الفهرس 
CONTENTS
General Features of Bony TissueOnly 14 pages are availabe for public view
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Abstract
This study was carried out on 154 fertilized chicken eggs(each egg weight45-50 g)of Gallus GallusDomestics. They were obtained from the Research Poultry Farm, Faculty of Agriculture, Assiut University.he eggs were randomly divided into two groups incubated at 37 ◦C in egg incubator (Brand: WQ-88 egg incubator) and 65% humidity turned three times daily.The first group(control group) consists of 77 fertilized eggs injected by saline solution into air space at 6th day of incubation.The second group (nicotine-treated group)consists of 77 fertilized eggs injected by a single dose of100 µl containing 2.5% of nicotine solution into the air space. Samples:The whole chick embryos from the control and treated groups were removed on day 8, 12, and 16th day of incubation and day of hatching then dissected to take the femur.Tissue preparation:Samples from the femur of control and nicotine -treated groups are prepared for light microscopic examination. Two different fixatives were used: 1- Neutral buffer formalin. pH 7.2 2- Bouin’s solution. Fixed tissue samples where dehydrated in ascending grades of alcohol, cleared in methyl benzoate and embedded in paraffin wax, then 5 µm paraffin sections were cut and immersed in xylene and rehydrated in a descending series of ethanol.Sections are examined by light microscope after they are stained by: • Hematoxylin and eosin (HE).•Crossman’s trichrome.• Mallory triple trichrome stain.•Periodic acid-Schiff reaction.•Combined Alcian blue (PH 2.5) –PAS stains.•Alizarin red Alcian blue.•Acridine orange Preparation of samples for SEM and TEM: Samples of chick embryos femur are fixed in a mixture of paraformaldehyde and glutaraldehyde and stained by osmic acid and embedded in resin. Semithin and ultrathin sections are cut and stained for examination and photography.Morphometrical studies:Morphometric studies and statistical analysis have been carried out including the followings: 1)Area of chondrocyte. Matrix area.3)Cortical bone thickness. 4) Single bone trabeculae.5)Area of trabecular bone fusion.6)Area percent of cortical bone Results of examination:
Macroscopic observation:Nicotine increasesthe mortality rate and causes embryos malformations; it has teratogenic effects in chick embryos. Also,it decreases thebody weight of embryos.Using alizarin red - Alcian blue,the stained area of ossification and deposition of bone matrix appear red, while that still occupied by cartilage tissue demonstrates blue color. In the diaphysis, shaft of long bone in control group, the area of bone formation is larger and containing homogeneous deposited red bone tissue, while in nicotine–treated group the area of ossification is small and contains non-homogeneously deposited red bone matrix.
Microscopic observation:At day 8 of incubation, the femur in chick embryos of control and nicotine-treated groupsis represented by cartilage template formed of hyaline cartilage containing small chondrocyte; hypertrophic chondrocytesare observed in the area at the mid-diaphysis.At mid-diaphysis, from the periosteum osteogenic cells; mesenchymal cells differentiated to osteoblast, synthesized a bone collar on the external surface of the cartilaginous hypertrophic tissue. Also, vascular invasion began to penetrate the bony collar.At day12 and 16 day of incubation and hatch day,cartilage canals are observed in epiphysis of control and treated groups, but they are few in treated group when compared with that in control group The process of endochondral ossification and bone formation expands gradually toward the day of hatching in both groups. The vascular invasions begin by formation periosteal bud formed of endothelial cells and osteoblasts. They penetrate the periosteal collar in mid-diaphysis and form irregular blood spaces of primitive marrow cavity of primary ossification center.At the day 12 of incubation, the features of endochondral ossification are like that occur at day 8 of embryogenesis, but there are increase in the area and thickness of bone matrix deposition by bone forming cells, osteoblasts, and formation of primitive marrow cavity of primary center of ossification at mid-diaphysis. Different types of cells and multinucleated chondroclasts are observed within the medullary primitive marrow cavity.
In general, the embryos at different stages of development show cortical bone that was thicker in control group. There are few osteoblasts cover the surface of the cortical bone trabeculae in treated group when compared with control group.Multinucleated chondroclasts are in close contact with the remnant of cartilage matrix, are found deeply engaged in resorption of the thin transverse partition of lacunae.The chondroclasts are located away from the cartilage tissue and don’t appear in direct contact or engaged in the process of cartilage resorptionin treated group when compared with control group. Examination of endochondral ossification at day 12, 16 of incubation and hatch day, revealsmorphologically differences in the amount of bone development, formation of trabecular bone, delay in the process of bone formation and process of replacement of cartilage template by bony tissue, as well as decrease in the activity of bone forming cellsbetween control and nicotine-treated groups. The cortical network appears thick in control group in comparison with the cortical trabecular network in samples of nicotine-treated embryos. By TEM at the day of hatching, in samples of control group osteoblastsare cuboidal in shape and have well developed rough ER filling most of cell cytoplasm.On the other hand, the fine structure of many osteoblasts in nicotine-treated group show characters of inactive secretory cells. They have few cisternae of rough endoplasmic reticulum, cytoplasmic vacuoles, and the volume of cytoplasm is greatly reduced. Their nuclei occupy most of the cellular cytoplasm.Osteocyte insamples of control group, exhibits ultra-structurally large rod -shaped nucleus located in the center of the elongated oval cell body. Small amount of cytoplasm surrounds the nucleus and contains very few cell organelles. While in nicotine-treated group, the fine structure of many osteocyte displays characters of the cell when undergo to cell death. By SEM the homogeneous electron density of smooth surface in the bony trabeculae reflects regular deposition of bone matrix in control group while this character is absent in nicotine- treated group, the surface of bone trabeculae appears irregular and rough that reflects an irregular deposition of bone matrix.The light microscopyat day of hatching reveals that epiphyseal growth plate demonstrates distinct zones in both groups; zone of epiphyseal cartilage, zone of proliferation, zone of hypertrophy and maturation, and zone of calcification and ossification.In samples from nicotine -treated group, the epiphyseal growth plate demonstrates within the zone of proliferation abnormal spaces or lacerations of irregular shape; elongatedor semi-circular in shape.By application of acridine - orange staining on paraffin sections of control and nicotine-treated group, large numbers of apoptotic chondrocytesin the proliferative and hypertrophic zones in samples of nicotine -treated group are differentiated. They emit yellowish or reddish color particularly around the cartilage canals.While in samples of control group the chondrocytes are viable cells and emit the green color. By TEM the apoptotic chondrocytes show vacuolated cytoplasm; vacuoles of variable sizes formed of thin threads of cytoplasm. The vacuolated or fragmented cytoplasm leaves in some cells condensed small C-shaped or oval nucleus embedded in small area of cytoplasm. paraffin sections of samples from control group and nicotine treated group at the day of hatching demonstrates variable affinities for alkaline phosphatasein the different components of endochondral ossification.Alkaline phosphatase expression in osteoblast, osteoclast, bone matrix and cartilage canals in samples of control groups is stronger than that in nicotine treated group. By morphometric analyses, area of chondrocyte was significantly decreased at day of hatch. The decrease in thickness became highly significant at day of hatching compared with that in control group, that reflects the delay effects of nicotine administration on bone formation.At day of hatching, the mean of the area percent that occupied by the cortical trabecular bone tissue in mid-diaphysis of femur in chick embryo, is decreased in nicotine treated group to reach about 42% while the percent is about 58% in control group of the total calculated area. Therefore, a large area in mid-diaphysis is occupied by marrow in-between the bone trabeculae in samples of nicotine treated group.