Ultrastructure of bone marrow prior to and after programmed freezing

Ultrastructure of bone marrow prior to and after programmed freezing M. Barkix# DEPARTMENTS UNIVERSITY

arid M. Newuza?t, B.A.,“” OF ORAL OF CALIFORNIA

BIOLOGY

AND

AT LOS

Los Angeles, ORAL

SURGERY,

Calif. SCHOOL

, OF DENTISTRY,

AKGELES

An electron microscopic study has east doubt on reports of 80 to 90 per cent cell viability after programmed freezing methods but lends support to the specific technique of programmed freezing of marrow for the purposes of osseous reconstruction. The reticular and blast cells which were observed to be viable support the present theory that these elements of osteogenic bone marrow are responsible for the formation of new bone.

T

he osteogenic potential of frozen marrow has previously been demonstrated L 2 This previous work established in the mandibles of dogs and rhesus monkeys. a foundation on which properly preserved marrow could be used for treatment of osseousdefects. There have been numerous reports on methods of cryobiologically preserving a.nd storing bone marrow for use in the treatment of total body exposure to radiation, but only a few have dealt with banked frozen marrow for use in osseousreconstruction.‘~ 3-6 Programmed freezing methods which allow an 80 to 90 per cent recovery of viable cells upon thawing have been reported, indicating that properly preserved marrow could have both osteogenic and hemopoietic potential. For the most part, enzymatic studies7-g have been the basis upon which cell viability was determined. The purpose of this report is to identify at the ultrastructural level the cellular elements remaining in specimens of rhesus monkey bone marrow before and after programmed freezing with liquid nitrogen. The marrow from these animals has been reported as exhibiting osteogenic activity both prior to and after programmed freezing when implanted in microporous-filter-lined chamberslo *Student, Class of 1973. *“Student, Class of 1972. 341

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The use of microporous-filter-lined chambers, first described by Goldhaber,ll has permitted investigators to study the dynamics of osteogenesis. Goldhaber has shown that the diffusion chambers are an immunologically privileged site.ll In addition, Boyne12 has demonstrated that the environment within the chamber is conducive to the formation of new bone by allowing tissue fluids to pass through the filter but inhibiting the ingress of fibroblasts with subsequent scar formation. Despite differences of opinion in regard to which particular cell type in the hemopoietic organ system represents the future bone cells, it is generally agreed that all differentiate from marrow precursors under the proper stimulus. Various authors have referred to these cells as osteogenic cells, preosteoblasts, or osteoprogenitor cells.13 Included among these are : mesenchymal cells, reticular cells, endosteal cells (identified by lo&ion, but a,ctually reticular cells), fibroblasts, and periosteal cells. The belief is that the examination of bone marrow which has been shown to be competent within microporous-filter-lined chambers presents a model which can be studied repeatedly. With this model it would be possible to indicate that the osteogenic activity observed was due to the interaction of the graft and tissue fluid passing through the filter, and not to a combination of several environmental factors. The ultrastructural identification of cellular elements before and after programmed freezing not only would demonstrate the possible osteogenic precursor cells, but also would add valuable information to our understanding of the phenomenon of bone induction. METHODS

AND

MATERIALS

Six adult rhesus monkeys were used. Each animal was anesthetized with intramuscular Sernylan’ at dosesof 0.10 mg. per kilogram of body weight. Several grams of marrow were removed from each animal through an osteotomy in the femoral diaphysis, by means of the procedure described by Newman and Boyne.14 The first portion was placed in a sterile tube containing dimethyl sulfoxide (DMSO) and Hank’s ba.lanced salt solution precooled to +4O C. The specimen of marrow was then placed in an ice-water bath in order to reduce the temperature and allow penetration of the DMSO. Programmed freezing of the specimen was then initiated with liquid nitrogen. The temperature of the marrow was lowered lo C. per minute until the latent heat of fusion was released (solidified). At that point the freezing chamber was flooded with liquid nitrogen. Within 5 minutes the programmed freezing rate of lo C. per minute was reestablished and continued until the specimens were cooled to -50° C. The tubes were quickly transferred to a liquid nitrogen storage tank (-196O C.). Two weeks after the marrow biopsy procedure, the marrow was quick-thawed in a. 37O C. water bath. Instead of implanting, the specimens were prepared for electron microscopy. The second portion was used as a control and prepared for electron micros*Phencyclidine

hydrochloride.

