Microanatomy_Cart_and_Bone_2024.08.30_Final_Copy.pptx

roanatomy of Cartilage and Bone: ne Formation, wth, d aling https://www.kcbj.com/im-getting-my-cast-off-today-what-should-i-e xpect/ Session Goals 1. Understand the processes that produce and alter bone tissue and, by extension, bo...

roanatomy of Cartilage and Bone: ne Formation, wth, d aling https://www.kcbj.com/im-getting-my-cast-off-today-what-should-i-e xpect/ Session Goals 1. Understand the processes that produce and alter bone tissue and, by extension, bone organs, during growth and development, through adult life, and during the healing process. 2. Gain a knowledge of how bone is normally maintained, and how an imbalance in this maintenance leads to pathological changes in bones and joints. Learning Objectives 1. Describe the molecular pathways by which the cells of bone react to mechanical signals and the resultant interplay between them that drives resorption and deposition to produce the structural changes in the bone tissue occurring throughout life.  Cellular Components of Cartilage Chondroblasts Immature cartilage cells Generally found in perichondrium Give rise to chondrocytes Undergo appositional growth (differentiation) Chondrocytes Mature cartilage cells Generally located deep in the tissue Reside in lacunae Divide and form isogenous groups Interstitial growth (cell division)  Chondrocyte Cell Lineage Pathway Mesenchymal stem cells (MSC) Serve as the precursor for chondroblasts (chondrocytes) Chondrocytes (chondroblasts) Synthesize and maintain the cartilaginous matrix Synthesize Type II collagens, GAGs, and proteoglycans Differentiation Regulated by BMPs (bone morphogenic proteins) BMPs Transforming Growth Factor-β (TGF-β) superfamily Growth factors that induce bone/cartilage formation  BMP Signaling Pathway BMP/TGF-β Receptors 2 types of receptors: Type I & II Activate signal transducers by the recruitment of Smad proteins Smad transcription modulation Smad complex upregulates the expression gene targets: Runx2 (osteoblasts) BMP Signaling Upregulates Sox9 (chondroblasts) Target Genes: Sox-9 found in all chondroprogenitors Runx2 Sox9  BMP Signaling Pathway Signaling Mechanism 1. Activation of Type II receptor by BMPs 2. Type II receptor activates Type I receptor 3. Type I recruits and activates receptor Smads 4. Formation of Smad complex Receptor-smads and Smad-4 complex together 5. Smad complex translocates into the nucleus 6. Forms a transcription regulatory complex 7. Upregulates expression of target genes BMP Signaling RunX2 Upregulates Sox9 Target Genes: Runx2 Sox9  Chondroblast Differentiation Sox-9 and Runx2/β-catenin transcription factors Serve as a molecular instruction for differentiation Histology and Cell Biology 5th Edition, Chondroblast BMPs  Sox-9 Osteoblast BMPs  Runx2 Wnt  β-catenin 2020 Sox9 - Chondroblast Sox-9 Sox Upregulated by BMPs 9/5/ 6 Runx MS Key transcription factor for chondroblast C 2 differentiation Runx2 Osteo- Also upregulates Sox-5 & -6 chondro- β-catenin Sox-9/5/6 Trio induces chondroblast progenitor Pre- Osteobl cell osteoblast ast differentiation Osteocy Cellular Components of Bone Osteoblasts Synthesize bone Osteoclasts Degrade bone Osteocytes Mechanosensor cells that control blasts and clasts Williams Textbook of Endocrinology (2016), Chapter 29: Osteoporosis and Bone Biology  MSCs Differentiate into Osteoblast Cells Mesenchymal stem cells: Located in bone marrow MSCs differentiate into progenitor cells Progenitors differentiate into osteoblasts Osteoblasts Synthesize bone Synthesize bone matrix and bone matrix proteins Differentiation Regulated by: Bone Morphogenic Protein-2 (BMP2) Wingless (Wnt) signaling HPC Scientifica. Nov. 21, 2013; 213: 1-17  Transcription Factors used during Bone Formation Bone morphogenic proteins (BMPs) BMP-2: Promotes differentiation of MSC into osteoprogenitors Upregulate Runx2 Wnt Pathway Stimulates differentiation and survival of osteoblasts and osteocytes Pathway Stabilizes β-catenin that causes upregulation of Runx2 Runx2 Key transcription factor for directing differentiation of osteoblasts Runx2 expression is upregulated by Wnt and BMP-2 pathways Key: High levels of Runx2 cause MSC differentiation into osteoblasts  Wnt/β-Catenin Pathway Frizzled receptor Structure similar to GPCRs Recruits disheveled protein Dishevelled Scaffolding protein that recruits axin β-catenin Transcription regulator Degraded by degradation complex (Axin, APC, GSK3, and CK1) Mechanism 1. Wnt activates Frizzled 2. Axin