Ossification of the Femur and Tibia of the Post-Hatching Japanese Quail
Yasser A. Ahmed,
Soha A. Soliman
Enas A. Abdel-Hafez
The current study aimed to describe the histological changes
of the femur and tibia of the post-hatching quail. Femur and tibia from 1-day-
to 6-weeks post-hatching quail were processed for light microscopy. Histological
examination revealed that endochondral ossification was a delayed process in
the development of femur and tibia preceded by periosteal ossification. Femur
and tibia of 1-day-post-hatching quail consisted of growth cartilage enclosed
in a tube of periosteal bone collar. The collar extended toward the epiphysis
dividing it into articular cartilage proper and lateral articular cartilage.
Down to the articular cartilage, there was a physeal growth cartilage, in which
the chondrocytes were organized into resting, proliferative and hypertrophic
zones. Focal areas of hypertrophic chondrocytes were observed in the epiphysis
of the tibia but not of the femur, which acted as a nidus for formation of the
secondary ossification centre after in 2-week-posthathcing quail. Primary ossification
centre was seen in both femur and tibia after 2 weeks and ossification continued
replacing the cartilage until the 6th week when only permanent articular cartilage
remained. Cartilage canals were present in both femur and tibia starting from
the day 1, but chondrified and completely disappeared after the 6th week. The
current study suggests that the periosteal ossification preceded the endochondral
ossification and plays an important role in quail long bones development.
Received: January 11, 2013;
Accepted: March 04, 2013;
Published: April 15, 2013
Long bone growth and development occurs through the process of endochondral
ossification in which a cartilage template is formed and gradually replaced
by bone tissue. During the embryonic life, condensed undifferentiated mesenchymal
cells differentiate into chondrocytes which secrete a specific collagen type-II
and proteoglycan- rich matrix of a cartilage template. The central chondrocytes
of the cartilage template undergo further differentiation into hypertrophic
cells and a periosteal bone collar is formed around them. The hypertrophic chondrocytes
physiologically die and the cartilage matrix at this area is invaded by bone
cells, blood cells and blood vessels forming a Primary Ossification Centre (POC)
at the middle of the cartilage template. Similarly, a Secondary Ossification
Centre (SOC) is established at each end of the template. Postnatally, remnants
of the cartilage template remain only as Physeal Growth Cartilage (PGC) between
the POC and SOC and around the SOC as articular cartilage. The ossification
spreads through the cartilage and at specific time when the animal matures,
the POC and SOC get together leaving only the permanent articular cartilage
(reviewed by Mackie et al., 2008, 2011;
Cicek et al., 2011). The chondrocytes of the
PGC are organized into three distinct zones; resting, proliferative and hypertrophic.
The resting chondrocytes are rounded and randomly distributed throughout the
cartilage matrix, the proliferative chondrocytes are flattened and arranged
in columns parallel to the longitudinal axis of the long bone, while the hypertrophic
chondrocytes are enlarged cells important for calcifying the cartilage matrix
(Jeffcott and Henson, 1998; Rivas
and Shapiro, 2002). Hypertrophic chondrocytes die by modes of physiological
non-apoptotic death before replacement of the cartilage remnants with bone tissue
(Roach and Clarke, 2000; Ahmed
et al., 2007; Chen et al., 2010). Although,
the permanent articular cartilage is avascular organ, the developing growth
cartilage are normally invaded by many vascular channels contributing to nourishment
of the developing long cartilage and thought to help in the formation of the
SOC (Burkus et al., 1993; Blumer
et al., 2005) but as the animal matures, cartilage canals of the growth
cartilage normally chondrify and disappear. The long bone development of mammals
has extensively been studied, however, information on the development of the
avian long bones is very poor (Pourlis et al., 2006;
Simsa and Ornan, 2007). The current study highlighted
the process of long bone ossification in quail.
MATERIALS AND METHODS
Raising of birds: Japanese quail (Coturnix japonica) were raised
in the quail research centre belonging to the Histology Department, Faculty
of Veterinary Medicine, South Valley University and used for the current experiment.
The birds after hatching were feed on poultry starter 28% protein with soybean
meal sorghum was added to the ration as an external source of protein. After
14 days, the ration was poultry starter with 21% protein until the end of the
experiment. Temperature was 38°C during the first week, but decreased to
24-30°C to the end of the experiment. Regarding the light program, the birds
after hatching till the first 3 days were exposed to 24 h light day-1
and decreased gradually to reach 12 h day-1 after 2 weeks to the
end of the experiment.
