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Oral Histology Lec.1 Lab.1 Preparation of Histological Specimens

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Oral Histology Lec.1 Lab.1 Preparation of Histological Specimens Oral Histology Lec.1 Lab.1 Dr.Munir Nasr Preparation of histological specimens Histology (compound of the Greek words: histo “tissue”, and logy “science”) is the study of the microscopic anatomy of cells and tissues of plants and animals. It is commonly performed by examining cells and tissues by sectioning and staining, followed by examination under a light or electron microscopes. Histological studies may be conducted via tissue culture, where live cells can be isolated and maintained in a proper environment outside the body for various research projects. The ability to visualize or differentially identify microscopic structures is frequently enhanced through the use of histological stains. The steps of sample preparations: 1. Tissue fixation 2.Tissue processing 3. Tissue cutting or sectioning 4. Tissue staining Tissue fixation Fixation is a complex series of chemical events that differ for the different groups of substance found in tissues. The aim of fixation: 1- To prevent autolysis and bacterial attack. 2- To fix the tissues so they will not change their volume and shape during processing. 3 - To prepare tissue and leave it in a condition which allow clear staining of sections. 1 4 . To leave tissue as close as their living state as possible, and no small molecules should be lost. Fixation is coming by reaction between the fixative and protein which form a gel, so keeping everything as their in vivo relation to each other. Factors affect fixation: -PH. -Temperature. -Penetration of fixative. -Volume of tissue. According to previous factors we can determine the concentration of fixative and fixation time. Types of fixative: Acetic acid, Formaldehyde, Ethanol, Glutaraldehyde, Methanol and Picric acid. Tissue processing the aim of tissue processing is to embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut , and yet soft enough not to damage the knife or tissue. Stages of processing: -1Dehydration. -2Clearing. -3Embedding. Dehydration To remove fixative and water from the tissue and replace them with dehydrating fluid. There are a variety of compounds many of which are alcohols. Several are hydrophilic so attract water from tissue. 2 Types of dehydrating agents: Ethanol, Methanol, Acetone. Clearing Some clearing agents: -Zylene. -Toluene. -Chloroform. -Benzene. - Petrol. Embedding Is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning. Paraffin wax properties: Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. It is about two thirds the density and slightly more elastic than dried protein. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C. The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax: - improve ribboning . - increase hardness. - decrease melting point - improve adhesion between specimen and wax 3 Embedding wax Processing machine 4 Cutting using the microtome A microtome is a mechanical instrument used to cut biological specimens into very thin segments for microscopic examination. Most microtomes use a steel blade and are used to prepare sections of animal or plant tissues for histology. Microtome knives steel knives non-corrosive knives for cryostats disposable blades glass knives diamond knives 5 Staining Hematoxylin and Eosin (H & E) H & E is a charge-based, general purpose stain. Hematoxylin stains acidic molecules shades of blue. Eosin stains basic materials shades of red, pink and orange. H & E stains are universally used for routine histological examination of tissue sections. Nuclei - blue - with some metachromasia Cytoplasm - various shades of pink-identifying different tissue components Staining machine 6 Dentistry Department; 2nd grade Dijlah University College Oral Histology Lec.2 Lab. 2 Dr. Munir Nasr Development and growth of the teeth The development of tooth involves many complex biological processes, including epithelial mesenchymal interaction, morphogenesis and mineralization. In human beings, 20 deciduous and 32 permanent teeth develop from the interaction between the oral epithelium cells and mesenchymal cells Fig (1). The basic developmental process is similar for all teeth but each developing tooth develops as an anatomically distinct unit. Vitamins, minerals and hormones affect tooth development. Vitamin A is important for epithelial growth, vitamin C for connective tissue development and vitamin D is essential for calcification. Tooth formation begins in the 6th week of intrauterine life(IUL) with the formation of a continuous horseshoe-band of thickened epithelium in the location of the upper and lower jaws called the primary epithelial band Fig. (2), at about 7th week the primary epithelial band divides into a lingual process called dental lamina & a buccal process called vestibular lamina Fig. (3) All deciduous teeth arise from dental lamina at the 8th week of fetal life, later the permanent successors