of degradation complex is recruited by disheveled Leads to destruction of the degradation complex Wnt Upregulates Target Genes: 3. β-catenin is stabilized and translocates to nucleus Runx2 4. Upregulates the expression of Runx2  Crosstalk Between Wnt and BMP Pathways during Osteogenesis Wnt: Wnt signaling helps to stabilize β-catenin Induces the expression of Runx2 BMP: BMP signaling activates formation of Smad complexes Smad complexes induce Runx2 expression Parallel effect: Wnt and BMP2  Runx2 which drives osteogenesis JBC (2005), 280(39); 33132-33140  Combination Effects of BMP Signaling and Wnt Drive Osteoblast Differentiation Wnt Runx2 Osteoblasts Differentiation pathway: BMP + Wnt  High Runx2  Osteoblasts MSC BMPs only  Sox9  Chondroblasts Wnt Sox9/5/6 Chondroblasts  Osteoclasts & HSC Differentiation Derived from hematopoietic stem cells (HSC) Activated by cytokines secreted by osteoblasts and osteocytes Osteoclasts Motile cells Degradation and resorption of bone Proper shaping of bone Williams Textbook of Endocrinology (2016), Chapter 29: Osteoporosis and Bone Biology Osteoblast Cytokines Macrophage colony stimulating factor (M-CSF) Receptor activator of nuclear factor κB ligand (RANKL) Both initiate differentiation and maturation of osteoclasts  Osteoclast Activation Signaling Molecules M-CSF RANK RANKL L RANKL/M-CSF Essential for osteoclast differentiation and maturation Stimulate transcription regulators that upregulate osteoclast genes Osteoprotegrin (OPG) Secreted by osteoblast lineage cells Inhibits RANKL signaling  Activated Osteoclasts Histology and Cell Biology 5th Edition, 2020 Activated osteoclasts Highly polarized Exhibit 3 specialized regions Cellular Regions 1. Sealing or Clear zone Site where osteoclasts adhere to bony matrix Highly dense in actin filaments Integrins help form a seal (act as suction cups) 2. Ruffled Border Contains microvillus-like structures (actin) Site of proton and digestive enzyme secretion Metalloproteinases, cathepsin K Digests bone matrix Endocytosis of degradation products and debris 3. Basolateral Region Exocytosis of digested material Histology 7th Ed. (Wolters Kluwer 2016)  Bone Homeostasis Interplay between deposition and resorption Resorption Formation Dynamic tissue (Osteoclast (Osteoblast s) s) Constantly being resorbed and formed Balance between osteoblasts and osteoclasts Disturbance of this balance Osteoporosis  “normal”  Osteopetrosis ?  Osteocytes Osteocytes An osteoblasts encase themselves in matrix and become osteocytes Form long dendritic processes (found in canaliculi) Used for communication Communicate through gap junctions Function Mechanosensory cells of the bone Histology and Cell Biology 5th Edition, 2020 Play a central role in the maintenance of the bony matrix by regulating Osteoblast and osteoclast activities Stimulus Osteoclast Osteocyte Osteoblast Osteocyte Bone in bony matrix  Osteocyte Respond to Mechanical Forces Compressive loading leads to mechano-adaptation Mechanical load is perceived as shear stress Primary cilia respond to mechanical forces Sense interstitial fluid (IF) flow (shear force) Benzel’s Spine Surgery (2017), Bone Modeling and Remodeling  Osteocyte Respond to Mechanical Forces cAMP/PKA pathway Adenylyl cyclase 6  cAMP  PKA PKA inhibits osteogenesis Primary Cilia (non-motile) One per cell Made of microtubules Serves as a chemosensory and mechanosensory organ Ca2+ inhibits Adenylyl cyclase (AC)6 Inhibition leads to activation of osteogenesis T Osteogenesis Bone 2013; 54(2): 196-204  Osteocytes Respond to Mechanical Forces Mechanical Load Mechanical loading: Mechanical load  IF Flow Ca2+ enters cilia Osteogenesis Inhibition of AC cAMP/PKA Reduction in active PKA Pathway Mechanical load  IF Flow Ca2+ does not enter cilia Inhibition of osteogenesis T AC remains functional Osteogenesis PKA remains active Bone 2013; 54(2): 196-204  Osteocytes Orchestrate Bone Formation or Resorption through Mechanotransduction mechanical stimuli  bone formation Wnt Activates expression of Runx2 Osteoprotegrin (OPG) Inhibits osteoclast differentiation mechanical stimuli  bone reduction RANKL and M-CSF Promote osteoclast differentiation and activation Sclerostin (Wnt inhibitor) Endocrine Reviews (2013), 34: 658–690 Inhibit Osteoblast differentiation Learning Objective 1: Application Describe the molecular pathways by which the cells of bone react to mechanical signals and the resultant interplay between them that drives resorption and deposition to produce the structural changes in the bone tissue occurring throughout life. Ask yourself: 1. What are the different pathways through which mechanical stimuli mediate bone formation? Bone resorption? 