J. Anat. (1994), 185, pp. 369-376, with 12 figures Printed in Great Britain 369

The vascular system of human fetal long : a scanning electron microscope study of corrosion casts

A. SKAWINA', J. A. LITWIN2, J. GORCZYCA' AND A. J. MIODONSKI3

Departments of 1 Anatomy, and 2Histology and 3 Laboratory of Scanning Electron Microscopy, Jagiellonian University School of Medicine, Cracow, Poland

(Accepted 15 March 1994)

ABSTRACT The vascular system of the femur and humerus was investigated in 17-24 wk human fetuses by scanning electron microscopy of corrosion casts. The number of nutrient foramina present in both bones ranged from 1 to 3 and the number of nutrient vessels associated with individual foramina also varied. The medullary supplied both the cortex and marrow. There was no arterial supply to the shaft cortex from the periosteal side, where only were found to enter the bone. The metaphyses were supplied and drained by conspicuous vascular triads composed of an and 2 . In the marrow cavity, 2 morphologically different areas of the fine vascular network could be distinguished: diaphyseal sinusoids and metaphyseal capillaries forming a 'vascular besom' which was abruptly demarcated by the growth plate cartilage. The cortical microvascular bed was composed of capillaries and more numerous irregular sinusoids. The 2 main vascular systems, nutrient and periosteal, were interconnected not only via the network of cortical capillaries/sinusoids, but also by larger arteries and veins traversing the cortex. The features of the vascular system of human fetal long bones suggest its considerable functional flexibility and its capacity to modify blood flow patterns depending on circumstances.

Key words: Bone; periosteum; vasculature; microvasculature.

approach combining corrosion casting with scanning INTRODUCTION electron microscopy now offers improved resolution The vascularisation pattern of long bones has been and quasi-3-dimensional images of the investigated well recognised since the classic studies of Langer vascular networks (Lametschwandtner et al. 1990). (1876) on human tubular bones, as well as the more This useful technique has so far only been applied to recent investigations of Trueta & Harrison (1953), a limited extent for the investigation of the vascular Brookes et al. (1961) and Trueta & Cavadias (1964). system of long bones, and only in laboratory animals: The shaft cortex is supplied and drained by 2 vascular rat femur (Irino et al. 1975; Ohtani et al. 1982) and rat systems: periosteal and nutrient (medullary). In (Draenert & Draenert, 1980). addition, the metaphyses and epiphyses have their Vascularisation of fetal bone presents an intriguing own blood supplies, which merge after closure of the problem, since it involves bone in the course of growth plate (Rhinelander, 1972). Controversies exist, development and remodelling. Fundamental papers however, concerning such issues as the extent of on this subject were published by Brookes (1958, participation of these systems in the nutrition of bone, 1963) who investigated long and short bones in the or the direction of blood flow through the cortex (for lower limbs in human fetuses using dye and contrast reviews see Brookes, 1971, and Whiteside, 1984). injection methods. It therefore seemed of interest to The vast majority of studies dealing with this examine the vascularisation ofhuman fetal long bones subject have been performed using dye injection using corrosion casting and scanning electron mi- techniques and light microscopy. A relatively novel croscopy.

Correspondence to Dr Andrzej Skawina, Department of Anatomy, Jagiellonian University School of Medicine, Kopernika 12, 31-034 Krak6w, Poland. 24 ANA 185 370 A. Skawina and others

