Case 3 - Bones PDF

CASE 3- Bones Overview - Structure, function and composition of bone - Bone development + growth - Maintenance of bone tissue Structure, function and composition of bone - Approx 206 bones. - Different types - Long bones, flat bones (shoulder blades, skull), irregular short bones...

CASE 3- Bones Overview - Structure, function and composition of bone - Bone development + growth - Maintenance of bone tissue Structure, function and composition of bone - Approx 206 bones. - Different types - Long bones, flat bones (shoulder blades, skull), irregular short bones (spinal column) - Various functions - Joints, attachment of muscles -> allow movement - Internal support and protection -> organs - Haematopoiesis (bone-marrow) - Triglyceride storage (bone-marrow) - Calcium/phosphate metabolism - Endocrine regulation (bone marrow) - Mechanical functions - Weight bearing - Flexibility Structure, function and composition of bone Bone definition -> Hard, calcified matrix containing many collagen fibers, osteocytes lie in lacunae (small hallows), and is very well vascularized. Two types - Outer layer -> smooth and solid -> Compact bone-> more thicker and more compact - Internal layer -> spongy bone -> contains trabeculae -> gives the inner space -> for red/yellow bone marrow.  Collagen reinforcement is required for strength! That's why bones are stronger Bone composition than teeth. Bone is composed of organic and inorganic components. - Question: What is the correct ratio between organic and inorganic components in bone tissue? - 65% inorganic matter (hydroxyapatite), 35% organic matter (collagens) - Bones are mostly composed of - Strong fibers -> Collagen type 1 and osteocalcin (30%) - Solid particles -> Calcium phosphate and minerals (60%) - Crystalline Hydroxyapatite -> strongest mineral - Cells -> osteoblasts (formation) and osteoclasts (degradation) (10%) - Collagens are needed for tensile strength! - Brittle calcium phosphate can break apart -> thus you need Collagen - Vascularization - Periosteum - There is a gap region is collagen -> - Important for the mineral growth Hydroxyapatite - Calcium and phosphate ions can interact -> to initiate - Mineralization Bone structure Diaphysis (middle) - Forms the long axis of bone. Constructed from thick collar of compact bone that surrounds central medullary cavity. In between compact bone and narrow cavity -> thin layer of spongy bone. Epiphysis (end) - Bone ends broader than diaphysis, outer shell of compact bones - Thin hyaline cartilage covers the joint surfaces of epiphysis, cushioning opposite bones. Between the diaphysis and epiphysis is the epiphyseal line, remnant of the epiphyseal growth plate. Cartilage There are 3 types of cartilage - Hyaline cartilage -> most abundant in the body, found in most ends of bone - Fibrocartilage -> Found in vertebrae - Elastic cartilage -> Found in ear and nose Structure, function and composition of bone Haversian system -> building blocks of long bones - The compact part is organized with osteons (structural unit of compact bone). - Osteons provide nutrients to bone cells - Surrounding this, are rings of osteocytes and bone tissue -> making up - Stacking multiple osteons -> create strong compact bone. - Surrounding bone is periosteum, fleece like structure, containing progenitor cells. - Endosteum also has progenitor cells. Periosteum -> White double layered membrane, covering external surface of bone. Outer-> is fibrous layer of dense irregular connective tissue. Inner layer -> primitive stem cells, osteogenic cells. Richly supplied with nerve fibers and blood vessels. Every cell except osteoclasts! Endosteum -> Delicate connective tissue covering internal bone surface. Covers trabeculae of spongy bone. Contains osteogenic cells -> differentiate into other bone cells. Hematopoietic tissue + Bone matrix - Red marrow found in the trabecular cavities of spongy bone of long bones, and in dipole of flat bones! - In infants -> most of bone marrow found produces RBC -> Red marrow - In adults -> most do not make RBC, but stores fats -> Yellow marrow Bone matrix - Calcified bone matrix - Uncalcified bone matrix -> osteoid Bone matrix is formed by osteoblast cells and reabsorbed by osteoclasts. Matrix is surrounded by osteocytes -> mature bone cells that maintain the bone matrix and act as mechanosensors. ECM is composed of organic compounds (collagen t1 fibers) and inorganic compounds (HA depositions) Osteon - Osteons are the structural unit of compact bone. It is an elongated cylinder. - Lamella surrounds the osteon. - Lacunae is where osteocytes are present. - Osteons are interconnected with each other. - Form bridges called the Volkmann’s channel. - Artery, vein and nerve fibers - Osteocytes are interconnected, helps in detected microdamage in bone tissue. - The black dots in the figure below are osteocytes. 