Cell and Molecular Biology with Genetics

Structures within the PeriodontiumThe 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

• Extracellular matrix

• Cells as below.

Cells of PDL1. 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

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. 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

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.

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.

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.

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.

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.

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

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.

• 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

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

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.

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

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 formationIn 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.

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.

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.

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.

• 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

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).

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.

• 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

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.

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