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copy before freezing. Both the frozen marrow and the unfrozen marrow were fixed in 2.5 per cent glutaraldehyde with eacodylate buffer (pH 7.2) for 1 hour. The tissue was then post-fixed in 0~0, for 30 minutes, dehydrated in a graded series of alcohol, and embedded in Epon. The embedded tissue was sectioned on a Portor Blum M-l ultramicrotome with glass knives. The sections were mounted on Parlodion-coa.ted grids and stained for 10 minutes in 5 per cent uranyl acetate followed by 5 minutes in lead citrate.15 Electron photomicrographs were made with a Siemens Elmiskop 1-A. RESULTS

The unfrozen bone marrow was found to contain all the stages of cell maturation and development expected in normal marrow. The primitive blast cell (Fig. 4) was identified by its classic round nucleus and cytoplasmic activity.16 The three major blood lines-leukocytes, erythrocytes, and lymphocytes in their various stages of cell development-could be identified (Figs. 1 and 4). Mitochondria with prominent cristae, endoplasmic reticulum, and intact cell and nuclear membranes (Figs. 1 and 4) were the criteria used to establish that the cells were normal and viable. The reticular cell, which is of particular interest, is seen associated with its network (Fig. 1). Our investigation failed to reveal any necrotic cells in this control marrow. In contrast to the control marrow, the programmed frozen marrow was filled with necrotic cells, many of which had breaks in their cell membranes, indicating considerable damage (Figs. 2, 3, and 6) .I71I8 A granulocyte with a destroyed cell membrane was seen (Fig. 2)) and several cells with pyknotic nuclei were present (Figs. 3 and 6). Myelin figures were also found in many areas (Fig. 3). There were various degrees of cellular destruction, depending upon the area observed. In Fig. 3, the granulocyte in the lower half of the field has intact cell and nuclear membranes, although its cytoplasm seemsto be grossly distorted when compared to the eosinophil in Fig. 1. The cells in the upper half of Fig. 3 have undergone autolysis, characterized by cells with clearing cytoplasm. Also observed was a wide range of destruction. The area in Fig. 3 is representative of the least amount of destruction found, where as Fig. 6 represents an area in which no viable cells can be identified. These areas are of comparable magnification and, in addition, are adjacent to each other within the same section. The two cells which appear to have survived the freezing without undergoing permanent damage are the reticular cell and primitive blast cell. A reticular cell can be seen next to a fat cell and a destroyed granulocyte (Fig. 2). The reticular cell has undergone internal changes. However, cell membranes as well as the nuclear membranes were carefully examined at this magnification and at higher ones and were seen to be intact. A mitochondrion, one of the first elements disturbed when cell damage occurs, is also present although few cristae can be seen.‘??I8 In comparison, the cell adjacent to the reticular cell has breaks in both cell and nuclear membranes. On the basis of our morphologic

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Fig. ( E), red x .16,000.)

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2. Normal marrow showing reticular blood cell, lymphocyte, and reticular

Fig. 2. Comparison freezing. Psrogrammed Pig. 3. A relatively ~5,000.) ( Magnification,

cell (R), near net (arrow)

Sin-g. 1972

fat cell (F), developing eosinophfi can also be seen. (Magnification,

of a reticular cell (R) near fat cell (P) and destroyed eosinophil after Arrow indicates break in cell membrane. (Magnification, x16,000.) viable area in frozen marrow, a myelin figure (arrow) can be seen.

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Fig. 4. Normal marrow showing various stages of blood cell development, and a round blast cell (B) is easily depicted. (Magnification, ~15,000.) Pig. 5. A primitive blast cell of frozen marrow with cell (single arrow) and nuclear ‘double arrow) membrane in tract. (Magnification x17,000.) Fig. 6’. An area of massive cellular destruction in frozen marrow. (Magnification, ~5,500.)

