Microvascular features and ossification process in the femoral head of growing rats

J. Anat. (1999) 195, pp. 225–233, with 10 figures Printed in the United Kingdom 225

Microvascular features and ossification process in the femoral

head of growing rats

SERGIO MORINI1, LUIGI PANNARALE2, ANTONIO FRANCHITTO2,3, SAARA DONATI2

AND EUGENIO GAUDIO1,3, 4

"University Campus Bio-Medico, Rome, # Institute of Anatomy, State University ‘La Sapienza ’ Rome, $ ISEF, L’Aquila,

and %Department of Experimental Medicine, State University, L’Aquila, Italy

(Accepted 4 May 1999)

In the epiphysis of long bones, different patterns of development of ossification processes have been

described in different species. The development of the vascularisation of the femoral head has not yet been

fully clarified, although its role in the ossification process is obvious. Our aim was to investigate ossification

and vascular proliferation and their relationship, in growing rat femoral heads. Male Wistar rats agedC 1, 5

and 8 wk and 4, 8 and 12 mo were used. Light microscopy frontal sections and vascular corrosion casts

observed by scanning electron microscopy were employed. In the rat proximal femoral epiphysis, ossification

develops from the medullary circulation of the diaphysis, quickly extending to the neck and the base of the

head. Hypertrophic chondrocytes occupy the epiphyseal cartilage, and a physeal plate with regular cell

columns is present. Starting from about the end of the third month one or more points of fibrovascular

outgrowth, above the physeal line, can be observed in each sample. They are often placed centrally or,

sometimes, peripherally. The fibrovascular outgrowths penetrate deeply into the cartilage and extend

laterally. At age 8 mo, large fibro-osseous peduncles connect the epiphysis to the diaphyseal tissue. At

12 mo, the entire epiphysis appears calcified with an almost total absence of residual cartilage islands. This

situation differs in man and in other mammals due both to differing thickness of the cartilage and to the

presence of more extensive sources of blood vessels other than the diaphyseal microcirculation, as supplied

by the teres ligament and Hunter’s circle. In young rats, subchondral vessels and the synovial fluid could

play a role in feeding the ossifying cartilage. Later, a loss of resistance of the physis due to marked

degeneration of the cell columns, and extensive chondrocyte hypertrophy permit fibrovascular penetration

starting from diaphyseal vessels rather than neighbouring vascular territories, such as those of the

periosteum and capsule.

Key words : Bone; cartilage ; epiphysis ; vasculature.

Ossification processes and their relationship to vas-

cular proliferation in the epiphysis of long bones is a

more complicated and debated subject than the

ossification mechanisms in the diaphysis (Trueta &

Morgan, 1960; Trueta, 1963; Visco et al. 1990; Brown

et al. 1993).

In the literature, different patterns of development

of secondary ossification centres have been described.

The one most frequently cited is ossification starting

around preexisting cartilage canals (connective tissue

Correspondence to Prof. Eugenio Gaudio, Dept. of Experimental Medicine, University of L’Aquila, via Vetoio, Coppito 2, 67100 L’Aquila,

Italy. Tel. : 39 (862) 433504; fax: 39 (862) 433523; e-mail : gaudio!univaq.it

spaces extending into the cartilage and containing an

arteriole) mainly vascularised by particular vessels

that are not directly connected with the diaphyseal

circulation. This pattern has been found in man

(Haines, 1933; Hurrel, 1934; Levene, 1964), dogs

(Levene, 1964; Wilsman & Van Sickle, 1972), mice

(Kugler et al. 1979), pigs (Visco et al. 1990) and

rabbits (Levene, 1964; Ganey et al. 1992). A second

type of ossification is the one that takes place in front

of a peripheral line of vascular proliferation without

involving preexisting cartilage canals, as demonstrated

in mice by Floyd et al. (1987). A third type, observed

in man, involves the formation of the so-called

pseudoepiphysis : a mushroom-like extension of the

shaft bone into the epiphyseal cartilage (Lee & Garn,

1967; Haines, 1974). This corresponds to an extension

of the primary ossification process from the shaft.