Sample processing for light microscopy: The birds were euthanized using
ether inhalation and then the femur and tibia of at least 5 male birds during
each stage of post-hatching development (1 day, 1 week, 2 weeks, 3 weeks, 4
weeks and 6 weeks) were carefully dissected without touching the articular surfaces
with the scalpel and washed thoroughly in Phosphate-buffered Saline (PBS). Specimens
were fixed in either 10% formalin (pH; 7.4) at 4°C for 3 days, then they
were decalcified in 0.33 M Ethylene-diamine-tetra-acetic Acid (EDTA). The whole
femur and tibia as a whole (before formation of the POC) or the growth cartilage
with some adjacent bone (when the POC was found) from the proximal ends of femur
and tibia were dissected, dehydrated in ascending grades of ethanol and finally
embedded in paraffin using standard methods. Paraffin sections (3-5 μm
thickness) were undertaken and stained with hematoxylin and eosin, safranin-O,
Crossmans trichrome and Periodic
Acid-Schiffs reagent (PAS) and
examined with light microscopy.
At day one, the femur and tibia were completely formed of cartilage enclosed
by a tube of periosteal bone collar. The bone collar was enclosed into the epiphysis
dividing articular growth cartilage above the Physeal Growth Cartilage (PGC)
into articular cartilage proper and lateral articular cartilage (Fig.
1a). The periosteal bone collar was separated from the PGC by a mixture
of haematopoietic cells concentrated at the centre of the femur and tibia, penetrated
through the peripherally of the cartilage and run parallel to the periosteal
bone collar (Fig. 1a,b). Zonation of PGC
into resting, proliferative and hypertrophic areas was present but not clear
(Fig. 1c). The chondrocytes of the articular cartilage was
overcrowded and randomly distributed throughout the cartilage extracellular
matrix. Cellular condensation and focal hypertrophy of chondrocytes were seen
in the epiphysis of tibia only but not femur of 1-day- and 1-week-post hatching
quail and was also penetrated with cartilage canals (Fig. 1d).
The SOC was seen for the first time in the epiphysis of the tibia (Fig.
1e). It was noticed that the proliferative zone of the femur and tibia noticeably
longer than of mammals and the proliferative zone of the tibia was much longer
than that of the femur. During this stage of development, large areas of the
PGC were degraded and replaced by bone trabeculae of the POC (Fig.
1e-f) and a bone marrow cavity filled with a mixture of
haematopoietic and fat cells (Fig. 1f). Thus the tibia from
2-week-post-hatching quail had 2 centres of ossification; POC and SOC and the
cartilage is divided into PGC between the POC and the SOC and the articular
cartilage between the articular surface and the SOC (Fig. 1e).
While, the femur at the same stage of development had only POC (Fig.
1f) and the cartilage is still divided into articular cartilage above PGC.
In older birds, the ossification gradually replaced the cartilage (Fig.
The chondrocytes of the PGC were organized into clear three zones, resting,
proliferative and hypertrophic after 2 weeks of hatching (Fig.
2a-b) and the zonal organization of the chondrocytes became
well defined as the development proceeded. The cartilage canals of 1-day-old
quail contained blood capillaries that were surrounded by many undifferentiated
mesenchymal cells (Fig. 2c). The cartilage canals of 2-week
and older post-hatching quail had well defined wall and contained arterioles
surrounded by many mesenchymal cells (Fig. 2d). After four
weeks, blood vessels of many of the cartilage canals diapered and the contents
of the canals were mostly fibrinoid and chondrified (Fig. 2e).
By the 6th week, the cartilage canals were no longer observed within the permanent
articular cartilage (Fig. 2f).
|| Periosteal and endochondral ossification of the femur and
tibia of the post-hatching Japanese quail. Paraffin sections from the femur
(a, b, c, f, g) and tibia (d, e, h) from 1-day (a, b, c), 1-week (d), 2-week
(e, f) and 4-week (g-h) -post-hatching quail stained with H and E (a, b,
c, f, g), safranin-O (d) and PAS (e). Note articular cartilage proper (ac),
lateral articular cartilage (lc), physeal growth cartilage (pgc), haematopoietic
cells (hc), resting (r), proliferative (p), hypertrophic (h) chondrocytes,
primary ossification centre (poc), secondary ossification centre (soc),
physeal growth cartilage (pgc) and bone marrow (bm). Arrows indicate periosteal
bone collar (a, b, f) and cartilage canals (a, c, d, e, g) and arrowheads
indicate haematopoietic cells (a). Bars = 200 μm in a, f, g, h, 80
μm in b, c, d, e and 140 μm in F
The aim of the current study was to illustrate the main histological changes
of the quail long bones during post-hatching development. To achieve this aim,
paraffin sections of the femur and tibia from 1 day to 6 weeks was examined
with light microscopy. The femur and tibia of 1-day-post-hatching quail was
formed of cartilage enclosed by a tube of periosteal bone collar. The bony tube
extended from the metaphysis into the epiphysis dividing the articular cartilage
into inner and outer parts. This picture is similar to the larval stages of
frogs (Mackie et al., 2008).
