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Cell and molecular biology with genetics by almuzian ok ok


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  • 1. Cell and Molecular Biology with Genetics Structures within the Periodontium The periodontium is the connective tissue connecting the teeth to the jaws. It is composed of the two fibrous tissues the periodontal ligament and the lamina propria of the gingiva, as well as the mineralised tissues of the cementum and the alveolar bone. Cementum  Mineralised tissue covering the roots of the teeth.  The cementum provides an attachment surface for the collagen fibres that bind the tooth to surrounding structures. Composition of the cementum  It is specialised CT with some similar characteristics with compact bone.  It consists of 45-50% inorganic substances (calcium and phosphate as hydroxyapatite), 50-55% organic material (collagen type I and proteoglycans) and water.  The cementum has two forms, cellular and acellular. The acellular cementum can cover the root dentine from the cementoenamel junction to the apex, but is often missing on the apical third of the root; this is why the apical third resorbed the most by orthodontic forces. Composition of PDL • Collage fibres which are either gingival gp from tooth to gum or dentoalveolar gp from cementum to lamina dura. PDL is composed of 80% type I collagen and 20% type II collagen • Between these fiber bundle there is blood vessel and lymphatic v
  • 2. • Extracellular matrix • Cells as below. Cells of PDL 1. Chondrocyte originate from mesenchymal tissues differentiate to produce minerlaized bone matrix 2. PDL fibroblast originate from mesenchymal tissues differentiate to produce PDL 3. Pulpal fibroblast originate from mesenchymal tissues differentiate to produce pulp tissues and odontoblast 4. Gingival fibroblast originate from mesenchymal tissues differentiate to produce gingival CT 5. Cementoblast originate from mesenchymal tissues differentiate to produce cementum 6. Odontoblast originate from mesenchymal tissues differentiate to produce dentine 7. Osteoblast originate from mesenchymal tissues differentiate to produce minerlaized bone matrix 8. Osteoclast originate from heamopetic tissues differentiate to produce bone resorption 9. Cementoclast originate from heamopetic tissues differentiate to produce root resorption
  • 3. Alveolar bone • Most bones have a basic architecture composed of outer cortical bone and inner trabecular or cancellous bone. • Cortical bone, which is 80% calcified, forms a rigid outer shell that resists deformation • The trabecular bone, only 20% calcified, provides its strength through a complex system of internal struts, which follow the principal stress trajectories for these sites. Spaces between the trabecular bone are filled with bone marrow (haematopoietic cells). Bone Composition 1. Inorganic mineral like CA, Ph and carbonate that form hydroxyapatite, 2/3 of weight. 2. Organic part involving type I collagen (1/3 by weight) 3. Non-collagenous component consists of glycoproteins 4. Cells distributed between them. Bone Cells Osteoprogenitor cells, or pre-osteoblasts • They are bone stem cells derived from mesenchymal cells that eventually differentiate into mature osteoblasts then steoblasts remain in the mineralised osteoid and become osteocyte. Osteoblasts The main functions of the osteoblasts is 1. Respond to this stain Hematoxylin and eosin, or H&E, staining, 2. The production of type I collagen 3. Mature osteoblast will turn to osteocyte
  • 4. 4. The control of osteoclast function by the production of a soluble factor that acts directly on the osteoclasts (OPG-RANKL-RANK) 5. In addition the osteoblasts also control resorption by forming a physical barrier to the bone surface against the osteoclasts, and when it stimulated by PTH it changes its shape to become more round and allow osteoclast to be in a direct contact with bone and to start resorption. Sandy, 1992. 6. Production of MMPs which are proteolytic enzymes which degrade the organic matrix of the bone (because the non-organic component removed by osteoclast while the organic part removed by the MMPs that produced by osteoblast. Osteoblast starts secretion of protease enzyme MMPs after interaction with osteoclast). 7. Part of osteoblast-osteocyte complex that maintain the integrity of the bone matrix and Prevent its hyper mineralisation, 8. Connect to osteocyte to maintain bone integrity. Osteoclast 1. In bone, osteoclasts are found in pits in the bone surface which are called resorption bays, or Howship's Lacunae. Osteoclasts are characterized by a cytoplasm with a homogeneous, "foamy" appearance. This appearance is due to a high concentration of vesicles and vacuoles. These vacuoles include lysosomes filled with acid phophatase. 2. The osteoclast is a large multinucleate cell that is derived from blood monocytes. 3. The principle function of osteoclasts is to mobilise mineralised bone through a combination of enzyme hydrolyses and acid hydrolases such as