Many factors, among which genetic, metabolic and

endocrine influences, probably contribute to deter-

mine the pattern of growth of the secondary ossifi-

cation centres (Resnick & Niwayama, 1988). How-

ever, special significance should be attributed to

metabolic factors because the development of ossifi-

cation is always strictly connected to the presence of

preexisting or newly formed vessels in the cartilage

(Trueta, 1963; Wilsman & Van Sickle, 1970; Reiden-

bach & Schmidt, 1994; Roach et al. 1998).

Although the importance of vascular processes

involved in the formation of secondary ossification

centres, especially with regard to the orientation of

epiphyseal growth, these processes are not completely

understood. Epiphyseal formation and development

have also clinical relevance in diseases where the

normal physiology of epiphyseal growth is altered,

such as in osteonecrosis of the femoral head in

children (Thompson & Salter, 1987; Chang et al.

1993).

Cartilage canals have been widely studied in many

species (Wilsman & Van Sickle, 1970; Haines, 1974;

Visco et al. 1990; Ganey et al. 1992) and the

morphology and ultrastructure of the vessels within

them have been also described (Stockwell, 1971;

Wilsman & Van Sickle, 1972; Skawina et al. 1994;

Ganey et al. 1995). Cartilage canals seem to be species

specific for analogue cartilages as shown in man

(Nelson et al. 1960; Levene, 1964), rabbit (Gothman,

1960; Ganey et al. 1992) and other species (Levene,

1964). Moreover, it is to be noted that different

patterns of epiphyseal ossification can also coexist in

the same bone, as in human metacarpals, metatarsals

and phalanges (Ogden et al. 1994).

The situation in the rat is somewhat confused due

to the report of large canals in some cartilages, such as

the proximal tibia in young animals (Levene, 1964)

but not in the proximal epiphysis of the femur (Thorp

& Dixon, 1991). Moreover, a supposed vascular

proliferation and ossification process in the femoral

head similar to that of man has been reported (Hirano

et al. 1992, 1994), but apparently it is not supported

by other studies (Thorp & Dixon, 1991).

Our aim was to investigate ossification and vascular

proliferation and their relationship, in growing rat

femoral heads, both by light microscopy (LM) and

vascular corrosion casts observed by scanning electron

microscopy (SEM).

Male Wistar rats agedC 1, 5 and 8 wk (150 g body

weight) and 4, 8 and 12 mo (350–750 g body weight)

were used, with the approval of the Ethical Committee

of the Institute of Human Anatomy of the University

of Rome ‘La Sapienza’.

Light microscopy

Four rats from each age group were killed by an

overdose of sodium thiopental and the hind limbs

were dissected. Hip muscles were removed in order to

expose the articular capsule which was cut open at its

acetabular insertion. Both femoral heads were dis-

located from their site in order to cut the teres

ligament. Samples were decalcified with Histodec and

cut into 2 symmetric pieces along a longitudinal

section plane including the greater trochanter and the

axis of the anatomical neck of the femur. Specimens

were embedded in paraffin and 5 µm sections cut

following the previous section plane. Finally, speci-

mens were stained with haematoxylin and eosin (HE)

and Masson’s trichrome.

SEM of vascular corrosion casts

Based on the data obtained by LM, 4 and 8 wk as well

as 4, 8 and 12-mo-old rats were employed. Four

animals from each age group were infused with resin to

obtain the casts. Animals were anaesthetised initially

with diethyl ether and immediately after with an

intraperitoneal injection of 15–20 I.U. sodium thio-

pental. In order to facilitate blood drainage, i.v.

heparin and acetylcholine were also injected. After

having performed a median abdominal incision, the

abdominal aorta was isolated, dissected and cannu-

lated using an Inpharven – Int cannula (diameter

1±7 mm). The vascular bed was cleared by prewarmed

0±9% normal saline and then injected with 50 ml

diluted Mercox resin (4:1) under a pressure of

200–250 mmHg forC 7 min. Pressure was monitored

in the cannula by means of a CONEL electronic

manometer. The pressure value was obtained by

trying increasing injection pressures until consistent

and satisfactory filling of the vessels was achieved,

according to our previous experience (Pannarale et al.

1991, 1997; Gaudio et al. 1993).