2. What role do osteocytes play in bone remodeling? Osteoclasts? Osteoblasts? Osteoprogenitor cells? Learning Objectives 2. Name, locate, and describe the structural parts and tissues found in a typical bone (organ) and explain their functional roles. PARTS OF A MATURE LONG BONE and its constituent structures and tissues Epiphysis (extremity) Note: blood vessels supply bones via: a) periosteal vessels b) nutrient vessels – their branches travel in a thin membrane known as the endosteum, which covers all interior surfaces of the bone. Diaphysis (shaft/body) Marrow Cavity and… LYMPHATICS! With lymphangiogenesis being a characteristic response of injury – both micro and microtrauma to bone tissue. From: Biswas et al., 2023, Cell 186, 382–397 January 19, 2023 ª 2022 The Authors. Published by Elsevier Inc. https://doi.org/10.1016/j.cell.2022.12.031  Macroscopic architecture of mature bone tissue compact Two types: 1. Compact aka: dense or cortical bone 2. Spongy aka: cancellous or trabecular bone spongy A trabecula, or spicule, is a prong of bone issue seen in spongy bone architecture compact  Bone tissue localization within a long bone Cancellous bone Bone tissue architecture Compact is a response to local bone mechanical stresses. Compact bone = tension resistant Cancellous bone =resistant to Cancellous bone compression (Femur) at joint surfaces (of spongy bone) Learning Objective 1: Application 2. Name, locate, and describe the structural parts and tissues found in a typical bone (organ) and explain their functional roles. Ask yourself: 1. Where is the blood supply of the bone located? How do the different regions of a bone receive their blood supply? Are any regions less well supplied than others? 2. What will the presence of compact bone tissue tell you about the mechanical stresses that a certain region of a bone organ experiences? Cancellous? Learning Objectives 3. Describe intramembranous osteogenesis, including the participant cells, tissues, and matrix components at each stage, and explain the functional reasons that intramembranously forming bones develop preferentially in certain specific locations of the developing skeleton.  Bone Tissue Formation (osteogenesis) on features of all bone formation: ne always develops in an already present, vascular CT. ne matrix is always laid down first as osteoid (organic matrix). ne always forms first as woven (aka, primary) bone.  Patterns of Bone Formation I. Intramembranous sponsible cells: mesenchymal cells and osteoblasts  osteogenic mesenchymal cells proliferate around a dense capillary networ begin to produce a meshwork of Collagen I fibers and GAGs (glycosaminog - this product is called osteoid; it is the organic component or phase of t - cells next secrete mineral content; retain their connections via gap jun as they become surrounded by matrix to become osteocytes 1 additional osteogenic cells arrive via capillaries - these line up on the osteoid (matrix) surfaces and become osteoblasts - osteoblasts begin to produce additional matrix Mesenchymal mass - as these osteoblasts become surrounded by matrix, they secrete mine forming skeletal primordium phase of the matrix - once completely surrounded, they too are now mature bone cells: oste 2  This initial bone formed is called woven bone, because the collagen fibers strengthening its matrix are arranged randomly, without any specific orientation creatingb)somewhat of a woven Differentiation appearance into of mesenchyme 1: cartilage and 2: perichondrium Modified from Sadler, 2006 Osteoblast seeding osteoid with mineral crystals Note: osteoblasts first secrete the organic components of the ECM, THEN seed it with mineral. The first phase of ECM secretion, produces what is known as osteoid, which is not yet calcified. The calcified phase forms next, completing the initial bone production at a given location. The first bone tissue produced anywhere is known as woven bone.  