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criteria, the primitive blast cells also have retained intact membranes (Fig. 5). Although the latter are affected by freezing, the damage that occurs to them may be reversible. DISCUSSION In recent years, technological demands for a tissue which has the capacity to restore osseous defects in multiple-stage surgical procedures has been developed. Programmed frozen marrow within millipore filter chamber systems was observed to have osteogenic potential.lO Other investigators have demonstrated that programmed frozen marrow contained as much as 80 to 90 per cent cell viability upon thawing; these observations were not based on morphologic criteria nor do they indicate which cells remained viable.‘Mn On the basis of morphologic criteria, this study failed to confirm previous research which reported an 80 to 90 per cent viability of thawed marrow cells. Although a quantitative study was not performed, many sections were taken; an area of least destruction, as seen in Fig. 3, has no more than sporadic viability. Figs. 3 and 6 represent the range of cell necrosis, Fig. 6 being an area of total destruction of marrow in which no viable cells are seen. Even though this study does not prove that reticular and blast cells are responsible for the osteogenic potential of frozen marrow, it does reveal that marrow which is implanted in millipore filter chambers exhibits gross destruction, with the exception of the two cell types previously discussed. Although a great deal more research is needed to explain clinical findings, the biologic explanation of the preservation of osteogenic marrow is growing in importance as more surgical procedures are being developed. SUMMARY This electron microscopic study has cast doubt on reports of 80 to 90 per cent cell viability after programmed freezing methods but lends support to the specific technique of programmed freezing of marrow for the purposes of osseous reconstructi0n.l The reticular and blast cells which were observed to be viable (on the basis of morphologic criteria) l,support the present theory that these elements of osteogenic bone marrow are responsible for the formation of new bone. This work was performed Boyne,

University

of

California

in the laboratories at Los Angeles

of Dr. George W. Bernard and Dr. @ilip School of Dentistry, Los Angeles, Calif.

J.

REFERENCES 1. Richter,

2. 3. 4. 5.

H. E., Sugg, W. E., and Boyne, P. J.: Stimulation of Osteogenesis in Dog Mandibles by Autogenous Bone Marrow Transplants, ORAL Sunu. 26: 396, 1968. Yeager, J. E., and Boyne, P. J.: The Use of Bone Homografte and Autogenous Marrow in Restoration of Ederitulous Alveolar Ridges, J. Oral Surg. 27: 185-189, 1969. Burkle. J. S.. Gresham. R. B.. Wheeler. T. E.. Brodine. C. E.. and Cranmore. D. : The Use of Frozen Autologous Bone ?&arrow for the Protection of Lethally Irradiated Dogs, Surg. Gynecol. Obstet. 116: 600, 1963. Pappas, A. M., Perry, U. P., Wheeler, T. E., and Hyatt, G. W.: Bone Marrow Storage: Current Concepts, Milit. Med. 126: 347, 1961. Pyle, II. M., and Boyer, H. F.: Preservation of Viability of Frozen Human Marrow, Bibl. Haematol. 19: 89, 1964.

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6. Pegg, D. E.: Freezing of Bone Marrow for Clinical Use, Cryobiology 1: 64-71, 1964. 7. Lavrik, 5. 5.: Conservation of Bone Marrow With Polyvinylpyrrolidone by Freezing in Liquid Nitrogen, Fed. Proc. ‘25: 978, 1966. 8. Kisselev, A. E.: Changing Aspects of Bone Marrow Transplantation, Bibl. Haematol. 23: 248. 1965. Per-ryiV.P.: Techniques of Viable Tissue Storage, Fed. Proe. 22: 102, 1963. 1:: Bovne. P. J.. and Yeaeer. J. E.: An Evaluation of the Osteoaenic Potential of Frozen Marrow, Or& SURG. 26 764-771, 1969. 11. Goldhaber, Induction Across Millipore Filters in Vivo, Science 133: P. : Osteogenic 20652067, 1961. 12. Bovne. P. J.: Regeneration of Alveolar Bone Beneath Cellulose Acetate Filter Imulants. I I J. Dent. Res. 43: g27, 1964. 13. McLean, F. C., and Urist, M.: Bone, Chicago, 1968, University of Chicago Press, p. 18. 14. Newman. M. cf.. and Bovne. P. J.: The Effect of Calcified Bone Matrix on the Osteoaenic 0 Potential of Hemopoietic Marrow, ORAL Suno. 32: 506-5X$ 1971. 15. Venable, J. II., and Coggeshall, R.: A Simplified Lead Crtrate Strain for Use in Electron Microscopy, J. Cell. Biol. 25: 407-408, 1965. 16. Bloom, W., and Fawcett, D.: A Textbook of Histology, Philadelphia, 1968, W. B. Saunders Co., pp. 111-128, 184-200. 17. Florey, H.: General Pathology, Philadelphia, 1962, W. B. Saunders Co., pp. 40-98, 197-234. 18. Robbins, S. L.: Pathology, ed. 3, Philadelphia, 1967, W. B. Saunders Co. Reprint requests to : Dr. M. Barkin Department of Oral Biology School of Dentistry University of California at Los Angeles Los Angeles, Calif. 90024