Perfused animals were kept for 24 h at room

temperature for better polymerisation of the resin.

Following this, the hindlimbs were dissected as

already described. Remnants of muscle and connective

tissue were removed by leaving bones in 20% NaOH

226 S. Morini and others

solution for 24 h. The femoral bones were then

processed with 5% HCl for decalcification. Despite of

the very long exposure of the samples to repeated

baths in HCl solution (up to 3 baths, for a total time

of 60 h), precipitates always spoiled the appearance of

the cast surface. This problem has already been

reported in the literature (Aharinejad et al. 1995;

Pannarale et al. 1997). Therefore, samples were placed

in 5% trichloroacetic acid for complete removal of

tissue remnants. Finally, the casts were frozen in

distilled water and freeze-dried (Lametschwandtner et

al. 1990). Specimens glued onto stubs and coated with

platinum were examined in a Hitachi S4000 field

emission SEM operating at an accelerating voltage of

7 kV.

1st week

The whole epiphysis was made up of hyaline cartilage.

Head, neck and greater trochanter were easily recog-

nised in frontal LM sections. Neither cartilage canals

nor secondary ossification centres were present

(Fig. 1).

1st month (4–5 wk)

Ossification of the diaphysis had already extended

into the epiphysis, occupying the whole femoral neck.

Less than one third of the head was made up of bone

or calcified cartilage. An ‘M-shaped’ tide line of

ossification bordered by a fine line of calcified cartilage

corresponded to the cell columns of the growing

cartilage (Fig. 2). No secondary ossification centres

were observed. A secondary ossification centre was

present at the tip of the greater trochanter together

with physeal ossification.

Fig. 1. Femoral head, 1-wk-old rat. Note the arrangement of

chondrocytes to constitute initial cell columns (arrows). Some

vessels are present near the insertion of the capsule on the neck

(arrowhead). Haematoxylin and eosin. Bar, 100 µm.

2nd month (8–9 wk)

The ossification front appeared as a straight line

situated at the bottom of the femoral head with initial

focal degeneration of the cell columns (Fig. 6a).

Secondary ossification centres were not present in the

head. The cartilage showed an extended zone of

chondrocyte hypertrophy which extended into the

whole core of the cartilaginous head, with lack of

isogenous groups and a multiple cell group pattern.

Chondrocytes tended to be flat and parallel to the

articular surface in the periphery. No notable vessels

were visible extending from periosteum into the

epiphyseal cartilage. The teres ligament showed only a

few small capillaries at its insertion on the femoral

head (Fig. 3). A secondary ossification centre was still

present in the greater trochanter. In the first 2 months

the epiphyseal end of the corrosion cast showed a

convex appearance corresponding to the lower surface

of the physis. At this level, terminal vessels, 10–15 µm

Fig. 2. Femoral head, 6-wk-old rat. Tide line of ossification with

calcified cartilage (arrows) and cell columns of growing cartilage

(*). A zone of chondrocyte hypertrophy is present above.

Haematoxylin and eosin. Bar, 100 µm.

Fig. 3. Insertion of the teres ligament on the femoral head in 9-wk-

old rat. No vessels larger than capillaries (arrowheads) are present.

Masson’s trichrome. Bar, 100 µm.

Microcirculation in rat femoral head 227

Fig. 4. Vascular corrosion cast of physeal surface. SEM. (a) Vessels

at the surface of the physis forming short vascular loops. Bar,

100 µm. (b) Small sprouts (arrowheads) are present at the tip of a

vascular loop. Bar, 20 µm.

Fig. 5. Femoral head, 4-mo-old rat. Clusters rather than columns of

partially degenerated chondrocytes are present above the ossifi-

cation tidemark. A fibrovascular outgrowth (arrows) is clearly

surrounded by calcified cartilage and degenerated chondrocytes.

Haematoxylin and eosin. Bar, 100 µm.

in diameter, formed short, most often curling loops.

At the tip of the loops small sprouts indicated

metaphyseal vascular proliferation (Fig. 4).

Fig. 6. Progressive stages in ossification in the physis. (a) 8-wk-old

rat. The cell columns of the growing cartilage show initial focal

degeneration and calcification (arrowheads). Masson’s trichrome.