  • 5. acid phosphatase. This is why serum acid phosphatase is an important marker of bone disease. 4. Indeed, osteoclast has no surface receptor for hormone like parathyroid hormone while osteoblast has. So osteoblast interact with hormone and send signal to osteoclast to activate bone resorption (OPG-RANKL- RANK). Osteocytes 1. Osteocytes are osteoblasts that have become surrounded by the forming bone. 2. They become smaller in size and lose their ability to form bone. 3. They communicate with one another and the surface osteoblasts via long radiating processes joined at gap junctions. 4. The function of the osteoblast-osteocyte complex is to:  Osteocytes act as mechanoreceptors identifying the loads placed on the individual bones (Mullender and Huiskes 1997).  Maintain the integrity of the bone matrix  Prevent its hyper mineralisation, NB: Other cells that are found within bone include: periosteal fibroblasts, chondrocytes, chondroblasts, epithelial cells, macrophages/monocytes, erythrocytes, leukocytes, and platelets.
  • 6. Bone Types 1. Collagen formed by osteoblasts is deposited in parallel or concentric layers to produce mature, lamellar bone 2. Collagen not deposited in a parallel array but in a basket-like weave and is called immature, primitive or woven bone Another classification 1. Cortical bone 2. Spongy bone Bone Functions • Mechanical support for the muscle • Site of muscle attachment for locomotion. • Protection for vital organs. • The marrow is a source of manufacture for blood cells. • A metabolic reservoir for ions especially calcium and phosphate. Bone Development • Skeletal formation begins when mesenchymal cells migrate to the site of skeletogenesis. The cells interact with epithelial cells, which triggers the mesenchymal cells to undergo condensation. Condensed cells undergo differentiation into chondrocytes, osteoblasts, adipocyte or myofibroblast. • Core binding factor-1 (CBFA-1) is an important bone specific gene, which is vital for mesenchymal differentiation into osteoblasts. • Bone morphogenetic proteins (BMP's) are important in skeletal patterning and skeletal cell differentiation.
  • 7. Process of bone resorbs • 1. hormone like PGE2 produced and bind to osteoblast • osteoblast activate osteoclast by OPG-RANKL-RANK • osteoblast secret MMP that remove organic part of the bone • Osteoclast remove mineral part of bone Ossification Intramembranous ossification is seen during embryonic development by direct transformation of mesenchymal cells into osteoblasts. Intramembranous ossification is seen in the cranial vault, some facial bones, parts of the mandible and the clavicle. Endochondral - Long bones develop by ossification of a cartilaginous precursor. Chondrocytes differentiate from the mesenchyme in response to genes and growth factors. These chondrocytes deposit matrix proteins at the middle of limb bud in the region known as the primary ossification centre The mesenchymal cells differentiate into osteoblasts, as previously described, and begin to secrete matrix proteins producing a primary bone collar around the circumference of the bone. The cartilaginous core within the bone collar is ossified to form a trabecular network of bone. During ossification of the bone, blood vessels develop from the limb vasculature and one of the vessels develops into the nutrient artery, which provides nourishment for the developing bone. A hyaline cartilaginous known as the perichondrium formed at the end of limb bud WHICH represent future joints.
  • 8. The bone collar and trabecular core make up the shaft of the limb known as the diaphysis. At birth, the diaphysis is completely ossified whereas the epiphyses (ends of the bone) are still cartilaginous. Postnatally, secondary ossification centres form within the epiphysis that gradually ossify the cartilage. A layer of cartilage known as the epiphyseal growth plate persists between the epiphysis and the metaphysis (the growth end of the diaphysis) until growth has finished (~20years). The growth plate allows the limb bones to grow longitudinally by a mechanism of continued proliferation of chondrocytes. Sutural Bone Growth  Sutural bone growth is found exclusively in the skull. It is a method of bone growth rather than development.
