TOPOGRAPmCAL PICTURE OF DIGIT OSSIFICATION IN THE , SHEEP IN THE LATE EMBRYONIC AND EARLY FOETAL PERIOD OF ' DEVELOPMENT

Cerny H.: Topographical Picture of Digit Ossification in the Sheep in the Late Embryonic and Early Foetal Period of DefJelopment. Acta vet. Bmo, 59,1990: 91-99. Using a model of acropodium skeleton of three embryos (CRL 18-35 mm) and 26 foetuses (eRL 41-210 mm) of Merino sheep we studied the development of the skeleton of digits during the late embryoniC and early foetal period of ontogeny using light microscopy. The material was processed by standard methods for the study of histological .. sections. These were stained with hematoxylin-eosin, van Gieson and alcian blue, together with a mixture of Ponceau 2 R acid fuchsine. From the histotopographical point of view we described the structural picture . of the developing acropodium skeleton in relation with the foetal age aild morphological determination of individual·phases of ossification. We confirmed the sequence of ossification of digits III and IV. The distal phalanx is the first to ossify, followed by the proximal and then middle phalanges. Ossification takes place at the age of 47 to 70 days of prenatal development. It begins on the 47th day with desmogenous ossification of the tip of the distal phalanx, progressing proximally and joining the endochondral ossification of the cartilaginous skeleton of the cartilaginous skeleton of the corpus. . The onset of ossification of the cartilaginous skeleton of diaphyses of the proxinial and middle phalanges takes place on axial facets and progresses abaxially .. From the 80th day we can notice marked proliferation of blood vessels to the skeleton of epiphyses and distal capitula of the proximal and middle phalanges. The spreading blood capillaries cause disintegration of cartilage, form numerous canals holding osteogenous mesenchyma besides the vessels. Capitula of both phalanges ossify from the diaphysis. Sheep, prenatal ontogeny, ossification of phalanges The cartilaginous embryonic skeleton, which subsequently ossifies to form a definitive skeleton, comes into being in the transformation of the prechondralinto chondral blastema. The complexity of the process of osteogeny is conditioned by the differentiation and growth of the structure in the course of ontogenetic development. Available literary references show that sheep skeleton was a frequent subject of investigation in the past. A number of authors contributed to general knowledge of the cartilaginous skeleton, its development and growth fJ:om the point of.view of its morphology or function in relation to its prenatal as well as postnatal ontogeny. . Although the development of the sheep foetal skeleton was described by Harris (1937), Benzie (1950), Smith (1956), Lascelles (1958), Rajtova (1972, 1973), more recently by Neiss (1982) and Cerny (1987) with a large number of detailed information,there is still enough room for a detailed description of ossification in individual developmental periods iIJ. view of the step-like character of this process, and histotopographical. identification. ., . We 'therefore decided to use the model of sheep acropodium to study the dynamics of changes in the morphological picture of ossification in the period of early foetal ontogeny. c.

The cartilaginous embryonic skeleton, which subsequently ossifies to form a definitive skeleton, comes into being in the transformation of the prechondralinto chondral blastema.The complexity of the process of osteogeny is conditioned by the differentiation and growth of the structure in the course of ontogenetic development.
Available literary references show that sheep skeleton was a frequent subject of investigation in the past.A number of authors contributed to general knowledge of the cartilaginous skeleton, its development and growth fJ:om the point of.view of its morphology or function in relation to its prenatal as well as postnatal ontogeny.
. Although the development of the sheep foetal skeleton was described by Harris (1937), Benzie (1950), Smith (1956), Lascelles (1958), Rajtova (1972Rajtova ( , 1973)), more recently by Neiss (1982) and Cerny (1987) with a large number of detailed information,there is still enough room for a detailed description of ossification in individual developmental periods iIJ.view of the step-like character of this process, and histotopographical.identification.
., .We 'therefore decided to use the model of sheep acropodium to study the dynamics of changes in the morphological picture of ossification in the period of early foetal ontogeny.c.