Bar, 100 µm. (b) 4-mo-old rat. Marked degeneration (arrowheads)

of the characteristic cell columns of the physis with initial

fibrovascular penetration (arrow). Haematoxylin and eosin. Bar,

100 µm. (c) 4-mo-old rat. More advanced phase of fibrovascular

penetration (arrows) through the cartilage. Note the absence of the

cell columns in the physeal plate. Haematoxylin and eosin. Bar,

100 µm.

4th month

The physeal line acquired an irregular front due to

fibrovascular penetration from the metaphysis into

the epiphyseal cartilage (Fig. 5) ; an increase of its

basophilic staining was also evident. The charac-

teristic cell columns appeared heterogeneous and

partially degenerated and replaced by clusters of cells


Fig. 7. Vascular corrosion cast of neck and head microvasculature at the 4th month. SEM. (a) Deeper network; P, periosteal network; Ap,

periosteal artery ; Vp, periosteal vein. Bar, 500 µm. (b) A, medullary arteriole ; S, sinusoids ; C, cortical vessels. Bar, 100 µm.

separated by extracellular matrix (Fig. 6b). An

irregular tidemark which extended into the cartilage

near the osseous penetration was evident. Calcified

cartilage and degenerated chondrocytes surrounded

the fibrovascular proliferation. One or more points of

fibrovascular outgrowth could be observed in each

sample (Fig. 6c). They were often placed centrally or,

sometimes, also peripherally. The fibrovascular out-

growths penetrated deeply into the cartilage and

extended laterally. The number of outgrowths and

their lateral extension was variable in subjects of the

same age. The corrosion casts showed 2 clearly

different well defined vascular networks in the epi-

physis (Fig. 7a). The periosteal microvasculature of

the femoral neck arose from arteries which gave off

collateral branches. Capillaries formed a delicate

network which ended with terminal arcades that

encircled the femoral neck just under the level

corresponding to the epiphyseal cartilage. No indi-

cations of proliferation from these vessels were

evident. Veins showed a more irregular pattern and

were more numerous than arteries. The deep circu-

lation was composed of vessels arising from the

diaphyseal vasculature, which ran along the main axis

of the femoral neck. Arterioles (30 µm) divided by

bifurcation (Fig. 7b), while venules (50 µm) had an

Fig. 8. Vascular corrosion cast, SEM. Marginal bundle (B) of

vessels arising from medullary vessels (M) of the femoral neck

showing small outgrowths (arrows) facing backwards with short

connections at their tips. Bar, 150 µm.

irregular branching pattern. Elongated sinusoids were

present in the depth; they were less densely arranged

than in the upper diaphysis. Fine cortical vessels

originating from the medullary circulation were visible

in the neck while sparse sprouts were still observable

in the surface of the head. A new feature was the

presence of vascular outgrowths made up of bunches

of vessels extending over the adjacent surface and

eventually mushrooming to different extents (Fig. 8).


Fig. 9. Femoral head, 8-mo-old rat. (a) Arrows, articular cartilage ;

Pd, fibro-osseous peduncle ; *, physeal plate. Masson’s trichrome.

Bar, 100 µm. (b) Vascular corrosion cast, SEM. Microvascular bed

of the femoral head stemming from a single vascular peduncle (Pd).

A deep furrow (*) is present in the cast between diaphyseal (D) and

femoral head (H) microvascular beds. Bar, 350 µm.

Smaller outgrowths were composed of branching

arterioles and venules with short connections at their

tips which, in turn, showed smaller vascular sprouts.

8th month

The osseous expansion deeply invaded the cartilage

proceeding towards the margins (Fig. 9a). A thin

layer of articular cartilage covered the femoral head.

