CELL STRUCTURE AND FUNCTION 2, 353-360 (1977)

C by Japan Society for Cell Biology

Hematopoiesis in Bovine Heart Bone

Masa-oki Yamada, Ken Fujimori, Hisashi Takeuchi, Keizo Yamamoto* and Akira Takakusu*

Laboratory for Cytochemistry, Department of Anatomy, Schoolof Medicine, Tokushima University, Tokushima 770, and *Laboratory of Cell Biology, Depart-ment of Anatomy, Nara Medical University, Kashihara, Nara 634, Japan

ABSTRACT. In the bovine heart, chondrification of the fibrous tissue occurs just after birth and proceeds to bony tissue. The ossification appears first in the right fibrous trigonum three months after birth and then in the left trigonum twelve months after birth. During ossification, the bone marrow structure appears to form a narrow space within the bony tissue and develops during the subsequent year. Light and electron microscopic examinations revealed development of typical hematopoiesis in the heart bone marrow. Hemoglobin was estimated in various erythroid cells by cytospectral analysis. The hematopoietic activity was retained for about 10 years in the heart bone.

The presence of cartilage in the heart has been known in man (2) and in vertebrates, e.g., the cow (6, 8), hamster (5), rat (3, 4), bird (8) andeven in the frog (1) and fish (8). In most animals, cartilage appears in peculiar loci of the heart. However, the heart bone was observed as an ossified plate in the fibrous annulus surrounding the base of the aorta (8, 9). In rare cases of avian heart (8), chondrification forms a bony piece involving bone marrow space. The details on the formation of heart bone marrow structure have yet not been described. In dissections of many bovine hearts, we found bone marrow structures and have elucidated the hematopoiesis in the structures. This paper also reports on heart bone hematopoiesis at various age periods by cytospectral analysis of erythroid cells.

MATERIALS AND METHODS

Twenty-seven hearts were dissected from domestic Bos at sacrifice. The examined age range was from newborn to 10 years old and consisted of males, females and castrated males (Table 1). The heart bones were carefully resected from the base of the aorta and pulmonary trunk. The resected bones were cut into small pieces and fixed with 2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH, 7.2, for 1 h at 4° C and examined by electron microscopy. For ordinary histologic examination, the bony pieces were fixed with 10% formalin in 0.1 M phosphate buffer, pH 7.2, then decalcified with 2 % citric acid solution saturated with EDTA-2Na for several days and postfixed with Zenker's fluid. The tissueswere embedded in celloidin-paraffin, sectioned at 15,u and stained with hematoxylin and eosin. The cut face of the raw bone was stamped on a glass slide, dried, fixed with methanol and prepared by Wright stain for blood cell diagnosis. Some samples were applied for cytospectral identification of immature ery-throcytes. Cytospectral analysis was carried out by the relative wavelength-scanning method,

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reported by us (13). This method for characterizing erythroid cell types was applied in combi-nation with Wright stain prepared specimens for blood cytodiagnosis. The specific absorption of hemoglobin was used as a cytospectral marker for erythroid cells. The relative absorption curve ( % ratio of absorption to maximum absorption) was obtained.

RESULTS

Localization and development of heart bone marrow. In the newborn one pair of cartilaginous tissue appeared in the right and left fibrous trigona of the heart. Three months after birth, the cartilage changed to bony tissue, and a pair of heart bones was located in the fibrous trigona a year later. A larger bone piece was formed in the right trigonum, and a smaller bone piece was formed later in the left trigonum, as shown in Fig. 1. These bone pieces obtained from a 1.5 year old cow appeared to form thin

arches surrounding the base of the aorta and kept their position and shape. Fig. 2

shows some dissected bone samples obtained from animals at different ages. Bone

marrow development with aging is shown in Table 1. Chondrification was detectable

in the heart at the postnatal period, then began to ossify from the right to left cartilage.