Materials and Methods
, A total of three embryos (CRL 18-35 mm) and 26 foetuses (CRL 41-210 mm) of Merino sheep whose age was determined according to Evans and Sack (1973) was used for the study of the development of the acropodium skeleton ,in the sheep.
The material was fixed in 10 % formal and processed in a standard manner for light microscopy.
Frontal, sagittal and horizontal sections made of tissue paraffin or celloidin blocks were stained with hematoxylin-eosin, van Gieson, alcian blue all:d a mixture of Ponceau 2 R and acid fuchsine.

Results
In the embryo of CRL 18 nun, the laminae of the hand and foot are clearly discernible (Fig. 2).Laminae are bowl-shaped, with the palmar and plantar facets slightly concave.The edges of the two laminae feature delicate indentation, shallow nicks between teeth marking the boundary between individual primordia.These are formed by prechondral cellular blastema (Fig. 1), between principal primordia of digit III and IV marked reduction of mesenchyma and formation of blood vessels can be observed (Fig. 1).Qearly discernible at this time are also reduced primordia of digits II and V all Figures are placed at the end of the volume.
In the next period in embryo of CRL 25 nun the change of the prechondral blastema into chondral blastema is taking place in the lamina of the hand with the formation of embryonal cartilage.
Also foot lamina contains digit primordia formed by prechondral blastema with gradual chondrogeny.
Hand and foot laminae in the embryo of CRL 35 nun are characterized by a cut between principal primordia and the lamina as a whole becomes elongated.Development of principal primordia takes place, primordia of digits II and V are disproportionally smaller.,The main part of the digit skeleton (phalanx proximalis and media) is already formed by embryonal cartilage, the corpus of the distal phalanx passing through the stage of differentiation of chondral blastema.Individual phalanx skeletons are separated from each other by narrow gaps containing in their central parts condensed material of the original mesenchymal blastema.
The appearance of joint cavities of digit joints can be observed, which came into being through the reduction of the blastema material.
In the foetus of CRL 60 mm (47 days), hypertrophy of the cartilage of the distal phalanx and desmogenous ossification of the tip have begun (Fig. 3).Phalanx media and proximalis are formed by normal cartilage at this stage.The above description applies to the thoracic as well as the pelvic limbs.
The period of CRL 108 mm (60 days) is characterized by gradual ossification of phalanges beginning with the phalanx distalis, then phalanx proximalis and media.The apex of the distal phalanx is markedly ossified, cortica1lamellae appear, the perichondrium differentiates, the activity of the osteogenous blastema at the , tip increases, and the surrounding cartilage is becoming hypertrophic.
Thin lamellae of the cortical bone appear on the surface of the corpus of phalanx proximalis as a result of the activity of perichondrium, and later periosteum.In the central part of the phalanx, endochondral ossification is taking place, while in the proximal part a continuous cartilage of the skeleton is being formed.In the epiphyseal skeleton, numerous canals occur proximally that contain blood vessels and cellular osteogenous material.
The last ,of the phalanges to ossify is the phalanx media.On the axial area of its corpus we can find the initial stage of the primary ossification centre which is manifested by hypertrophy of chondrocytes and metachromasia of the ground matter of the hypertrophic cartilage.A more detailed investigation shows a gradual invasion of cellular osteogenous elements into the centre.The cells are derivatives of the osteogenous layer of the perichondrium.In view of the fact that at this stage the structure of the ossification centre shows no mineralization, radiographic imaging is not possible.
