Macrostructure of bone

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Macrostructure of bone

The human skeleton can be subdivided into two parts, the axial skeleton, which includes the skull, vertebral column and rib cage, and is particularly important for protecting vital organs, and the appendicular skeleton which includes the limbs. The axial skeleton is composed mainly of flat bones, such as the skull and scapula, while the appendicular skeleton contains long bones such as the femur, tibia and radius. Long bones consist of a hollow tube called the diaphysis, with flared ends known as epiphyses as depicted in 528HFigure 1.1. There are two types of bone, cortical bone, which is very hard and dense, and trabecular or spongy bone which has a more variable and porous structure. All bone is made up of osteons, which are composed of an organic matrix, primarily composed of type I collagen, and mineralised with calcium salts that form hydroxyapatite crystals.

Endochondral ossification

In many parts of the skeleton, bone is formed by endochondral ossification, a process that involves synthesis of a cartilage template before bone formation can occur (529HFigure 1.2). Mesenchymal condensations differentiate into chondrocytes that rapidly produce cartilage structures resembling the final shape of the bone. Chondrocytes in the centre or diaphysial region of the structure eventually become hypertrophic and undergo apoptosis, causing cartilage mineralisation, and allowing vascular invasion. This allows infiltration of cells which differentiate into osteoblasts, and form bone in the degraded cartilage matrix. Later in development, secondary ossification centres form in the epiphysial regions of the bone, leaving the growth plates in between as a site of chondrogenesis.

Bone remodelling

In the adult skeleton bone is continually turned over in a process known as remodelling. The skeleton is completely replaced every 11 years, although bone turnover occurs much more quickly in the trabecular bone [4]. Remodelling is a coupled process where bone resorption is closely followed by bone formation, so in a healthy skeleton there is no net loss or gain of bone mass. Bone remodelling occurs in distinct areas by groups of sequentially acting cells known as the basic multicellular unit (BMU). At any one time there are millions of BMUs in the skeleton at different stages, and the duration of BMU activity may be up to eight months [5]. Bone remodelling originates at sites of microdamage, which is likely to involve signals originating from osteocytes, or resulting from osteocyte apoptosis.

Bone mass

Bones grow throughout childhood until their adult form is reached at the end of puberty. Peak bone mass and strength of the skeleton is attained during the third decade of life. Genetic factors are the most important determinants of peak bone mass in humans. Environmental factors also contribute, including nutrition and body weight, physical activity, and levels of various hormones [7]. Later in life, bone mass begins to decline. Women, in particular, lose bone rapidly after the menopause. There are a number of diseases that affect bone mass. Clinically, reduced bone mass is known as osteopaenia, and more severely reduced bone mass as osteoporosis. Conditions with excessively high bone density are known as osteopetrosis. In the clinic, bone mass can be estimated using bone mineral density (BMD) measurements performed using DEXA scans.

Bone matrix

Bone makes up the largest proportion of the body’s connective tissue, and is unique in being mineralised. The organic, collagen-based matrix provides elasticity and flexibility while the mineral gives the tissue rigidity and strength. The most important and abundant component of the organic matrix is type I collagen, which comprises around 90% of bone protein content. Type I collagen is a triple helical molecule made up of two α1 chains and an α2 chain. With appropriate posttranslational modifications, the chains form a left-handed helix [8]. Fibrils form and assemble in a staggered fashion, and inter-molecular cross-links are added to increase the strength of the matrix. Type I collagen is produced as a propeptide, but the ends are rapidly cleaved producing the N-terminal and C-terminal propeptides of collagen (P1NP and P1CP) which can be used clinically as serum markers of bone formation.

TABLE OF CONTENTS :

• 0ABSTRACT
• 1HACKNOWLEDGEMENTS
• 2HLIST OF TABLES
• 3HLIST OF
• CHAPTER 1: INTRODUCTION
• Part A. BONE BIOLOGY
  • 1.1 Macrostructure of bone
    • 1.1.1 Cortical bone
    • 1.1.2 Trabecular bone
  • 1.2 Bone formation and modelling
    • 1.2.1 Intramembranous ossification
    • 1.2.2 Endochondral ossification
  • 1.3 Bone remodelling
  • 1.4 Bone mass
  • 1.5 Bone matrix
    • 1.5.1 Mineralisation
    • 1.6 Bone cells
    • 1.6.1 Osteocytes
    • 1.6.2 Osteoblasts 
    • 1.6.3 Osteoclasts 
  • 1.7 Hormones and growth factors involved in bone metabolism
    • 1.7.1 Parathyroid hormone and parathyroid hormone related protein
    • 1.7.2 Vitamin D.
  • 1.8 Common disorders of bone metabolism
    • 1.8.1 Osteoporosis
    • 1.8.2 Hyperparathyroidism
    • 1.8.3 Cancer and bone
• Part B. PAGET’S DISEASE OF BONE
  • 1.9 Epidemiology 
  • 1.10 Clinical features
  • 1.11 Diagnosis and treatment
  • 1.12 Pathophysiology
  • 1.13 Aetiology
    • 1.13.1 Genetics
    • 1.13.2 Paramyxoviruses and Paget’s disease
  • 1.14 Genetic disorders similar to Paget’s disease
    • 1.14.1 Familial expansile osteolysis and other diseases caused by RANK mutations
    • 1.14.2 Idiopathic hyperphosphatasia
    • 1.14.3 Inclusion body myopathy, Paget’s disease and frontotemporal dementia
• 4HPart C. AIM
  • 1.15 Aims of this study|
• CHAPTER 2: METHODS 
  • 2.1 Materials
    • 2.1.1 Cell culture
    • 2.1.2 Molecular biology
    • 2.1.3 Protein detection
    • 2.1.4 Solutions
  • 2.2 Cell culture 
    • 2.2.1 Human osteoblasts 
    • 2.2.2 Human bone marrow cells 
    • 2.2.3 Primary rat osteoblasts 
    • 2.2.4 Cell lines
    • 2.2.5 Proliferation assay.
    • 2.2.6 Mineralisation assay.
  • 2.2.7 Mouse bone marrow osteoclastogenesis culture
    • 2.2.8 Luciferase assay 
    • 2.2.9 Cultures in three dimensional scaffolds 
  • 2.3 Molecular biology 
    • 2.3.1 RNA extraction 
    • 2.3.2 RT-PCR
    • 2.3.3 Sequencing
• CHAPTER 3: GLOBAL ANALYSIS OF GENE EXPRESSION IN PAGETIC OSTEOBLASTS
  • 3.1 Introduction
  • 3.2 Methods
    • 3.2.1 Sample details
    • 3.2.2 Cell characteristics 
  • 3.3 Microarray analysis results
    • 3.3.1 Overview of Affymetrix microarrays
    • 3.3.2 Quality control and normalisation
  • 3.4 Discussion
• CHAPTER 4: DIFFERENTIAL GENE EXPRESSION IN PAGETIC CELLS
• CHAPTER 5: EFFECTS OF DICKKOPF 1 AND KERATIN 18 ON BONE CELLS
• CHAPTER 6: EFFECT OF WILD-TYPE AND MUTANT SQSTM1 IN OSTEOBLASTS40H
• CHAPTER 7: DETECTION OF MEASLES VIRUS RNA IN PAGETIC CELLS
• CHAPTER 8: DETECTION OF SOMATIC SQSTM1 MUTATIONS IN PAGETIC CELLS
• 160HCHAPTER 9: GENERAL DISCUSSION AND CONCLUSIONS

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Involvement of the Osteoblast in Paget’s Disease of Bone