RESULTS MATERIALS AND METHODS The nutrient arterial system Twelve fetuses (7 male, 5 female) with crown-rump lengths ranging from 160 to 240 mm (17-24 wk of The majority of the investigated bones were supplied intrauterine life) were used for the study. The fetuses by a single nutrient artery perforating the shaft cortex, were obtained after spontaneous abortions from the together with an accompanying , through the Institute of Gynaecology and Obstetrics, Jagiellonian nutrient foramen located roughly at mid-shaft level University School of Medicine, Cracow. All abortions (Fig. 1). Occasionally 2 or 3 nutrient foramina were were due to maternal disorders and on inspection the observed in femurs and 2 foramina in humeruses. In fetuses showed no malformations. bones with 2 foramina, 1 contained an artery and About 2 h after the abortion, the thorax of each vein, the other only an artery. When 3 foramina were fetus was opened to expose the and large vessels. present, a solitary artery passed through one of the The heart apex was cut off and a cannula inserted via lateral foramina, another contained both an artery the left ventricle into the ascending and secured and a vein, while the central foramen contained a by a ligature. The vascular system of the fetus was solitary vein (Fig. 2). then perfused with a sequence of solutions, efflux After entering the cavity, the single occurring via the umbilical veins and incised posterior nutrient artery divided into ascending and descending tibial vessels. branches, both running along and near the long axis The perfusion began with heparinised, prewarmed of the shaft (central arteries). When 2 nutrient arteries (37 °C) saline and was continued until the effluent were present, the solitary one made a sharp turn saline was devoid of any visible traces of blood. towards the respective metaphysis whereas the one Perfusion fixation was then carried out with 300- accompanied by a vein gave rise to 2 branches of 600 ml of 0.66 % paraformaldehyde/0.08 % glutar- similar size, running in opposite directions (Fig. 2). aldehyde in 0.2 M cacodylate buffer, pH 7.3 (Paine & These branches further divided into longitudinal Low, 1975) containing 0.2 % lidocaine, at 37 'C. medullary arteries which supplied both bone marrow Finally, 60 ml of casting medium consisting of 8 ml sinusoids and the cortical vessels via radial rami- Mercox CL-2B (Vilene, Tokyo) and 2 ml methyl- fications, short twigs communicating with the sinu- methacrylate (Fluka) containing 0.2 g initiator per soids and longer cortical entering the bone 10 ml of the final casting medium was injected. tissue. The latter arterioles were occasionally seen to Following the injection, the fetuses were kept over- cross the entire thickness of the cortex and to supply night in water at 55 'C in order to accelerate and the periosteal bed (Figs 3, 4). complete resin polymerisation (Miodon'ski et al. 1981). After polymerisation of the resin, the femoral and The periosteal arterial system humeral bones were dissected out, the surrounding The dense vascular network of the periosteum muscles were removed and the bones were softened by supplied the bone cortex of the diaphysis only via preliminary decalcification in 5 % trichloroacetic acid capillaries. A different pattern was observed in the for 6-12 h. Some were cut longitudinally into halves metaphyses where conspicuous triads of larger vessels using a steel microtome knife in order to expose the composed of an artery and 2 veins dispersed into the marrow cavity. All bone samples were further decal- periosteal capillary bed. The triads were connected by cified in several passages of 5 % trichloroacetic acid arcade-like anastomoses (Fig. 5). Some of the triad for 7 d. Following rinsing in distilled water, the arteries, as well as occasional solitary arteries entered specimens were macerated in 10- 15 % potassium the bone cortex (Fig. 6). hydroxide for several days, the solution being changed every day after careful washing in running hot tap The capillary beds and venous system water. The resulting vascular casts were carefully and The cortical capillaries were interconnected with thoroughly cleaned in 5 % trichloroacetic acid, fol- numerous postcapillary also located in the lowed by washing in distilled water for the next few cortex and often displaying a sinusoidal character days. The casts were then freeze-dried, mounted onto (Fig. 7). They formed a network resulting from a specimen stubs using colloidal silver and 'conductive combination of longitudinal and radiating vessels. At bridges' (Lametschwandtner et al. 1980), coated with the endosteal border, the radiating vessels com- gold and examined in a JEOL JSM 35-CF scanning municated with the bone marrow sinusoids via short electron microscope at 20-25 kV. funnel-shaped segments (Fig. 8). In the metaphyseal Vasculature of human fetal long bones 371