The onset of the osteogenous phase, however, comes very soon because at the next stage"i.e. 10 days later in the foetus of CRL 150 mm (70 days), the middle part of the phalanx is completely ossified.Documents from this period show that ossification begins axially and progresses in the abaxial direction (Fig. 10).On the axial area, a thin edge of cortical bone due to periosteal ossification appears.Perichondrium in this area differentiates completely into periosteum (Fig. 11).
Light microscopy shows that ossification of the tip of the distal phalanx continues together with the differentiation of the periosteum and the endochondral ossification of its corpus.Further development of endochondral ossification can also be demonstrated in the proximal phalanx.
In foetuses of CRL 210 mm (80 days) the diaphyses of the proximal and middle phalanges are completely ossified, individual beams of the spongious bone form a compact anastomosing system.The medullary cavity is usually only hinted at in the central parts of diaphyses.Because these are monoepiphysea1 bones, the epiphysodiaphyseal, boundary is formed only in proximal parts of of the bone.Distal capitula of both phalanges ossify in later developmental stages from the diaphysis.In this period, however, capitula are completely made up of cartilage.
C'..ontrary to the cylindrical shape of proximal and middle phalanges, the distal phalanx is markedly conical.Its skeleton is formed by cartilage, which arches in a prominent skeleton of the processus extensorius at the dorsal crown-shaped edge.The corpus and apex are formed by beams of a spongy bone, which are mainly formed by periosteal ossification (Figs. 7,8).Differentiation of osteogenous cells and their secretion activity lead to the formation of new bone tissue, attached to the surface of the cortical bone.The cortical bone is interrupted with communication to intertrabecular space.Osteogenous cellular material of the periosteum penetrates to the centre of the skeleton both independently and with blood vessels, where it differentiates into cellular elements of bone marrow.The apex phalangis distalis, just as the distal part of the corpus, originates from the osteogenous mesenchymal blastema.First, the woven bone is formed, which is then restructured into a definitive bony apex.
In epiphyseal cartilages, as well as in distal capitula of the cartilaginous model, we can observe numerous osteogenous canals (Fig. 12) which contain blood vessels and abundant cellular material.In the case of hypophyses, this osteogenous cellular material comes from perichondrium or periosteum, its basic cell is the fibroblast which in subsequent stages of ossification differentiates into a cellular element with a significant osteogenous potential -the osteoblast.
On the other hand, capitula are penetrated by osteogenous cells from the forming primary cavity of the ossifying diaphysis.The cellular material filling central parts of the diaphysis undergoes differentiation into osteogenous cells and cellular elements of the medulla (Figs. 12,13,14).
From the viewpoint of morphology, the existence of blood vessels with pericapillary cells in the proliferative and hypertrophic zone of the ossifying cartilage (Fig. 15) and vessels penetrating from the epiphyseal perichondrium transchondrally to the diaphyseal region can be considered an interesting finding.In this respect, we noted isolated cases of blood vessels penetrating from the proliferative zone of the cartilage as far as the osteoid zone of endochondral ossification.
Morphological picture of the developmental stage studied manifests a pronounced proliferation of blood vessels and cellular elements to cartilages of epiphyses and cartilaginous skeletons of capitula of the proximal and middle phalanges.It was here that we observed penetrating blood capillaries accompanied with pericapillary cells.They were medullar capillaries of the diaphysis which invade the capitulum cartilage and initiate its gradual ossification and the appearance of the subchondral bone.The original skeleton is reduced to articular cartilage.