  • 9.  Associated disorder: Craniosynostosis is the umbrella term used to describe a collection of disorders that are caused by a premature fusion of the cranial sutures. Some of the most common craniosynostosis syndromes include Crouzon and Apert. Mutations in the gene coding for fibroblast growth factor receptor (FGFR) 2 have been shown to be responsible for these syndromes. Factors affecting bone turn over • Local Factors like 1. Prostaglandins - potent mediators of bone resorption. They can be found in sites of inflammation,. 2. Cytokines - soluble mediators released from cells, which modulate activity of the same cells, or other cells. 3. Interleukin-1 (IL-1) -potent stimulator of bone resorption, acting both directly and by increasing prostaglandin synthesis. IL-1 is also an inhibitor of bone formation. 4. Tumour necrosis factor (alpha, beta) (TNF) - stimulates bone resorption and inhibits bone collagen and non-collagenous protein synthesis. 5. Growth Factors 6. Transforming growth factor B (TGFB) 7. Fibroblast growth factor (FGF) 8. Bone morphogenic proteins (BMP'S) • Systemic Factors 1. Parathyroid hormone - released from the parathyroid gland in response to low serum calcium, phosphate or vitamin D3.
  • 10. 2. Calcitonin - released from thyroid C-cells in response to high serum calcium. Acts as an antagonist to the effects of PTH and inhibits bone resorption by osteoclasts. 3. Thyroid hormones 4. Insulin 5. 6. Growth hormone, important regulator of calcium absorption from the intestine and renal tubules. Recruitment. 7. Glucocorticoids 8. Sex steroids • Testosterone-High levels increase maturation of cells: decrease bone growth. • Oestrogens- Elevated levels of oestrogen increase maturation of cells and decrease bone resorption by osteoclasts. Bone disorders of relevance to an orthodontist 1. Congenital and hereditary disorders 2. Bone infections 3. Metabolic bone diseases 4. Fractures 5. Other non-neoplastic disorders of bone 6. Bone tumours
  • 11. Examples of Metabolic bone diseases • Excess vitamin D3 leads to increased absorption of calcium from the gut, hypercalcemia, increased bone production and tissue mineralisation and nephrotoxicity. • Osteomalacia and Vit D dependent rickets, is a disorder involving softening and weakening of the bones. The condition is most common in children. Cause: The condition is caused primarily by a lack of vitamin D and/or calcium and phosphate. Treatment: Vitamin D deficiency is treated with supplementation. • Excessive secretions of PTH lead to primary hyperparathyroidism. This relatively common condition is associated with elevated serum and urinary calcium. • Chronically elevated levels of growth hormone, frequently caused by an adenoma within the anterior pituitary, lead to acromegaly. This disease is associated with continued growth of the bones of the jaw, hands, and feet. • A decline in sex steroids, as found in post-menopausal females, is thought to be one of the major factors responsible for osteoporosis. Osteoporosis is associated with either a loss of bone or a decrease in bone formation. Example of bone infections • Paget's disease (Osteitis deformans), characterised by excessive bone turnover in which bones become enlarged and deformed. Complications include fractures, neoplasia, nerve compression and high output cardiac failure. • Cause: Paget's disease may be caused by a slow virus infection, present for many years before symptoms appear. There is also a hereditary factor since the disease may appear in more than one family member.