Ossification was always found in postnatal samples. Thus, chondrification proceeded

ossification, which differed from calcification. The development of the heart bones was

completed after one year or more and was maintained for at least 10 years. The red

Fig. 1. Localization of heart bone at the fibrous trigona, illustrated by hatched lines. R, right

fibrous trigonum; L, left fibrous trigonum; a, anterior cusp; rp, right posterior cusp; Ip, left posterior

cusp; m, mitral valve; hatched lines, right and left heart bones.

Fig. 2. Heart bone pieces dissected from heart of various age bovines. II, Left bone of 18 month

old male; Ir, right bone from the same heart; IIr, right bone of 20 month old male; IIIr, right bone of 2 year old male; IVr, right bone of 2 year old male; Vr, right bone of 3 year old female; VIr, right

bone of 5 year old female; VII, left bone from the same heart.

Hematopoiesis in Bovine Heart Bone 355

marrow appeared in parallel with bone formation and it was replaced gradually withfatty marrow with aging.

Histologic findings. In fibrous trigona of newborn animals, a vasculated space ap-

peared to accompany chondroid tissues and the surrounding perichondroid cells. A hyaline-like cartilage developed in the chondroid, and then a darkly crescent ossified area was seen in cartilaginous tissue surrounding the typical bone marrow structure

(Fig. 3a). In older sample bones many fatty cells were found, but the hematopoiesis was still active (Fig. 3b). At higher magnification (Fig. 3c), various erythroblastic cells formed an islet with young erythroid cells, such as basophilic erythroblasts, orthochromatic erythroblasts and erythroid cells in denucleating stages. Granulo-

poiesis was also distinctly observed, as shown in Fig. 4a. Azurophil granules were found in the neutrophil promyelocyte (Fig. 4b) and metamyelocyte (Fig. 4c). Eosinophil

granules were well-developed in the eosinophil promyelocyte (Fig. 4d). Various other young agranulocytes were also present, such as the monoblast (Fig. 4e), lymphoid cell (Fig. 4f) and megakaryocyte (Fig. 3b, Fig. 4g).

TABLE 1. BONE MARROW OF BOVINE HEART BONE BY AGE AND SEX

356M. Yamada et al.

Fig. 4a. Various granulated cell types in the heart of 18 month old. •~ 2000

Fig. 4b. Neutrophil promyelocyte containing azurophil granules of high electron density in

the heart of 18 month old. •~ 5,000.

Fig. 4c. Neutrophil metamyelocyte containing both azurophil and specific granules in the

heart of 18 month old. •~5,000.

Fig. 4d. Eosinophil promyelocyte containing eosinophil granules with well-developed Golgi

vesicles. •~ 5,000.

Fig. 4e. Monoblast showing a fibrous cytoplasmic structure with mitochondria and granules

assembling to the concave space of the nucleus in the heart of 18 month old. •~ 5,000.

Fig. 4f. Lymphoid cell in the heart of a year old. •~ 3,300.

Fig. 4g. A cytoplasmic area of megakaryocyte showing a multiple endoplasmic membrane

system attached to numerous small granules of high electron density in the heart of 18 month old,

•~ 2,600.

Fig. 3a. An ossified area is stained

darkly crescent in the hyaline-like

cartilage. In the upper area, a blood

forming cell population is seen. From

the heart of 18 month old. Stained with

hematoxylin-eosin. •~ 200.

Fig. 3b. A cell population in hema-

topoiesis is compressed with large

spaces of fatty cells. A megakaryocyte

is seen in the middle. From the heart of

20 month old. Stained with hematoxy-

lin-eosin. •~ 200.

Fig. 3c. Normoblasts (B) in cyto-

spectral analysis in comparison with

normocytes (C) in the heart of 20 month

old. •~ 800.