Discussion
The development of appendicular skeleton!takes place in four stages.In the late embryonal period, a conversion of prechondral mesenchymal skeleton into cartilaginous embryonal skeleton takes place.Osteogeny proper begins with the appearance of primary ossification centres in diaphyses of cartilaginous skeletons.The third stage is characterized by the appearance of secondary ossification centres, ossification of epiphyses and the formation of epiphysodiaphysary lamellae.And finally the fourth stage is connected with the ossification of epiphyseal plate and the appearance of the definitive skeleton.
The first two stages take place in the prenatal period, the third is a transitory stage between the pre-and postnatal development, and the fourth stage is linked with the postnatal period of development.Our observations concern the morphological picture of the first two stages and partially also the third.
Ossification is a complex process of development taking place in phases, connected with tissue mineralization.Gradual mineralization of the basic bone tissue material increases its density and allows its radiographic imaging.An advanced degree of mineralization is an necessary condition for the use of the radiographical method.
In early stages of ossification of the cartilaginous foetal skeleton, important morphological changes of structure take place.In view of insufficient tissue mineralization, radiography cannot be successfully used to study these changes in their full extent.We therefore consider it necessary to employ histological methods to study, the situation in the late embryonal and early foetal" period using tissue section." When we look into the relation between the foetal age and the number 'of ossification; centres of the appendicular skeleton, we will conclude that asi~fica~t development of the skeleton does not take place before the age "of 90-100"aaysl From this point of view, Lascelles (1958) therofore divides the prenatal development in the sheep into two periods: 37-92 days and 92-141 days.
In the first period, structural differentiation mainly predominates, which is clearly discernible histologically.The radiographic method, on the other hand> would not yield unambiguous results, because structure imaging at the beginning of ossification is insufficient.
The other period, from 90 to 100 days of age, is characterized by an absolute increase in the number of ossification centres of the appendicular skeleton, which comes as a result of ossification of epiphyses.This period is further characterized by increased mineralization and general maturation of bone tissue; It is therefore appropriate to use the radiographic method, which not only allows the study of the skeletal development, but also provides the possibility of its further morphometrical and densitometrica1 investigation.
When we compare our results concerning ossification of the acropodium with literary data, we find some differences not only in the determination of the appearance of ossification centres weith regard to the foetal age, but also in the sequence of ossification of individual phalanges.
Harries (1937) reports that all phalanges on the pelvic and thoracic limbs ossify on the 61st day simultaneously, Lascelles (1959) believes that ossification of the 1st, 2nd and 3rd phalanges takes place in that order between the 59th and 71st day, the thoracic limb being 2 days ahead of the pelvic limb.According to Rajtova (1972), the first and second phalanges ossify from two ossification centres, while the third phalanx from only one.Ossification begins on 47th day in phalanx distalis> the corpus of the proximal phalanx ossifies on the 56th day and on the 58th day an ossification centre appears in the cartilaginous skeleton of the middle phalanx corpus.According to this author, the ossification of epiphyses and thus the formation of secondary ossification centres does not take place before the 145th day.Rajtova (1972) reports the same ossification sequence in individual phalanges as Smith (1956), Neiss (1982) and Cerny (1987), while differring with them in time intervals of their ossification.The range reported by Rajtova (1972) is 47th -58th day, while Harris (1937) puts it on the 61st day.Neiss (1982) claims that the first calcium in the distal phalanx is deposited in the foetus of CRL 67.5 mm, and that mineralization can be constantly demonstrated from the CRL of 71 mm.The CRL reported by Evans and Sack (1973) represents the 48th day of foetal age, the CRL of 71 mm the 50th day post conceptionem.Ossification of diaphysis of the proximal phalanx begins at 76 mm, occurs regularly at 94 mm> the middle phalanx ossifies at the lenght of 94 mm.In foetuses of CRL of 110 mm> diaphyses of middle phalanges are ossified regularly.According to our previous findings (Cerny 1987) as well as our recent observations, the onset of ossification of phalanges III and IV is linked with the period of the 47th to 70th day.It is evident that the histological method of the study of tissue sections can be used a detailed documentation of the preossification stage, when ossification from the structural point of view has already begun but the deposition of mineral substances in the basic cartilaginous material is still insuffcient for radiographical purposes.Our findings show that the beginning of the ossification proper, connected with the mineralization of the basic material occurs in fact between the 50th and 62ad day of prenatal development.
In agreement with other authors, we can report that ossification of the diaphysis of the proximal and middle phalanges always begins in their axial facets and progresses gradually in the abaxial direction.From diaphyses of these phalanges> ossification progresses distally, causing ossification of cartilaginous skeletons of capitula.We observed intensive vascular penetration of medullar capillaries from the diaphysis to the capitulum, which not only participates in cartilage resorption but also leads to the appearance of distinct canals in the cartilage which are used by osteogenous cells in their penetration from the primary medullar cavity to the capitulum.
A slightly different type of ossification was observed in the distal phalanx.Its cartilaginous skeleton is not fully replaced with bone tissue.Its apex and distal part of the corpus is formed by desmogenous ossification from osteogenous mesenchymal blastema.The remaining part of the corpus ossifies endochondrally.Ossification progresses proximally, i. e. in the opposite direction than in the case of proximal and middle phalanges.
Our results concerning the time of ossification of phalanges are in closest agreement with the interval proposed by Neiss (1982).As regards their mutual comparison, our results show only insignificant differences with the data pesented by Rajtova (1972).
More substantial are the differences with results of the other authors quoted.These may be caused by breed variability of the material used, or by methods of its processing.Neiss (1982) also endorses an opinion that any differences that may occur in an interpretation of conclusions concerning sheep skeleton ossification in prenatal ontogeny could be attributed to existing breed differences, which the author considers a kind of intraspecies variability.
Plate IV.
Plate v.

Table 1
Are and Number of Embryos and Foetuses Used

Table 2 Topographical
Picture of Ossification Related to the Age of Embryo and Foetus