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Fig. 1. Nutnent artery (NA) and vein (NV) entering the femur through a single nutrient foramen. pc, periosteal capillaries. Bar, 100 gm. Fig. 2. Longitudinal section through femoral shaft with 3 nutrient foramina, revealing the course of the nutrient system vessels in the marrow cavity. Note that the foramen on the left contains a nutrient artery and vein (NA, NV), the middle one only a vein (emissory vein, EV), and the one on the right only an artery. CV, central venous sinus; ms, marrow sinusoids. Bar, 1000 gim. Fig. 3. Longitudinal section through femoral shaft showing the interconnected vasculature of bone marrow, cortex and periosteum (separated by dashed lines). CA, central artery; CV, central venous sinus; ma, medullary artery; mv, medullary vein; ms, marrow sinusoids; ca, cortical . Bar, 100 jim. Fig. 4. Part of a longitudinal section through femoral shaft showing a cortical arteriole (ca) which traverses the cortex and reaches the periosteal bed (pc). mv, medullary vein; ms, marrow sinusoids; cv, cortical ; cc, cortical capillaries. Bar, 100 gm. regions, sinusoids and venules took the form of a The medullary sinusoids and capillaries were 'vascular besom', abruptly demarcated by the zone of drained by small venules, predominantly originating growth plate cartilage (Fig. 9). The peripheral area of in and near the metaphyseal region. They combined to the 'besom' was composed of very densely packed form medullary veins which ran towards the centre of capillaries which extended to the bone/cartilage the diaphysis and emptied into the central venous interface and looped back towards the diaphysis (Fig. sinus, a very large vessel with irregular contours 9, inset). In older fetuses, however, some vessels were aligned along the long axis of the shaft (Fig. 3). The seen to invade the growth plate cartilage and to form central sinus was drained by the nutrient vein. local intrachondral vascular systems (Skawina et al. Apart from the centripetal venous drainage to the 1994). nutrient vein, the cortical capillaries from the per- 24-2 372 A. Skawina and others

Fig. 5. Periosteal vasculature of the metaphysis with 2 characteristic triads composed of an artery (Ma) and 2 veins (Mv). The triads are interconnected by arcade-like anastomoses (arrowheads). Bar, 1000 gm. Vasculature of human fetal long bones 373 ipheral areas of the bone communicated with small The variability of the number and localisation of periosteal venules. The periosteal venous vessels as the nutrient foramina and the associated vessels well as rare venules coming from the cortex left the observed in this study confirmed our previous findings bone surroundings exclusively at muscle attachment made in a larger material (Skawina & Miaskiewicz, sites (Fig. 10) or, in the case of the humerus, directly 1982; Skawina & Wyczolkowski, 1987). In general, to the deep brachial vein (Fig. 11). the results of the present study are consistent with the The 2 draining systems displayed occasional con- observations of Brookes (1958, 1963) who investigated nections. Some veins originating in the periosteal the vascularisation of human fetal long bones using network were observed to cross the cortex and dye injection. We were able to confirm that apart from communicate with the medullary veins, or even occasional metaphyseal arteries, no arterial vessels directly with the central medullary sinus (Fig. 12). entered fetal bone from the periosteal side, although blood supply to the cortex could be provided by numerous periosteal capillaries invading the bone. We DISCUSSION also observed the 2 types of cortical vessel described The corrosion casting technique adapted to scanning by Brookes (1963): wider sinusoid-like ones which electron microscopy has both advantages and short- were prevalent in our material and narrower vessels, comings. On one hand, the microscopical images probably representing typical capillaries rather than obtained provide a clear view of the spatial ar- precapillaries, as suggested by Brookes. Similar rangement of the vessels, on the other the casts are sinusoid-like intraosseous vessels were also described extremely fragile and can easily be damaged during by Draenert & Draenert (1980) in trabecular bone. final preparation, resulting in incomplete reconstruc- The presence of such vessels is typical of fetal tions of the vasculature. Additionally, for very angiogenesis and can be regarded as one of the fetal complex vascular systems such as that of bone, whole- features of bone vasculature. mount preparations do not permit a clear view of the We could not confirm Brookes's (1958) observation entire vasculature since the dense superficial (peri- that the metaphyseal 'vascular cone' is largely venous osteal and/or cortical) vessels obscure the deeper in nature. The area called by us the 'vascular besom' vessels of the bone marrow. Thus longitudinal was in fact mainly composed of capillaries, although sectioning of the bone, possible only after softening by the majority of veins belonging to the medullary at least partial demineralisation, was a prerequisite to system (draining to the central venous sinus and then reveal the vascularisation of the bone marrow. This in to the nutrient vein) found their origin there. In turn further contributed to mechanical damage to the addition, we could not clearly discriminate the 3 casts. vascular fields described by Brookes (1963) in the Nevertheless, the examination both of the super- human fetal bone cortex, but this could be due to the ficial vascularisation and the sectioned casts provides limitations of our technique, allowing us to view the a relatively good insight into the organisation of the cortical capillary network mostly as the profile vascular system. Although the direction of revealed by a longitudinally sectioned bone. Such blood flow cannot be followed as closely as in some fields, however, were observed in our previous dye dye/contrast injection techniques, the arterial and injection studies on human fetal bones (Skawina & venous vessels can be discriminated on the basis of the Gorczyca, 1984). appearance of endothelial nuclear imprints which are Some findings by other authors who have investi- elongated and often spindle-shaped in arteries and gated bone vascularisation in animals are discrepant roundish or ellipsoid in veins (Miodonski et al. 1976). with our observations, but this may result from The character of the nuclear imprints helps especially interspecies differences. In particular, the medullary in the identification of smaller vessels and their arteries in human fetal bone did not show a spiral arrangement in the system enables blood flow patterns course, reported as typical for rat, rabbit and guinea to be deduced. pig bone (De Bruyn et al. 1970; Draenert & Draenert,