|| Histology of the growth cartilage of the post-hatching Japanese
quail, Paraffin sections from the 2-week (a, b), 1-day (c), 3-week (d),
4 week (e) and 6-week (f)- post-hatching quail stained with H&E (a,
b, e), PAS (c, d) and Crossmans trichrome (f), Note, resting (r),
proliferative (p), hypertrophic (h) zones and articular cartilage (ac).
Arrows indicate cartilage canals and arrowhead indicates arterioles within
the canals. Bars = 80 μm in a, b and 20 μm in c, d, e, f
The periosteal bone collar was reported to firstly observed around the long
bone at 6-day of the pre-hatching quail and is formed of bone rings surrounding
the central hypertrophic zone of the developing cartilaginous long bone, then
increases in thickness and expands radially toward the epiphysis with development
of the future bone (Pourlis et al., 2006). No
sign of endochondral ossification was seen in the developing femur or tibia
of the quail before the 2nd week of the post-hatching development, when bone
trabeculae was firstly seen forming the POC within the metaphysis of the bone.
Thus the development of the periosteal bone in such way is likely to limit the
diameter expansion of the developing bone especially until the first 2-post-hatching
weeks. Unlike mammals (Rivas and Shapiro, 2002), endochondral
ossification in the femur and tibia of quail may be a delayed process and long
bone growth and development in the quail may depend more on the periosteal ossification
than endochondral ossification at least during the early stages of post-hatching
development. Although no endochondral ossification occurred within the PGC,
the chondrocytes were undergoing proliferation and hypertrophy as mammals (Miralles-Flores
and Delgado-Baeza, 1990; Rivas and Shapiro, 2002).
This supports the idea that chondrocyte proliferation and hypertrophy may not
related to the matrix calcification, however, it is important for radial expansion
associated with bone elongation through periosteal ossification (Felisbino
and Carvalho, 1999). A mixture of haematopoietic cells was observed at the
middle of the femur and tibia, which extended on both sides of the cartilage
under the periosteal bone collar. These cells are likely are important for cartilage
erosion and formation of marrow cavity when the development advanced. A striking
feature of the bone development in quail was the observation of foci of hypertrophic
chondrocytes in the epiphysis of the tibia but not the femur at 1-day and 1-week-post-hatching
and later on the SOC was seen in the tibia but not femur at the 2nd week of
post-hatching development. These hypertrophic chondrocytes acts as a nucleus
for the formation of the SOC and that is similar to what is happening in mammals
(Rivas and Shapiro, 2002). It was noticeable that the
proliferative zone of the femur and tibia was usually long in comparison to
mammals. One of the main histological differences between mammalian and avian
long bone PGC is that the avian cartilage has longer proliferative column and
larger blood vessels that penetrate further into the hypertrophic zone (Leach
and Gay, 1987). Further more, the proliferative zone of the tibia was longer
than that of the femur. Taken this with our result that the tibia is longer
than the femur and the high weight gain of the quail after the second week of
post-hatching development (not published), that may indicate the presence of
the SOC in the tibia during this time.
Although, it is known that the cartilage is avascular tissue, the growth cartilage
was invaded with a vascular cartilage canals. We described the development of
the cartilage canals during embryonic development in another paper (submitted
for publication). In this study we found the cartilage canals were found within
the cartilage starting from one-day-post-hatching and within the SOC. Cartilage
canals provide not only the vasculature and nutrition to the chondrocytes but
also the osteoblast precursors, the undifferentiated mesenchymal cells that
may contribute to formation of the SOC (Burkus et al.,
1993; Rivas and Shapiro, 2002; Blumer
et al., 2004; 2005; 2006).
The cartilage canals were completely disappeared from the cartilage of mature
bird after 6 weeks of post-natal development. The cartilage canals undergo a
normal regression, chondrification (Haines, 1974) and
retention of the cartilage canals may lead to dyschondroplasia in many species
(Ytrehus et al., 2007).
In conclusion, the current study showed that, endochondral ossification is
a late process in the post-hatching quail, however the bone elongation occur
mostly due to the periosteal ossification. The femur, unlike the tibia, has
no SOC and the cartilage canals normally disappeared at maturity.
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