Generally, one or more fibro-osseous peduncles

connected the epiphyseal growth to the diaphyseal

tissue, leaving a region of cartilage analogue to the

physis. Signs of provisional calcification and chondro-

cyte degeneration were present diffusely in the

epiphysis, and calcified cartilage islands were still

traceable within it. A few small cavities containing

vessels and bone marrow could be detected. Vascular

corrosion casts showed that the microvascular bed

extended widely into the femoral head stemming from

one or more restricted peduncles (Fig. 9b). A deep

furrow was always present in the cast between the

diaphyseal and femoral head microvascular beds. It

Fig. 10. Femoral head, 12-mo-old rat. Note the reduced presence of

residual cartilage islands (arrowheads) and a more mature ap-

pearance of the calcified tissue. Masson’s trichrome. Bar, 100 µm.

corresponded to the physeal space and divided the 2

microcirculatory districts of the femoral neck and

head. The microvasculature underlying the articular

surface was almost completely composed of slender

cortical vessels connected with elongated bone mar-

row sinusoids.

12th month

The entire epiphysis appeared calcified with an almost

total absence of residual cartilage islands. Sinuses

were better shaped and bone marrow together with

sinusoidal vessels stood out clearly on this background

(Fig. 10). The microvascular pattern corresponded to

that established at 8 mo, except for a less evident

furrow at the physeal level.

The development of the vascularisation of the femoral

head has not yet been fully established, although its

role in the ossification process is clearly evident

(Trueta, 1963; Wilsman & Van Sickle, 1970).

As carefully described by Trueta (1957) in man, one

or more secondary ossification centres develop and

eventually join into a larger ossification centre which

occupies the whole femoral head. The secondary

ossification centres in man are fed by blood vessels

arising from the teres ligament, and by capsular and

periosteal vessels. When the cartilage growth plate

begins to disappear during the adolescent period,

vascular connections with the diaphysis develop. Our

LM and SEM observations on vascular corrosion

casts in the rat show that vascularisation as well as

ossification differ from those in man. The processes do

not start before the 3rd month, and take origin from


the deep diaphyseal network. The aspects of vascular

proliferation under the physeal plate (sprouting from

capillary loops) correspond to the microvascular

patterns observed in growing long bone metaphysis

(Arsenault, 1987; Hunter & Arsenault, 1990; Stanka

et al. 1991; Aharinejad et al. 1995) and also in other

bones (Pannarale et al. 1997). No vessels arising from

other regions, such as the periosteum and teres

ligament, run into the cartilage. As seen in the

vascular corrosion casts, the periosteal capillaries end

with terminal arcades that run in a circle around the

femoral neck. At this site no vascular proliferation is

evident. The teres ligament shows only a few small

capillaries at the junction with the femoral head,

probably insufficient to supply the metabolic needs for

ossification. Moreover, in agreement with other

authors (Koshino, 1975; Thorp & Dixon, 1991),

blood vessels are only present in the rat femoral head

after calcification of the matrix and are associated

with ossification of the cartilage.

The SEM vascular corrosion cast studies of Hirano

et al. (1994) showed secondary ossification centres in

the femoral heads of growing Wistar rats as early as

the 5th and 6th weeks. This clearly contrasts with

our observations as far as age and ossification are

concerned. Hirano and coworkers did not specify

which substrain of Wistar rats was employed in their

1994 paper, but it is likely that it was the SHR

substrain as it was used in the previous studies from

this group (Hirano et al. 1989, 1992). This substrain

shows ossification modalities for the femoral head

that are very different both in the number and

location of the secondary ossification centres. More-

over, it does suffer from vascular disturbances,

including a high frequency of osteonecrosis (Yamane,

1987; Iwasaki & Hirano, 1988; Iwasaki et al. 1992).

The ossification process we found could be con-

sidered very similar in some aspects to that dem-

onstrated in human phalanges and metacarpal and

metatarsal bones. In these, pseudoepiphyses have

also been documented (Haines, 1974). The pseudo-

epiphysis has been described as an extension of the

diaphysis into the epiphysis (Thomson, 1869; Pfitzner,

1890; Freund, 1904; Bailleul 1911, 1914; Haines,

1974) and appears as a variably sized secondary

ossification centre connected through the metaphysis

by one or more osseous bridges. The blood supply

always came from cortical or medullary vessels.

Haines (1974) and Ogden et al. (1994) carefully

described the pseudoepiphysis from histological

samples. Ogden et al. (1994) identified in man 3 basic

patterns of pseudoepiphysis formation: central, per-

ipheral, and multiple. The present study yielded only

the first 2 patterns. Nevertheless, some samples

showed small fibro-osseous outgrowths from the

diaphysis into the metaphyseal cartilage, suggesting

that a multiple pattern growth could be starting,

although was not yet evident at the time of ob-

servation.