arise from its lingual extension which is called successional lamina & permanent molars from its distal extension. Fig.1 Primary epithelial band and the mesenchymal cell. 1 Dentistry Department; 2nd grade Dijlah University College Maxillary primary epithelial band Mandibular primary epithelial band Fig.2 early oral cavity. Dental Lamina The dental lamina begins to function at the 6th week of gestation period, this lamina invaginates as a sheet of epithelial cells to underlying mesenchyme cells around the perimeter of both the maxillary and mandibular jaws. Along the leading edges of the lamina, 20 areas of enlargement which are forming the buds of 20 primary teeth. Which then develop into primary teeth and the leading edge of the lamina continues to develop the 32 permanent tooth buds. 2 Dentistry Department; 2nd grade Dijlah University College Fig.3. Each band of epithelium will give rise to 2 sub divisions: Dental lamina and vestibular lamina The initiation of the permanent 1st molar occurs at the 4th month of intrauterine life, the 2nd molar one year after birth and the 3rd molar five years after birth. Successional lamina is responsible for the development of permanent incisors, canine and premolars. The successional lamina is active from the 5th month in utero (permanent central incisor) to the 10th month of age (second premolar). Fate of dental lamina The total activity of dental lamina extends over a period of at least 5 years. As the teeth continue to develop, they lose their connection with dental lamina. They later break by mesenchymal invasion of the jaw. Remnants of dental lamina persist as epithelial islands within jaw and gingiva. In the incisors area the dental lamina is first to appear and disappear after initiation of tooth development, and in the third molar area it is the last to appear and disappear. Vestibular lamina Facial (labial &buccal) to dental lamina another thick band of epithelium develops in the maxillary and mandibular dental arches. It is called the vestibular lamina or the lip furrow band. It later hollows out and forms the oral vestibule between the alveolar portions of the jaws and the lips and cheeks. The oral 3 Dentistry Department; 2nd grade Dijlah University College epithelium in the lower jaw forms an epithelial invagination separating the tongue from the developing alveolar process termed the linguoalveolar sulcus. Stages of tooth development Tooth formation is a continuous process by a series of stages. Each tooth develops through successive bud, cap and bell stages. During these stages, the tooth germs grow and develop into specialized cells which form the enamel, dentin and the cementum. Dental lamina plays an important role in the development of teeth. At certain points along the dental lamina each representing the location of one of the 10 mandibular & 10 maxillary teeth, ectodermal cells multiply rapidly & little knobs that grow into the underlying mesenchyme. Each of this little down growths from the dental lamina represents the beginning of the enamel organ of the tooth bud of a deciduous tooth. First to appear are those of anterior mandibular region. As the cell proliferation occurs each enamel organ takes a shape that resembles a cap. 4 Dentistry Department; 2nd grade Dijlah University College On the inside of the cap, the ectomesenchymal cells increase in number. The tissue appears denser than the surrounding mesenchyme and represents the beginning of the dental papilla. Surrounding the combined enamel organ or dental papilla, the third part of the tooth bud forms. It is known as dental sac/follicle and it consists of ectomesenchymal cells and fibers that surround the dental papilla and the enamel organ. Thus the tooth germ consists of ectodermal component- the enamel organ, the ectomesenchymal components- the dental papilla & the dental follicle. The enamel is formed from the enamel organ, the dentin and the pulp from the dental papilla and the supporting tissues namely the cementum, periodontal ligament & the alveolar bone from the dental follicle. During & after these developments the shape of the enamel organ continues to change. The depression occupied by the dental papilla deepens until the enamel organ assumes a shape resembling a bell. The dental lamina becomes longer, thinner & finally loses its connection with the epithelium of the primitive oral cavity. Tooth development is a continuous process, however can be divided into 3 stages: 1.Bud Stage: Initiation 2.Cap Stage: Proliferation 3. Bell Stage: (a) Early- Histodifferentiatin (b) Advanced- Morfodifferentiation The name of the stages is based on the shape of epithelial part of the tooth germ which is the enamel organ. Bud stage (1st stage): This is the initial stage of tooth formation where enamel organ resembles a small bud. It is rounded localized growth of the epithelial cells; it consists of peripherally located columnar cells and centrally located polygonal cells. Many cells of the tooth and surrounding mesenchyme undergo mitosis.
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