Application of Stains-All for Demarcation of Cement Lines in Methacrylate Embedded

Bone Helen E. Gruber and Pertchoui Mekikian

Medical Genetics-Birth Defects Center and Department of Pediatrics, Cedars-Sinai Medical Center, SSB Third Floor, 8700 Beverly Blvd., Los Angeles, California 90048 and Department of Pediatrics, University of

California at Los Angeles, Los Angeles, California

ABSTRACT. Cement lines provide a record of sites of past remodeling buried in the matrix of bone. A method is reported for application of Stains-all, a cationic carbocyanine dye, for de- marcation of cement lines in bone. The method, which is simple, works well for both glycol meth- acrylate and methyl methacrylate undemineral- ized embedments and produces good concomitant staining of cytoplasm and nuclei of osteoblasts, osteoclasts and marrow cells.

Key words: bone, cement lines, Stains-all

tains-all is a cationic carbocyanine dye S for which a variety of staining appli- cations have been reported by Green and Pastewka (1 974) in paraffin embedded tis- sues. Application of Stains-all’s diverse metachromatic staining capabilities have been utilized previously by our group in specialized application to cartilage matrix to evaluate mucopolysaccharides in meth- acrylate embedded specimens of the growth plate (Horton and Rimoin 1978). We report here a further specialized appli- cation of use of this stain to demarcate cement lines in undecalcified methacry- late embedded bone specimens.

Cement lines are specialized regions of the bone matrix which mark past changes in bone remodeling. Two types of cement lines are recognized: reversal lines occur when bone formation follows bone resorp-

1052-2095/91/6604-181/$3.00/0 BIOTECHNIC & HISTOCHEMISTRY Copyright 0 1991 by Wi!liams & Wilkins

tion and arrest lines are believed to form at sites where bone formation begins again after a period of arrest (Jee 1983). Cement lines are useful in clinical inter- pretation of bone biopsies and are a hall- mark of the mixed phase stage of Pagets’ disease of bone (Milligram 1977).

The application of Stains-all we report here for demarcation of cement lines in bone is simple and, as an advantage over our previously reported methods (Gruber et al. 1985), does not require prior section demineralization.

MATERIALS AND METHODS

Fixation, Embedment of Specimens and Sectioning Bone specimens were fixed in 10% neutral buffered formalin and embedded in glycol methacrylate or dehydrated and em- bedded in methyl methacrylate as previ- ously described (Gruber et al. 1985, 1988). Briefly, the glycol methacrylate method used the JB-4 embedding system (Polysci- ences, Inc., Warrington, PA) and speci- mens were infiltrated in a graded series of distilled HzO-glycol methacrylate and em- bedded on plastic block holders. The methyl methacrylate method, described previously (Gruber et al. 1988), used a fi- nal embedment mixture of 85% methyl methacrylate (Polysciences, 25 ppm, 10% hydroxyquinone), glycol methacrylate

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182 Biotechnic & Histochemistry

(Polysciences, ME 130.14), 5% dibutyl phthalate (Eastman Kodak) and 5% (w/v) polyethylene glycol (Polysciences, polyeth- ylene glycol 1540 distearate). For this embedment, coverslips are applied using Polymount (Polysciences).

Glycol methacrylate sections were pre- pared at 2.5 pm using glass knives and a Sorvall JB-4 microtome. Sections of methyl methacrylate embedment were prepared at 5 pm using a Reichert-Jung 2050 Supercut microtome. Methyl meth- acrylate sections were stained free-float- ing in groups of three to five in 35 X 10 mm dishes. Glycol sections were floated onto water and affixed to glass slides prior to staining. Slides were not pretreated with any material. For this embedment, coverslips are applied using Permaslip (American Histology Reagent Co., Stock- ton, CA).

Staining Procedure Stains-all: Stock solutions: Veronal buffer (Bancroft and Stevens 1977) so- dium acetate (Mallinckrodt, Paris, KY), 1.943 g and sodium barbiturate (Sigma, St. Louis, MO), 2.943 g in 100 ml distilled water. Working Veronal buffer: 5 ml stock Ve- ronal buffer, 0.1 M concentrated HC1 (Mal- linckrodt, Paris, KY), l l ml; 7 ml distilled water. Adjust to pH 4.3 with 0.1 M HC1. Stock Stains-a21 solution: 0.1 % Stains-all (Eastman Kodak, Rochester, NY) in 100% formamide ( N ,N-dimethylformamide, Fisher Scientific Co., Fair Lawn, N J ) pre- pared under a fume hood. Working Stains-a11 solution: 7 ml stock Stains-all, 2.1 ml Veronal buffer, 40 ml distilled water. Final pH of working solu- tion is 4.3-4.4

