,23

A standard binocular medical microscopewith a Zeiss Integrationsplatte II Micrometer at160x with a 0.4 N.A. objective is used for thesemeasurements. This eyepiece micrometer con-tains 10 series parallel-horizontal and ten se-ries superimposed parallel-vertical test lines and100 possible hits (Figs. 6-9 and 6-10). Duringmeasurement of the sections, the micrometereyepiece is optically superimposed upon theimage of the section being evaluated in incre-ments of one field. When a reticule line crossesthe image of the bone surface, it is recorded asan "intersect" with respect to (1) which of thefour types of osteoblasts adjacent to an osteoidseam it crossed, (2) which of the four types ofosteoblasts corresponds to a labeled or unlabeledosteoid seam, (3) which inert surface it crossed,(4) which of the five kinds of cell types it crossedadjacent to Howship's lacunae, and (5) the timethat two reticule lines intersect immediatelyupon the image of a bone surface, whereby

the intersects record a "hit" with respect to allpoints hitting upon an osteoid seam, haversianor Volkmann's canals (cortex only), bone, andmarrow cavity. Proceed to the next field, record-ing all the information observed, until every sec-tion area is scanned (see Table 6-2). Cover thesection systematically so that about 400 to 500intersects and 2000 to 5000 hits (for bone andVolkmann's and haversian canals) accumulateat the haversian surface and 500 to 1000 inter-

Table 6-2. Tabulated data fromFigs. 6-9 and 6-10

Haversian Trabecrrlarsurface surface

Intersects

Cell types of osteoid seamsI-Mesenchymal pre-

osteoblastsTenacycline labelNo tetracycline label

Il-Cuboid osteoblastsTetracycline labelNo teracycline label

Ill-Intermediate os-teoblastsTetracycline labelNo tetracycline label

IV-Flat, elongatedlining cellsTetracycline labelNo tetracycline label

Unidentifiable cells orabsenee ofosteo-blastsTetracycline labelNo tetracycline label

Inert surfaceCell types of Howship's

lacunaeMultinucleated osteo.

clastsMononuclear osteo'

clastsFlat, elongated lining

cellsMesenchymal pre-

osteoblastsUnidentifiable cellsAbsence of cells

Hits

Porosity (Haversian orVolkmann's cands)

BoneOsteoid

04

00

20

50

0I6

33

00

l4

3

I

2

0

22

l6

363

t44

Copyright © 1987 by Battelle Memorial Institute 

BONE 1 13

sects and 3000 to 5000 hits (for bone, osteoid millimeters, days, micrometers per day, mmr'/seams, and marrow) accumulate at the trabec- mmr]/year, and so forth, of a given surface ac-ular surface per section. cording to the formulas and examples described

Convert all raw data to percent, micrometers, in this text.

Trabecular surfaceCircumterence tormula (Fig. 6-9)

H + V 3 . 1 4 1 6N;of throws

* ,:

x a : Circumference, or perimeter (Zeiss Intergrationsplatte II)

H, Total horizontal intersects; V, total vertical intersects; a, width between parallel spacing testlines.

Example (Fig. 6-9)Given:

Horizontalintersects 12 Numberof throws : 2a : 1 m m1 8

30Substituting from above formula:

H + V 3 .1416 30 3 .1416NoiiErows " -

x 4'ori " 2

X I : 23'6mm

Surface measurements of trabecula

r. -/o orosteoro seams of totar surface - Total intersects of osteoid seam

Total intersects - x loo : fi: nt.t ,

Vertical intersectsTOTAL INTERSECTS

2. Vo oflabel of total surface :

6. Vo of osteoblastic activitv =

7. Vo of osteoclastic activitv =

Total intersects of labelTotal intersects

x 1oo : $: rrr"

s. vooffuactionallabeloftotalsurface - -- ? of lalelof total surface 20zo of osteoid seams mA;m;

x 100 : ffi: 46'2vo

4. va thatis inert of total surface - Total intersects of inert strfaee A

Total intersects

s. vo of Howship's lacunae of total surfac Total intersects of Howship's lacunae

, e : *

Total intersects of cuboid + intermediate osteoblasts

Total intersects of osteoid seams

Total intersects of multinucleated osteoclasts

l ' l100 : ) * :36.6Vo

JU

F

x 100 : *: 41.7Votz

loo: fr: u.urr"

B. Vo of relative osteoid volume :

Total intersects

Total osteoid hits

Total bone + Osteoid hits

4x l 0 0 - - - l 4 . 3 % o

I U

9. vo of ftactionar osteoid volume = eSffitr x r00 = #

= n o

to. voof fractional bone volum" : %ffP x r00 : 14 = 144n100

I l .