Fig. 6. Metaphyseal region viewed internally (longitudinal section). A solitary periosteal artery (Ma) emerges after penetrating the cortex from the periosteal side. mv, medullary vein. Bar, 1000 gm. Fig. 7. A network of cortical vessels. Note their sinusoidal character. Bar, 100 gm. Fig. 8. The endosteal border (dashed line). Note connections between the cortical vessels and marrow sinusoids. C, cortex; M, marrow. Bar, 100 gim. Fig. 9. Longitudinal section through the metaphysis. The very dense network of capillaries is abruptly demarcated by the avascular growth plate cartilage (C). Bar 1000 gm. Inset: capillary loops (arrows) at the bone/metaphyseal cartilage interface. Bar, 10 gm. 374 A. Skawina and others

Fig. 10. A venule leaving the periosteal capillary bed (pc) at the site of muscle attachment. Bar, 100 gm. Fig. 11. Humerus. Three periosteal veins and a cortical vein (arrow) joining the deep brachial vein (BV). A part of the brachial vein cast with attached periosteal vein broke offduring sample preparation (asterisk). BA, deep brachial artery; pc, periosteal capillaries. Bar, 1000 gm. Fig. 12. A relatively large vein (cv) traversing the cortex from the periosteum and emptying into the central venous sinus (CV) in the marrow cavity. NA, nutrient artery; NV, nutrient vein; pc, periosteal capillary bed. Bar, 500 gm.

1980). Bundles of arterioles entering the endosteal nor did we observe separate non-anastomosing ca- bone surface (Lopez-Curto et al. 1980) and 'bush- pillary beds in the bone marrow and cortex, as like' vascular profiles associated with the vessels described in adult dog tibia, in which blood vessels exiting from the transverse cortical canals (De Saint were injected with a silicone elastomere (Lopez-Curto Georges et al. 1992) were not evident in our material, et al. 1980). On the other hand, connections between Vasculature of human fetal long bones 375 the nutrient vessels and bone marrow sinusoids were primitive woven type, and its subsequent remodelling quite frequent in our material, whereas De Saint to the haversian (osteonal) structure will be ac- Georges & Miller (1992) reported that they were companied by a corresponding rearrangement of the absent in the rat. vascular system. It is now a widely accepted view that blood flow in the long bone cortex is transosteal and centrifugal ACKNOWLEDGEMENTS (Brookes, 1958; Brookes et al. 1961; Ohtani et al. 1982) and that the periosteal blood supply plays only The authors wish to thank Dr Maria Nowogrodzka- a minor, if any, role in the nutrition of the shaft. Some Zagorska for skilled technical assistance. authors, however, have suggested that the cortical flow may be bidirectional (Lopez-Curto et al. 1980), REFERENCES with the outer third of the cortex supplied by the BROOKES M (1958) The vascularization of long bones in the human periosteal and the inner two thirds by the nutrient foetus. Journal of Anatomy 92, 261-267. system (Johnson, 1927; Trueta & Cavadias, 1964). BROOKES M (1963) Cortical vascularization and growth in foetal Moreover, a centripetal direction for the cortical tubular bones. Journal ofAnatomy 97, 597-609. 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