2 canals - Central/Haversian canal -> filled with blood vessels + nerve fibers that nourish cells within lamella. - Volkmann's canal -> connects medullary cavity to central canals in osteons. Mineralization of Collagen type 1 - Mineral growth passes along the fiber of the collagen -> reinforced concrete - Initiated in the gap region! How do bones develop? - Different types of bones have distinct embryonic origin. - Two methods to make bone tissue - Intramembranous ossification - Osteoblasts only-> deposit collagen + mineral! - Cranial bones of skull - Endochondral ossification -> forms lamellar bone - Osteoblasts - Involves growth plate - Mainly for long bones Endochondral Ossification - Hyaline Cartilage is the precursor for for bone in endochondral ossification! - Cartilage tissue - Thin , avascular, resistant to compressive forces (absorptive function) - Bone tissue - Thick, Highly vascularized, strong calcified matrix How does cartilage become bone tissue? - Mesenchymal progenitor cell -> builds cartilage tissue -> make collagen t2 matrix with proteoglycans - Chondrocytes dye -> programmed cell death - Leave a cartilage scaffold behind - Osteoblasts remodel the cartilage matrix -> build bone tissue  Endochondral Ossification - Proliferation stage - Mesenchymal progenitor cells express Sox9 -> chondrocytes formation - Cartilage matrix + cartilage tissue formation - Chondrocytes differentiate, enlarge by hypertrophy -> trigger to attract blood vessels + bone progenitor cells. - Too much O2 -> Chondrocytes die - Bone cells take over, starts to remodel and mineralize the bone. - The hole in the center is space for the bone marrow. - Long bones will later create secondary ossification center - In between the middle part and SOC, is the cartilage called the growth plate. - Epiphyseal plate is required for bone growth till you are an adult. - The ends become permanent cartilage -> - Sox9 promotes the production of cartilage matrix proteins, such as collagen type II, ensuring the formation and maintenance of proliferative chondrocytes during early stages of cartilage articular cartilage. development. - Runx2 promotes the hypertrophy of chondrocytes and supports the recruitment of osteoblast precursors, facilitating the formation of the bone collar and vascularization, which are crucial for bone development. Endochondral ossification At late 2nd month of development. Starts from the hyaline cartilage 1. Bone collar forms around diaphysis of hyaline cartilage. a. Osteoblasts secrete osteoid against the hyaline cartilage diaphysis, encasing it in a collar of bones -> periosteal bone collar. 2. Cartilage in the center if diaphysis calcifies + develops cavities a. As bone collar forms, chondrocytes enlarge and signal surrounding matrix to calcify. b. Calcified matrix is impermeable to nutrients -> chondrocytes die -> making cavities c. Cartilage on bone ends will elongate and enlarge the bone. 3. Periosteal bud invades the internal cavity and spongy bone forms a. The cavities are invaded by periosteal bud. b. Contains the nutrient artery, vein, nerve fibers, red marrow element, osteogenic cells, osteoclasts. c. Osteogenic becomes osteoblasts which secrete osteoid -> forms trabeculae. Endochondral ossification 4. Diaphysis elongates and medullary cavity forms a. Primary ossification center enlarges, osteoclasts break down formed spongy bone, opens medullary cavity in the center of diaphysis. b. Until birth, epiphysis only has cartilage. c. Last 3 months, is where there is major mineralization. 5. Epiphysis ossify a. Shortly before birth, secondary ossification centers form! b. SOC -> protects chondrocytes in the growth plate from the mechanical stress. Intramembranous Ossification Forms the cranial bones of the skull. Intramembranous ossification occurs in the early embryo to create flat bones such as the skull and clavicle. 1. Mesenchymal cells cluster and differentiate into osteoblasts, forming the ossification center within the fibrous connective tissue membrane. 2. Osteoblasts secrete osteoid, leading to calcification. The trapped osteoblast becomes osteocytes. 3. Accumulating osteoid between the embryonic blood vessel -> form immature spongy bone. Vascularized mesenchyme condense on exterior forming periosteum. 