12.

TooftotaJbonevolume = Vooffractionalbonevolume * Vooffractionalosteoidvolume : l4%o * 4Vo: lSVo

M e a n r r a b e c u l a r t h i c k n e s s - 2 x a x ( T o q ? l b g n e h i t s + o s t e o i d h i t s ) - 2 x 1 - x 1 8 : l . 2 m m'lbtal intersects 30

Rates involved with bone remodeling

7. Assuming mea.sured mean uidth betueen two tetracycline labels is 9.1 pm and number of daysbetween two labeling episodes is 14 days, one may calculate the appositional rate, p.mlday (M),according to the formula below:

M Q.mtday) : = t*

: 0.G5 p.mlday(uncorrecred)

Copyright © 1987 by Battelle Memorial Institute 

114 THEoRY AND PRAcTICE oF HISToTEcHNoLoGY

Corrected M1(pmlday) = UncorrectedM xVo of fractionallabelof total surface = 0.65 x O.462 = 0.3 p.m/day

2. Assuming mean wall thickness of inert surface's cementing line is 50 pm (Fig. 6-9, arrows), onecan calculate formation time, o1, os follows:

oy(day) - Meqr rvall-thic-kless - 50 pm

corrected My dffi = 167 daYs

3. Assuming mean width of osteoid seam as 10 pm, one may now calculate mineralization lag time,M7,, &s follows:

M tt ffi : osffi+ = 15.4 days (M based)

ML,: ffi = o:##fia:33.3 days (My based)

4. Bone formation rate (volume based, mm3/mm3/year) :

Appositional rate (M) x ffi

x Total teracycline intersecm 0.6b x 0.365:

i ' o ' ' " u : 0 '079mm3/mm3/year

Haversian surface (Fig. 6-10)Cortical area, mm2 (A.)

1. The number of hits within the cortical matrix is 55. To calculate the area, the formula is givenbelow:

A"(mm2):

Substitutin*: %#TA" : 55 mmz

2. No. of osteoid seams/mm' : (Ar)No. of osteoid seams : 3Cortical area (Ar) : 55 mmz

?Ar : J= : 0 .054 /mmz" b b

3. Mean circumference of osteoid seams, mm (Si)Two osteoid seams with a total horizontal and vertical intersects of 14. To calculate S,. theequation is similar to the formula used at the trabecular surface:

Sy(mm): * l l f f*o

t 4 3 . 1 4 1 6: z x z x 2

x r

: 5.50 mm

4. Appositional rate, pr.m/day, uncorrected. If 0.75 p,mlday is the uncorrected calculated appositionalrate (M), the corrected appositional rate (M) using Parfitt's formulazz would be as follows:

M x Fractionallabeled seams : Mletmlday)O.75 x 0.785: 0.59 pmlday

using Frost's formulalr

M (p'mldav,. ffi

: MlQrmlday)

0.75 x I

= O.tO pmlday (Mlbased in pr.m/day)

Copyright © 1987 by Battelle Memorial Institute 

BONE 115

365SO x

ffi : 0. 18 mm/year (Mybased in mm/year)

5. Mineralization lag time, days (M1,). If the mean width of osteoid seams is.9 pm, the mineralization

lag time may now be calculated:

^n _ width of osteoid seam (pm)M t ' t M (t"^rd^y)

: oh:

12 days (M based)

\n _ width of osteoid seam (g,m) _ otYrLt

tr4r(pcm/day) -

o;9- : l5'2 days (Mi based)

6. Formation time, days (oy). If mean osteon wall thickness, MWT, is 68 g.m, this can be calculatedaccording to the equation below:

Mwr = =#-g+, = 11b.2 days, or 0.32 yearso': IvI,

= osg;tnldt

: t

7. Activation frequency, foci/year (pc)

A, 0.054. . _ : - - - - - - - -L*t- or(y.ars)

- 0.32

P 4 : Q . 7 7

8. Bone formation rate, mmslmmslyear (Vt)"

rr M x 0.365 x No. of tetracyclineintersectsV t :

r , 0 .75 x 0 .365 x 11t r : - -

5b x l -

Vr : 0.0548 mm3/mmslyear

Using Frostianrl dynamics (V), one can also calculate according to the formula:

Vr: No. of osteoid seams/mm'(A) x Circumference of osteoid seams (S) (mm) xRadial closure rute (M) mm/year