  • 12. • Treatment: Treatment surgery may include; medication that inhibits abnormal bone resorption such as bisphosphonates, Frequency: ~5 in 100 people over 50 years of age in the UK Congenital and Hereditary Disorders • Cleidocranial dysplasia (CCD) Phenotype: CCD is characterised by skeletal anomalies such as opened fontanels, late closure of cranial sutures with Wormian bones (isolated bones within the suture), rudimentary clavicles, and short stature. Dental features: The maturation of the primary dentition is normal, but permanent teeth are delayed from 1 to 4 years. Most patients have supernumerary permanent teeth. Inheritance: Autosomal dominant disorder • Craniosynostosis A term used to describe a collection of approximately 70 congenital disorders that are caused by a premature fusion of the cranial sutures. Some of the most prevalent craniosynostosis include Aperts , Crouzon , and Pfeiffer . Most of these syndromes are due to gain of function mutations in the fibroblast growth factor receptor 2 (FGFR2) Inheritance: autosomal dominant trait. Population frequency: ~0.4/1000 • Aperts Phenotype: Aperts syndrome is characterised by craniosynostosis of several sutures, symmetric syndactyly of the hands and feet
  • 13. dental features: a retrusive middle third of the face, and a V-shaped maxillary dental arch with crowded teeth. Cleft palate (25%) and mental handicap (mild in 31%, severe in 7%) are also associated • Crouzon Phenotype: Crouzon syndrome is characterised by craniosynostosis, bulging eyeballs, shallow orbits, and maxillary hypoplasia. They do not have syndactyly or cleft palate • Achondroplasia Phenotype: the commonest form of skeletal dysplasia, leading to a mean final height of 132 cm for males and 123 cm for females. Inheritance: Autosomal dominant trait Mutation: Due to point mutations within fibroblast growth factor receptor (FGFR) 3 genes. Birth rate: Frequency 1 in 15000-77000 live births in different survey • Osteopetrosis Phenotype: Osteopetroses are rare disorders caused by a marked decrease in bone resorption characterised by a lack of osteoclast function. There is
  • 14. excessive bone formation but the bones have an increase in density and are mechanically weak and prone to fracture. Dental complications may include: delayed eruption of teeth, missing and/or smaller teeth. There may also be evidence of enamel hypermineralisation and hypoplasia. The jaws are composed of dense bone and the mandible is more frequently affected than the maxilla Inheritance: Autosomal recessive. • Osteogenesis imperfecta (OI) Phenotype: Type I: Childhood type. This is the most common form of OI and is associated with brittle bones composed of immature woven bone. The sclerae are thin and may appear blue due to the visible pigmented choroids. Other abnormalities may include: conductive deafness due to otosclerosis, discoloured teeth due to dentinogenesis imperfecta (DI), hypermobility with lax ligaments, Life span is normal and the condition is often mild in severity. Type II: Congenital/Perinatal type, lethal with multiple fractures at birth. Newborns have soft calvarial bones, distinctive triangular face, bluish sclerae, beaked nose, narrow thorax and short and deformed limbs Type III: Infants born with fractures. Dental related abnormalities may include reduced craniofacial size measurements and a posteriorly inclined maxilla.
  • 15. Type IV: Similar to type I OI except that the sclerea are white, stature is slightly shorter and DI is common. Birth rate: Combined birth frequency of 1 in 20,000 • Vitamin D Resistant Rickets (familial hypophosphataemic rickets) Phenotype: Growth retardation and childhood rickets, reduced serum phosphate. Treatable with large doses of vitamin D (or its active metabolite calcitriol) and oral phosphate. Mutation: Mutations to the vitamin D receptor (VDR) Birth Rate: 1 in 20000
  • 16. Early Tooth Development and Basic Histology Prenatal development of the dentition • Teeth form on the frontonasal process and on the paired maxillary and mandibular processes of the first pharyngeal arch. • The basic histology of tooth development suggests that this process derives from two principle cell types, 1. the oral epithelium (gives rise to ameloblasts and the enamel of the tooth crown) and 2. the underlying neural crest-derived ectomesenchyme of the first branchial arch (contributes to the formation of the dental papilla and follicle and therefore, to the odontoblasts, dentine matrix, pulp tissue, cementum and periodontal ligament of the fully formed tooth). Embryological primary tooth formation In the human embryo, development of the deciduous dentition begins at around 6 weeks with the formation of a continuous horseshoe-shaped band of thickened epithelium around the lateral margins of the primitive oral cavity. The free margin of this band gives rise to two processes, which invaginate into the underlying mesenchyme: 1. The outer process or vestibular lamina is initially continuous, but soon breaks down to form a vestibule that demarcates the cheeks and lips from the tooth-bearing regions. 2. The inner process or dental lamina gives rise to the enamel organs of the future developing teeth which is called later tooth bud.