4. Trabeculae beneath the periosteum remodelled/replaced by compact bone. Immature spongy bone remodelled into mature and filled with red bone marrow. Why are babies so flexible? - There is a gradual disappearance in the cartilage of the hip joint, as the baby grows. - This is why babies are so flexible! - Cartilage is very flexible, but as it disappears you lose the flexibility. Growth curve- endochondral Ossi- - The 2 black lines show the epiphyseal growth plate which needs to be closed, before you turn into an adult. Postnatal growth - New cartilage is formed on the top, and is pushed down. - At the bottom, cartilage cells die and, and tissue becomes bones. - Resting zone - Mesenchymal progenitors that can go under chondrogenic differentiation. - Proliferation zone - Progenitor cells proliferate (cartilage cells) - Hypertrophic zone - Cell growth in larger size, Terminal differentiation - Calcification zone - Matrix calcifies, cartilage cells die, matrix deteriorates, blood vessels invade. - Cells go into programmed cell death - Ossification zone - Bone cells take place, to remodel and mineralize - From collagen type 2 + 10 to type 1 -> then mineralize it! Bone growth - Appositional growth + remodelling - Bone needs to grow in width and in length + remodelling needs to occur! - Length (interstitial growth) -> endochondral ossification - Width (Appositional growth) -> intramembranous ossification - Bone on top -> grows by osteoblasts - Bone on side -> bone reabsorbed through osteoclasts - Thickening of bone occurs through intramembranous ossification in the periosteum. - Outside -> take away bone mineral - Inside -> add bone mineral - Osteoblasts in the periosteum secrete bone matrix on the external surface of bone, as osteoclasts within the endosteal remove bone via resorption. - More building that breaking down -> bones are more thicker and stronger without being heavy. Regulation of bones Growth hormones - The stimulus of the epiphyseal plate activity is growth hormone by the anterior pituitary gland. - Thyroid hormones modulate the activity of growth hormone, ensuring proper skeleton growth. Male + Female Sex hormones - Testosterone and estrogen (slows bone growth) at puberty are released in increasing amounts. - Initially they promote growth spurt. - Later, the hormones induce epiphyseal closure, ending the longitudinal bone growth. Other hormones - Indian hedgehog, parathyroid hormone related protein, BMP, WNT signalling, FGF, IGF-1  Sox9 and Runx2 - Sox9 -> Hypertrophic chondrocyte - Runx2 -> Osteoprogenitor cell -> mature osteoblast -> osteocyte - Don’t need to know all the factors!!! Like Sp7 blah blah Wnt/β-catenin: Regulates transition from chondrocytes to osteoblast lineage. Ihh and Sox9: Drive chondrocyte proliferation and hypertrophy. Runx2 and Sp7: Essential for osteoblast differentiation and bone matrix production. Growth plate contribute to growth 4 growth processes - Cell growth -> hypertrophy - Cell division -> proliferation - Cell death - Matrix deposition -> ECM formation How does the growth plate contribute to growth? - Larger (cell growth) and more chondrocytes (cell division) Chondrocytes -> Growth plate - Why do chondrocytes die? - Make place for bone cells - Contribute to mineralize - Make enzyme alkaline phosphatase, which increase conc of phosphate, that helps in mineralization. - When cells die -> calcium is released -> used for mineralization - Due to hormonal changes -> Estrogen is responsible for the closure! - Epiphyseal growth plate closure occurs at different times - 18 years for females - 21 years for males - Estrogen induces growth plate closure due to progenitor cell depletion. - Induce terminal differentiation -> STOP  Cells in bone tissue - Osteogenic cells - Stem cells/ progenitor cell -> can differentiate into osteoblast/bone lining - Osteoblast 5 % -> 12 days - Matrix-synthesizing cell responsible for bone growth - Can become osteocytes - Cube shape when active, flat when inactive - Osteocyte 95% of bone cells -> 10-20 years - Mature bone cell that monitors and maintains the mineralized bone matrix. - Takes care - Osteoclast (large) 1% , days - Bone resorbing cell - Have many nuclei, to maintain size - From bone marrow - Bone lining cells - Protect the bones, by maintaining matrix - External -> periosteal cells - Internal -> endosteal What cells reside in bone tissue? Bone formation -> osteoblasts Bone matrix - Organic -> collagen type 1 - inorganic : hydroxyapatite deposition - Enzyme that helps mineralization is - Alkaline phosphatase enzyme How do osteoblasts make bone matrix? - Hydroxyapatite (HA) is the main inorganic building block of bone. - Osteoblasts excrete collagen type 1 matrix (osteoid) and ALP enzyme in extracellular vesicles. - ALP from the vesicles cleaves phosphate groups from nearby proteins and acts as foci on the collagen matrix for calcium and phosphate deposition and crystallisation of the HA. Osteoclasts - Many nuclei, ruffle border -> attach to bones - Large cell with multiple nuclei - Originate from bone-marrow progenitor cells How do Osteoclasts resorb? 