= 0.054 x 5.50 x 0.18

Vr : 0.0535 mm3/mm3/year

9. Porosity, Vo

P :Total no. of haversian + Volkmann's canal hits

Total cortical hitsx 100

o - 1 6ao

P : 29.l7o

Measurements of haversian surface

7 . v o o f o s t e o i d s e a m s o f t o t a l s u r f a c e : x 1 0 0 : # : 3 6 , 8 v oI OtaI rnters

2. vo orraber of totar surface : T**lifP?!l! l{l"b"l x 100 : * : Zl.evo

I OtaI rntersects JU

3 ' v o o f f r a c t i o n a l l a b e l o f t o t a l s u r f a c e : x l 0 0 = 3 + = 7 B . 5 v o70 OI Osteolcl Seams of total surface Jti.b

4. Vo thatis inerr of total surface - Total inlgrse-cls of inert surface

x 100 : 3 : 36.gvororal tntersects 56

s. vo of Howship's lacunae of total surfa Total intersects of Howship's lacun'o 1n

r " " : x 1 0 0 = ; i : 2 6 . 3 v o

6. Vo of osteoblastic activity - Total intersect-s of cuboid +-intermediate osteoblasts

x 100 : g: 57.1voTotal intersects of osteoid seams 14

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116 THEoRY AND PRAcTIcE oF HISToTEcHNoLoGY

7. Vo ofosteoclastic activitY : Total intersects of multinucleated osteoclastsToral intersects

Total osteoid hits

x 100 : *:

7.eWo

8. Vo of relative osteoid volume :Tota] bone hits * osteoid hits

9. Vo of ftactional osteoid volume =Total osteoid hits

Tota] hits

REFERENCES (Histomorphometric quantitativeanalysis of bone)

l. Bordier, P.J., and Tun-Chot, S.: Quantitative his-tology of metabolic bone disease, J. Clin. Endocri-nol. Metab. t:l97-215, 1972.

2. Chalkley, H. W., Cornfield, J., and Park, H.: Amethod for estimating volume-surface ratios. Sci-ence 110:295-297, 1949.

3. Duncan, H., Frost, H. M., Villanueva, A. R., andSigler, J.: The osteoporosis of rheumatoid arthri-tis, Arthritis Rheum. 8:843-954, 1965.

4. Epker, B. N., and Frost, H. M.: A histologicalstudy of remodelling at the periosteal, haversiancanal, cortical endosteal, and trabecular endostealsurfaces in human rib, Anat. Rec. 152:129-136,1965.

5. Frost, H. M., Villanueva, A. R., and Roth, H.;Measurement of bone formation in a 57 year oldman by means of tetracycline, Henry Ford Hosp.Med. Bull. 8(2):230-2il, 1960.

6. Frost, H. M., and Villanueva, A. R.: Measurementof osteoblastic activity in diaphysed bone, StainTechnol. 35: 179-189, 1960.

7. Frost, H. M., Villanueva, A. R., and Roth, H.;Qualitative method for measuring osteoclastic ac-tivity, Henry Ford Hosp. Med. Bull. 10:217-ZZB,March. 1962.

8. Frost, H. M., and Villanueva, A. R.; Human os-teoclastic activity: qualitative histological mea-surement, Henry Ford Hosp. Med. Bull. 10:229-236, March 1962.

9. Frost, H. M,: Bone dynamics in metabolic bonediseases, J. Bone Joint Surg. 48-A:1192-1203,1966.

10. Frost, H. M.: Tetracycline-based histologic andanalysis of bone remodelling, Calcif. Tis. Res. 3:211-217. 1969.

11. Frost, H. M.: Mathematical elements in lamellarbone remodelling, Springfield, Ill., 1973, CharlesC Thomas, Publisher.

12. Frost, H. M.: Orthopedic lecture series, vol. III:Bone remodelling and its relationship to metabol-ic bone diseases, Springfield, Ill., 1973, CharlesC Thomas, Publisher.

13. Frost, H. M.: Bone remodelling dynamics,Springfield, Ill., 1963, Charles C Thomas, Pub-lisher.

14. Heaney, R. P., and Recker, R. R.: Estrogen ef-

x 1 0 0 :

fects on bone remodelling at menopause, Clin.Res. 23:5354, 1975.

15. Henning, A.: A critical survey of volume and sur-face measurement in microscopy, Zeiss Werk-zeitschrift 20:3-12, 1958.

16. Jaworski, Z. F., Meunier, P., and Frost, H. M.rObservations on two types of resorption in humanlamellar corticd bone, Clin. Orthop. 83:279-285,1972.