  • 17. Stages 1. The dental papilla is formed by localized condensation of neural crest- derived ectomesenchymal cells around the dental lamina. Then the dental papilla extend around the enamel organ to form the dental follicle or tooth bud. Together, these tissues constitute the tooth germ and will give rise to all structures that make up the mature tooth. This is called bud stage. 2. At the cap stage, the tooth bud folds to demarcate the early morphology of the crown, which is modified by further folding at the bell stage. 3. During the bell stage, the innermost layer of cells within the epithelial component of the tooth organ, the inner enamel epithelium, induce adjacent cells of the dental papilla to differentiate into odontoblasts, responsible for the formation and mineralization of dentine. Dentine formation is preceded by the formation of predentine. The first layer of predentine acts as a signal to the overlying inner enamel epithelial cells to differentiate into ameloblasts and begin secreting the enamel matrix. At the margins of the enamel organ, cells of the inner enamel epithelium are confluent with the outer enamel epithelial cells at the cervical loop. Growth of these cells in an apical direction forms a skirt-like sheet called Hertwig’s epithelial root sheath, which maps out the future root morphology of the developing tooth and induces the further differentiation of root odontoblasts. Degeneration of this root sheath leads to exposure of the cells of the dental follicle to the newly formed root dentine and differentiation into cementoblasts, which begin to deposit cementum onto the root surface. Surrounding the enamel organ, the cells of the dental follicle produce the alveolar bone and collagen fibres of the periodontium.
  • 18. Formation of permanent teeth • Successional teeth have deciduous predecessors and consist of the incisors, canines and premolars. Successional teeth form as a result of localized proliferation within the dental lamina associated with each deciduous tooth germ . • Accessional teeth have no deciduous predecessors and consist of the three permanent molars. In contrast, accessional teeth form as a result of backward extension of the dental lamina into the posterior region of the jaws. Tissue and Molecular Interactions The genes that participate in different stages of tooth formation: Pax; Msx; Barx , Shh; FGF and BMPs signalling protein Tissue Recombination Experiments (important Mohammed) A series of highly informative recombination experiments carried out by Andrew Lumsden at Guys Dental Hospital in the 1980's demonstrated for the first time that not only does cranial neural crest participate in mammalian odontogenesis, it only expresses its odontogenic potential when combined with oral epithelium. • By recombining early first arch (oral) epithelium with cranial neural crest cells, trunk neural crest cells or non-neural crest-derived limb mesenchyme and then allowing these explants to continue their development in vivo, he demonstrated that tooth development only occurred when the oral epithelium was combined with cranial or trunk neural crest.
  • 19. When limb epithelium was combined with mandibular arch ectomesenchyme, no teeth formed. • Recombination experiments have also been performed to suggest the dominance of ectomesenchyme in the specification of tooth shape, once tooth development has been initiated. After the bud stage, the recombination of molar epithelium with incisor dental papilla results in the formation of an incisiform tooth. Similarly, at the same stage the recombination of incisor epithelium with molar dental papilla produces a molariform tooth. Therefore, after the tooth has reached the bud stage, the ectomesenchyme of the dental papilla is responsible for dictating what type of tooth will develop. Conclusion: Taken together, all these early recombination experiments have indicated that during: • the initial stages of odontogenesis, the inductive capacity for tooth development resides within the oral epithelium which under the influences of specific signalling molecules (such as Bmp-4, Fgf-8 and Shh). • later stages (from the bud stage of development) the underlying ectomesenchyme retains the capacity to dictate shape. How does the Genetic Control of Tooth Patterning Come About? 1. Either by clone theory as mentioned in the above including • the initial stages of odontogenesis, the inductive capacity for tooth development resides within the oral epithelium.