1. Attachment to bone 2. Seal bone - ruffles membrane (acBeta3 integrin) 3. Acidification to ph4.5 by protons and chloride by H+ ions 4. Mineral mobilisation and removal by TRAP 5. Degradation of ECM collagens by Cathepsin K 6. Removal of Fragments by TRAP 7. Bone disappeared Cathepsin K is a potent matrix degrading enzyme with specific activity towards connective tissue matrix molecules (collagen, elastin) Maintenance - Upto 10% of all bone mass is undergoing remodelling at any point in time! - No bone lining cells -> osteoclasts come and are resorbing -> increase concentration of calcium and phosphate -> makes it stop -> attracts osteoblasts -> mineralize and rebuild the bone. Parathyroid hormone regulating Ca2+ availability - The parathyroid hormone, regulates the Calcium ions availability. - Works of how many osteoclasts are active. - Osteoclasts degrade bone matrix and release calcium and phosphate into the blood. - Wolf’s law -> you stress your bone, it becomes stronger! - A tennis player has a more mineralized bone, more stronger bone - Space! - You lose a lot of bone mass - Every month 1.5% bone density is decreased - Osteoclasts find cracks and resolves it, but there is no stimulation, that encourage osteoblasts to remake the bone! :( Fractures! - Periosteum gets ruptures, blood vessels get ruptured, progenitor cells gets activated. - Cambium and the fibrous layer (closer to skin) - Cambium layer contains mesenchymal progenitor cells How does healing happen? 1. Hematoma formation from bleeding, due to blood clot! 2. Fibroblasts-connective tissues, chondrocytes make cartilage tissue -> forms small callus 3. Bony callus formation -> From small callus to bony callus 4. Bone remodelling occurs -> Chondrocytes are replaced by bone cells, and start to remodel. Fractures Hematoma - Forms due to ruptured blood vessel. - Pro-inflammatory cytokines released -> TNF-a, IL-1, IL6, IL-11 & IL-23. - Pro-inflammatory cytokines stimulate recruitment of WBC. This induces removal of necrotic tissue and further cytokines are released. - Leads to the activation of vascular endothelial growth factors, which stimulate healing. Fibrocartilaginous callus formation - Expression of VEGF leads to angiogenesis at the site of hematoma. - New blood vessels form on pre-existing blood vessels, and fibrin rich granulation tissue developed. - They recruit mesenchymal stem cells which differentiate to fibroblasts, chondrocytes and osteoblasts. -> Chondrogenesis (cartilage formation) -> make collagen rich fibrocartilaginous network. Fractures Bony callus formation - Cartilaginous callus begins to undergo endochondral ossification. - Rank-L expression sets, while further chondrocytes differentiate into chondroblasts, chondroclasts and osteoblasts and osteoclasts. - Cartilaginous callus gets resorbed and calcified, while bone continues to form. - New blood vessels proliferate -> further migration of progenitor cells. - Forming the calcifies callus of immature bone -> replaced by fibrocartilaginous callus Bone remodelling - Continued migration of osteoclasts & osteoblasts. - Hard callus undergoes repeated remodelling. - Center of callus is replaced by compact bone and inside is filled with spongy bone and marrow cavity. - Balance of resorption by osteoclasts and new bone formation by osteoblasts -> fracture is healed. Summary Bone tissue consists of: - Trabecular and cortical bone. - Bone cells, extracellular matrix (Collagen type I) and hydroxy apatite crystals (Calcium phosphate) - Haversian system in long bones. Bone initially forms from a cartilage scaffold (endochondral ossification) - Condensation, hypertrophy, primary / secondary ossification. Intramembranous ossification in the skull and appositional growth - Osteoblasts secrete osteoid that leads to mineralization of collagen Summary Bone growth occurs until adulthood. - Happens in the growth plate (resting, proliferation, hypertrophy, ossification zone) - Growth plate chondrocytes make a scaffold for bone cells and go into apoptosis. - Regulated by systemic factors (hormones) and local growth factor gradients. Bone is continuously remodeled and adapts to stress (Wolff's law) - Interplay osteocytes (sensing damage), osteoclasts (degrading bone) and osteoblasts (making bone). - Fracture healing re-iterates endochondral ossification from dedicated periosteal progenitor cells.