Stock Stains-all solution can be stored for up to two months in a brown bottle a t room temperature. Working solution should be made fresh for each staining run and should be thoroughly filtered prior to use with Whatman #41 filter paper. Glycol methacrylate staining: Staining is carried out in the dark for 1 hr at room temperature. Staining performed in the

light produces colors which fade during the staining process. This light sensitivity was noted previously by Green and Pas- tewka ( 1974). Following staining, glycol sections are rinsed briefly in distilled water, air dried, and coverslips applied. Methyl methacrylate staining: Due to the harder nature of this embedment medium, staining is carried out a t 50-60 C for 20 min in the dark. (Staining of cement lines can also be achieved at 40 C for 20 min, but cellular detail is poorer than that pres- ent after the 50-60 C staining procedure.) Working solution stain should be pre- heated prior to use (also in the dark; this is easily achieved by covering a beaker or other container with aluminum foil). Small stai’ning dishes containing the sec- tions are then filled with the preheated working solution and placed on a pre- heated slide warmer (with the lid also cov- ered with aluminum foil). Methyl methac- rylate sections tend to have fading of stain during the rinsing process with distilled water. We recommend that for this embed- ment the rinse utilize 1% acetic acid (pH 2.48) and be performed very quickly (less than 5 sec), sections removed individually, blotted dry on bibulous paper, and then mounted.

RESULTS AND DISCUSSION Cement line localization has received

considerable attention in recent years with the advent of new undemineralized bone preparations (Villanueva et al. 1986; Gruber et al. 1985; Bain et al. 1990). The objective is to achieve crisp cement line staining while retaining cytologic/nuclear features and definition of bone matrix and osteoid. The Stains-all method we present here achieves these objectives in both gly- col and methyl methacrylate sections of undemineralized bone. We have used the method for several years and found it to be reliable. Both thick, dense and thin cement lines can be visualized with this technique. We do not know if arrest lines also can be stained with our method since our data were collected on clinical speci- mens and we have not yet had the oppor-

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Bone Cement Lines 183

tunity to perform animal model studies in which perturbations produce arrest lines.

Figure 1 shows a representative photo- micrograph of glycol methacrylate em- bedded bone stained with Stains-all. In addition to light bone matrix staining, os- teoiding stain is much more pale than bone. Cement lines stain dark blue. Good

cellular/nuclear staining can be seen in osteoblasts, osteoclasts and marrow cells; cytoplasm stains pink, nuclei blue.

Figure 2 illustrates a Stains-all prepa- ration from methyl methacrylate em- bedded bone. Staining color patterns are similar to those described above.

In summary, Stains-all is a useful stain

Fig. 1. Cement lines stained with Stains-all, metaphyseal region, newborn child with Kniest dysplasia. Glycol methacrylate embedment. x 220.

Fig. 2. Cement lines stained with Stains-all, iliac crest bone biopsy from an adult patient with renal failure. Methyl methacrylate embedment. x 180.

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184 Biotechnic & Histochemistry

Bancroft, J. D. and Stevens, A. 1977. Theory and Practice of Histological Techniques. Churchill Livingstone, London. p. 4 1 1.

Green, M. R. and Pastewka, J. V. 1974. Simulta- neous differential staining by a cationic carbo- cyanine dye of nucleic acids, proteins and con- jugated proteins. 11. Carbohydrate and sulfated carbohydrate-containing proteins. J . Histo- chem. Cytochem. 22: 774-781.

Gruber, H. E., Marshall, G. J., Kirchen, M. E., Kang, J. and Massry, S. G. 1985. Improvements in dehydration and cement line staining for meth- acrylate embedded human bone biopsies. Stain Technol. 60: 337-344.

Gruber, H. E., Marshall, G. J., Nolasco, L. M., Kirchen, M. E., and Rimoin, D. L. 1988. Alka- line and acid phosphatase demonstration in hu- man bone and cartilage: effects of fixation in- terval and methacrylate embedments. Stain Technol. 63: 299-306.

Horton, W. A. and Rimoin, D. L. 1978. Histochemi- cal characterization of the endochondral growth plate: a new approach to the study of the chondrodystrophies. Birth Defects: Original Article Series XIV: 8 1-93.

Jee, W. S. S. 1983. The skeletal tissues. In: Histol- ogy: Cell and Tissue Biology (5th ed.), Weiss, L., ed. Elsevier Biomedical, New York. pp. 200- 255.

Milligram, J. W. 1977. Radiographical and patho- logical assessment of the activity of Paget’s dis- ease of bone. Clin. Orthop. Relat. Res. 127: 43- 54.

Villanueva, A. R., Sypitkowski, C. and Parfitt, A. M. 1986. A new method for identification of ce- ment lines in undecalcified, plastic embedded sections of bone. Stain Technol. 61: 83-88.

for bone cement line demarcation in ad- dition to good soft tissue and cartilage ma- trix staining. The procedure is technically not difficult and has the advantage of omitting demineralization of single sec- tions, (Gruber et al. 1985). The user should note the light sensitive nature of Stains- all (as mentioned by Green and Pastewka 1974) and be aware of the value of careful filtration and thorough rinsing to prevent formation of potential precipitates upon glycol embedded sections.

A C K N O W L E D G M E N T S The author thanks David Rimoin for pro- viding skeletal dysplasia specimens and for his encouragement, Loyda Nolasco for assistance with printing the photomicro- graphs, and Ms. Gail Reyburn for help with preparation of the manuscript. This work was supported in part by NIH grant lPOlHD22657-05GT.

REFERENCES Bain, S. D., Impeduglia, T. M. and Rubin, C. T. 1990.

Cement line staining in undecalcified thin sections of cortical bone. Stain Technol. 65: 159- 163.

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