Hematopoiesis in Bovine Heart Bone 357

Fig. 4

358 M. Yamada et al.

Cytospectral examination in erythroid cells. The specific absorption of hemoglobin was measured and distinguished from other absorption to identify growing erythroid cells. Fig. 3c shows the relative absorption curves (Fig. 5) of normoblasts and normo-cytes. There were marked spectral differences between normoblasts and normocytes. Absorption at 410 nm (A410) was lower in normoblasts than erythroblasts, and absorption at 600 nm (A600) was much higher in erythroblasts than normocytes. These differences were demonstrated in nucleated erythroid cells of heart bone mar-row. A600 is regarded as a parameter of cytoplasmic basophilia (11). Thus, the A600 value seems applicable in distinguishing young erythroid elements from normocytes. This cytospectral analysis may elucidate the presence of immature erythroid elements in heart bone marrow.

DISCUSSION

An extraordinary hematopoiesis may be initiated by stem cell emigration from the original bone marrow blood-forming site, and differentiation follows in cooperation with reticulum cells in a newly blood-formed site. A stem cell was not identified in the fibrous trigona of the bovine heart. However, reticulum cells appeared in vasculated loci, which accompanied chondroid tissue just after birth, and then ossified after three months, and one pair of heart bone was completed at one year. As ossification pro-ceeded, a narrow space developed to form the bone marrow, with various blood cell types in active hematopoiesis. The ossification occurred ina strictly restricted loci of the heart with hematopoiesis without exception. Active erythropoiesis was also con-firmed by cytospectral analysis for hemoglobin and basophilia of erythroid cell ele-ments. This erythropoietic activity does not appear only after birth, but appears three months later and is retained for 10 years or more. The bonemarrow does not seem to be an embryonal rudiment, because no trace was observed on blood cell reproduction immediately after birth. A sequential change is present in the postnatal development of the heart bone marrow. The sequence may consist of chondrification, ossification,

Fig. 5. Relative absorption of erythroid cells in bovine heart bone marrow stained with the Wright method for blood cytodiagnosis (13).

Hematopoiesis in Bovine Heart Bone 359

initial migration of stem cells, formation of hematopoieticislets, completion of red marrow and appearance of yellow marrow. This scheme is likethe ordinary sequence of blood formation. Matumoto (8) observed a small space filled with blood in avian heart bone. He described it as a bone marrow space and regarded it as a rare case. A similar description was made on the rat heart by Takazaki(10). However, no details have been reported on the ordinary formation of blood in the heart bone.

On the other hand, the presence of chondrification has been found in the heart of hamsters (5), birds (8) and even frogs (1), and bony tissues have been found in the heart of cows, birds (8) and humans (2, 9). Most cases are reported as cartilage pieces, and reports on bone formation are exceptional. But a cartilaginous focus was regularly found in rats as young as one-week old (3). Similarly in our bovine heart observations at different ages, the presence of bone does not seem rare,but common, and the usual sequential changes involving ossification with preliminary chondrification were found. Thus, some animal hearts may remain as chondrified tissues and the others may ossify. There is, however, insufficient observation on hearts at different ages in the same species to come to definitive conclusions.

Another problem may be related to pathologic changes of the heart. Calcification of animal hearts is known as a pathology (1, 4, 7, 12), whereas partial calcification is described in various areas of the heart, especially in the aortic wall. In the intima of an aged human aorta, the presence of a calcified piece with sclerotic complications was described (9). On seven human autopsy cases after traffic deaths (21-61 years old) no trace of heart bone was observed. Thus, sclerotic calcifiedchanges do not seem to be a part of the ordinary process of ossification. Moreover, there was no sign of blood formation. In a rare case (7), chondroid metaplastic changes overlapped with ather-osclerosis. In such peculiar cases, the pathologic changes deviated from the hemato-

poiesis commonly found in all heart bones examined in this study.

Acknowledgments. The authors are indebted to Prof. Michihiko Maeiwa of the Department of

Legal Medicine of this university and the Veterinary Society of Tokushima for preparing the ma-

terials and Messrs. Masayuki Shono and Yoichi Takaue and Misses Sayo Inui, Yuko Enomoto and

Kiyoko Hanaoka for their help.

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(Received for publication, May 30, 1977)