A relevant difference was found with respect to the

previously described pseudoepiphysis, i.e. the absence

of vascular canals into the epiphyseal cartilage. Haines

(1974) associated the development of a pseudo-

epiphysis in human bones to the observation that

preexisting cartilage canals frequently degenerated

and underwent chondrification, finally resulting in

‘ghost canals ’ that did not contain vessels able to feed

the cartilage. In addition, Ogden et al. (1994) found in

man fewer cartilage canals in the extremity of bones

that display a pseudoepiphysis. In our study, the

absence of cartilage canals could be justified by the

limited thickness of the epiphyseal cartilage (Wilsman

& Van Sickle, 1970; Floyd et al. 1987) ; in addition, the

epiphyseal cartilage could receive adequate nutrition

from the subchondral vessels and the synovial fluid.

Extensive hypertrophy in and around the areas of

osseous formation characterises the epiphyseal car-

tilage, and the formation of hypertrophic chondro-

cytes clearly precedes vascular invasion (Wilsman &

Van Sickle, 1970; Haines, 1974; Floyd et al. 1987;

Visco et al. 1990; Ganey et al. 1992; Ogden et al.

1994). However, the role of chondrocytes in per-

mitting the vascularisation of cartilage and the

formation of bone has still to be elucidated. Several

investigators thought that the formation of hyper-

trophic chondrocytes could play a key role by

releasing angiotrophic factors and thus inducing

angiogenesis in connective tissues (Bridge & Pritchard,

1958; Trueta, 1963; Kugler et al. 1979; Floyd et al.

1987). However, as demonstrated during bone for-

mation under tension (Ganey et al. 1995), basement

membrane deposition is present without an inter-

mediary hyaline matrix hypertrophic chondrocyte

phase, suggesting that chondrocyte hypertrophy at

the growth plate may be a reaction to vascular

invasion that, in turn, stimulates adjacent chondrocyte

proliferation. A recent in vitro study (Roach et al.

1998) showed that when at least 2 vessels are in

apposition, chondrocytes become hypertrophic and

the matrix calcified, suggesting that cumulative release

of factors from many vessels might play a trigger role

for chondrocyte hypertrophy.

The formation of the pseudoepiphysis is associated

with loss of cell column formation at the physis and

development of a tide line especially near the areas of

osseous bridging (Haines, 1974; Ogden et al. 1994). In


agreement with the literature, the temporarily re-

maining intact physeal cartilage loses the significant

hypertrophic cell columns capable of contributing to

the longitudinal growth of the involved bone; although

some minor cell columns are evident, finally only islets

of degenerated cartilage remain.

In conclusion, in the rat proximal femoral epiphysis

the ossification process develops from the diaphysis,

quickly extending to the neck and the base of the

head. A physeal plate with regular cell columns is

present at this stage. Epiphyseal cartilage probably

does not need vascular canals because it could receive

a sufficient supply from the subchondral vessels and

synovial fluid. Within the cartilage, hypertrophic

chondrocytes begin to develop; a loss of resistance

with degeneration of the physis (van den Hooff, 1964;

Haines, 1974; Brown & McFarland 1992) permits

fibrovascular penetration starting from metaphyseal

vessels rather than neighbouring vascular territories

such as the periosteal or capsular regions. In man

(Trueta, 1957; Skawina et al. 1994) and in other

animals (Thorp & Dixon, 1991; Hayashi, 1992; Brown

et al. 1993) the development of the vasculature and

ossification process in the femoral head differs, due to

blood vessel penetration into the cartilage in an earlier

age when a critical mass of cartilaginous tissue

develops (Ganey et al. 1992, 1995). The sources of

blood vessels in the femoral head are correlated with

the presence of various neighbouring vascular terri-

tories, as supplied by the diaphyseal microcirculation

in the rat, or also by the teres ligament and Hunter’s

circle in other larger animals.

This work was supported by grants from Italian

MURST (40% funds) and from ISEF of L’Aquila.

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Microvascular features and ossification process in the femoral head of growing rats

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