17. Jowsey, J.: The bone biopsy, New York, 1977,Plenum Medical Book Co.

18. Jowsey, J.: Microradiography. A morphologc ap-proach to quantitative bone turnover. In Frame,B., Parfitt, A. M., and Duncan, H., editors: CUni-cal aspects of metabolic bone disease, Amster-dam, 1973, Excerpta Medica Foundation.

19. Jett, S., Ramser, J. R., Frost, H. M., and Villa-nueva, A. R.: Bone turnover and osteogenesis im-perfecta, Arch. Pathol. 81:l 12-l 16, 1966.

20. Meunier, P., and Edward, C.: Quantification of' osteoid tissue in trabecular bone methodology

and results in normal iliac bone. In Jaworski,Z. F. G., editor: Proceedings of the First Inter-national Workshop on Bone Morphometry, Otta-wa, 1976, 0ttawa Press.

21. Morgan, B.: Osteomalacia, renal osteodysnophyand osteoporosis, Springfield, Ill., 1973, CharlesC Thomas, Publisher.

22. Parfitt, A. M.: The physiologic and clinical sig-nificance of bone histomorphometric data. InRecker, R., editor: Bone histomorphometry, tech-niques and interpretations, Boca Raton, Florida,C.R.C. Press. (In press.)

23, Parfitt, A. M.: The stereologic basis of bone his-tomorphometry, theory of quantitative micros-copy and reconstruction of the third dimension.In Recker, R., editor: Bone histomorphometry,techniques and interpretations, Boca Raton, Flori-da, C.R.C. Press. (In press.)

24. Parfitt, A. M., Villanueva, A. R., Crouch, M. M.,Mathews, C. H. E., and Duncan, H.; Classifica-tion of osteoid seams by combined use of cellmorphology and tenacycline labeling. Evidencefor intermittency of mineralization. In Meunier,P. J.: Proceedings of Second Workshop on BoneMorphometry, Paris, 1977, Armour Montagu.

x r o o : * : 7 . 6 w o

tr = 5.46voDD

10.

I t .

vo of fracnonal bone Tol4l fsnp h*c ?A

volume: ffi x 100: H: 65.4Vo

Vo of total bone volume : 7o of fractional bone volume *Vo of fracoond osteoid volume : 6b.4 + S.4G: TA.9%o

Copyright © 1987 by Battelle Memorial Institute 

25.

26.

Pirok, D., Ramser, J. R., Takahashi, H., Villanue-va, A. R., and Frost, H. M.: Normal histologic,tehacycline, and dynamic parameters in humanmineralized bone sections, Henry Ford Hosp.Med. Bull. 14:195-218, 1966.Schulz, A., and Delling, G.: Age-related changesof new bone formation-determination of histo-morphometric parameters of the iliac crest trabec-ular bone. In Jaworski, Z. F. G., editor: Proceed-ings of the First Intemational Workshop on BoneMorphometry, Ottawa, 1976, Ottawa UniversityPress.Sedlin, E. D., Frost, H. M., and Villanueva, A. R.:The eleventh rib biopsy in the study of metabolicbone disease, Henry Ford Hosp. Med. Bull. 11:217-219, June 1963.Takahashi, H,, and Frost, H. M.: A tetracyclinebased comparison of the number of cortical boneforming sites in normal diabetic persons, J. Jpn.Orthop. Assoc. 39:13-23, 1966.

BONE 117

29. Teitelbaum, S. L., Rosenberg, E. M., Richardson,C. A., and Avioli, L. V.: Histological studies ofbone from normocalcemic postmenopausal pa-tients with increased circulating parathyroid hor-mone, J. CUn. Endocrinol.422537-543, 1976.

30. Villanueva, A. R., Frost, H. M.,Ilnicki, L., Frame,8., Smith, R., and Arnstein, A. R.: Cortical bonedynamics in 2l cases of osteoporosis measured bymeans of teracycline labeling, J. Lab. Clin.Med. 5E:599-616, 1966.

31. Villanueva, A. R.: Quantirative histology of boneremodelling dynamics. In Frame, B., Parfitt, A.M., and Duncan, H., editors: Clinical aspects ofmetabolic bone disease, Amsterdam, 1g73, Ex-cerpta Medica Foundation, ICS ser. no.27O.

32. Villanueva, A. R.: A bone stain for osteoid seamsin fresh unembedded, mineralized bone, StainTechnol. 49zt-8,1974.

33. Villanueva, A. R.: Unpublished data, 1978.

27.

28.

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