  • 20. • later stages (from the bud stage of development) the underlying ectomesenchyme retains the capacity to dictate shape.. 2. the regional field theory, is that the shape of the tooth is determined from the moment its development has been initiated Abnormalities of tooth structure Enamel defects Localized factors 1. Infection 2. Trauma Systemic factors 1. Endocrine disorders 2. Infections 3. Drugs 4. Nutritional deficiency 5. Haematological disorders 6. Neonatal illness 7. Postnatal illness 8. Fluoride ingestion Dentine defects
  • 21. Localized factors 1. Infection 2. Trauma Systemic factors 1. Rickets 2. Ehlers-Danlos syndrome 3. Hypophosphatasia 4. Nutritional deficiency 5. Drugs (Tetracycline) Amelogenesis imperfecta Amelogenesis imperfecta (AI) is a collective term for a group of inherited conditions characterized primarily by abnormal enamel formation in either dentition AI can be inherited as an autosomal dominant, autosomal recessive or sex- linked trait It has a prevalence that can range from 1 : 1,000 to 1 : 14,000, depending upon the population. The predominant enamel phenotype is either: • Hypoplastic • Hypomineralized (either hypomature or hypocalcified, or a combination of the two).
  • 22. Dentine defects can be classified as: • Dentine dysplasia; • Dentinogenesis imperfecta. Dentinogenesis imperfecta (DGI) represents the most common group of inherited dentine disorders and there are three essential subgroups: 1. Type I is associated with osteogenesis imperfecta with type I collagen abnormaility. The deciduous and permanent teeth are affected by discolouration, attrition and pulp canal obliteration. 2. Type II is seen in around 1 : 7,000 Caucasians causing translucent, amber and bluish grey discolouration, enamel chipping and marked attrition. The crowns are bulbous and the pulp canals also become obliterated. 3. Type III affects certain subpopulations of people, including Native American Indians and European Caucasians. Both dentitions can be affected by so-called ‘shell teeth’, which lose enamel and have poorly mineralized dentine, leading to multiple pulp exposures. Orthodontic management of AI and DGI • Children affected by AI or DGI will require long-term multidisciplinary dental management.
  • 23. • early loss of enamel leading to dentine exposure and subsequent sensitivity, which in turn can result in poor oral hygiene and a significant caries risk. • there is known association between AI and the presence of anterior open bite. • When considering orthodontic treatment for the more severe cases: 1. Removable appliances should be used where possible; 2. Care needs to be taken if direct bonding is undertaken because bracket failure or removal can lead to enamel fracture; 3. Orthodontic bands can be used where possible; and 4. Oral hygiene and diet control must be carefully monitored during treatment. Questions and answers From your knowledge of the functions of osteoblasts and osteoclasts why do osteoblasts have prominent endoplasmic reticulum (ER) whereas osteoclasts have few ER Osteoblasts have prominent endoplasmic reticulum (ER) because their role is to actively produce large amounts of protein, particularly type I collagen and other bone matrix proteins. In contrast, osteoclasts do not require active ER as they
  • 24. produce little protein. The main function of the osteoclast is to resorb bone by acid and enzymic hydrolysis. Which structures in the mature tooth are formed from the enamel organ, dental papilla and dental follicle? The enamel organ is derived from epithelium and forms the ameloblasts, which produce the enamel of the tooth crown. The dental papilla and dental follicle are derived from neural crest cells and form the remainder of the tooth. The papilla forms the odontoblasts, which produce dentine and the pulp. The follicle produces the periodontium, consisting of the odontoblasts that form root dentine, cementoblasts that produce cementum and cells that produce the periodontal ligament. Give an outline of the early mechanisms involved during hard tissue formation in the developing tooth. Hard tissue formation in the tooth relies upon inductive interactions between the internal enamel epithelium and adjacent ectomesenchymal cells of the dental papilla. Morphological changes in the cells of the internal enamel epithelium precede cues from these cells to the adjacent dental papilla cells to differentiate into odontoblasts. Odontoblasts begin secreting predentine, which itself seves as a cue for the internal enamel epithelium to differentiate into ameloblasts. Odontoblasts and ameloblasts migrate away from each other and produce the enamel and dentine of the tooth crown.
  • 25. Why are ectomesenchymal cells so important during early tooth development? Ectomesenchymal cells are essentially a specialised form of embryonic connective tissue. They are derived from the neural crest, which are really a form of embryonic stem cells. They can therefore be induced to differentiate into a variety of cell types. In the case of the developing tooth, this includes odontoblasts, pulp cells and cementoblasts. In other words, the connective tissue components of the tooth