O= osteoblast Intramembranously forming bone B= bone M=mesenchyme CM=cellular mesenchyme P=periosteum V= vein Junqueira, 14th ed., 2016 Fig. 8-13  Intramembranously forming flat bone of the skull Numerous osteoblasts seen lined up on surfaces of newly produced bone tissue (i.e., woven bone) bone osteoblasts  tramembranous bone formation occurs irly quickly but does not provide good support. hus, it is ideal for the flat bones of the rapidly owing cranial vault which grows quickly early in e to accommodate the brain which also grows ost rapidly early in development. here there is a need for support, bones form rough a second process in what is known as ndochondral formation. Bones forming ndochondrally – go through an intermediate, artilaginous, stage in their development. This is e type of bone formation seen in the cranial base nd elsewhere support is needed in the developing keleton. Williams, et al., 1995 Yellow=intramembranously forming bones Embryonic Human Skull Blue= endochondrally forming bones Learning Objective 3: Application Describe intramembranous osteogenesis, including the participant cells, tissues, and matrix components at each stage, and explain the functional reasons that intramembranously forming bones develop preferentially in certain specific locations of the developing skeleton. Ask yourself: 1. Would a defect in the collagen II gene have an adverse effect on growth of the cranial vault? On the cranial base? Learning Objectives 4. Describe endochondral bone formation, including the participant cells, tissues, and matrix components at each stage, and explain the functional reasons that endochondrally forming bones develop preferentially in certain specific locations of the developing skeleton. Histogenesis of Hyaline Cartilage 1. Condensation of embryonic mesenchyme 2. Differentiation to chondrogenic cells 3. Chondroblasts begin to produce cartilage matrix. 4. Embedded chondroblasts become chondrocytes. Patterns of Bone Formation II. Endochondral Beginning with a mesenchymal mass, initial skeletal primordium differentiates into cartilage, forming a cartilaginous model of skeletal element – a cartilaginous miniature of the future bone Responsible cells: mesenchymal cells and chond AND, cartilage doesn’t provide enough weight-bearing support! Necessary for the cartilage to be replaced by bone tissue Responsible cells: chondrocytes, osteoblasts, clas  ENDOCHONDRAL OSSIFICATION AND GROWTH OF A LONG BONE. Cartilaginous miniature grows ntil becomes too large for cells o be maintained by the diffusion radient. – 3. Chondrocytes enlarge, to increase membrane surface area, and secrete kaline phosphatase, which calcifies ECM. hondrocytes die, resulting in empty lacunae n their calcified matrix. – 9.. Vascular bud brings in osteogenic ells which begin to secrete bone matrix on the scaffolding left behind y the vacated calcified cartilage matrix. alcified cartilage removed by clasts s increasing amounts of bone tissue ormed. 0. All cartilage replaced by bone tissue omplete (except at articular surfaces). Ross, Romrell, and Kaye, 1995.  Endochondral Ossification characterized by hyaline cartilage differentiating from a mesenchymal precursor to form models of skeletal elements cartilage subsequently replaced by bone tissue as it is removed by phagocytic cells (clasts*) of the skeletal system Note: the majority of the skeleton is formed endochondrally. *cells that phagocytize bone are called “osteoclasts;” those phagocytizing Fetal cartilage are called “chondroclasts,” etc. hand Developing Long Bone Deposition of woven bone on residual calcified cartilage spicules in marrow cavity Pawlina, p. 231 Learning Objective 4: Application Describe endochondral bone formation, including the participant cells, tissues, and matrix components at each stage, and explain the functional reasons that endochondrally forming bones develop preferentially in certain specific locations of the developing skeleton. Ask yourself: 1. Why does a cartilaginous miniature of a developing bone only get so large before the cells in its interior die? 2. Which cells are brought into the hollowed-out interior of an endochondrally forming bone at the primary marrow cavity to begin the process of osteogenesis? Learning Objectives 5. Describe the component parts and tissues of sesamoid bones, explain their relationships to tendons and the mechanical force(s) predicting where they most commonly occur in the body.  Sesamoid bone formation skeletal elements forming within the substance of a tendon lack a periosteum consistent sesamoids of the human skeleton: patella pisiform lateral & medial pollicial (thumb) sesamoids lateral & medial hallucial (great toe) sesamoids there are numerous variably occurring sesamoids as well; these may or may not ossify begin as condensations of fibrocartilage at locations subject to excessive shear; sometimes calcify & sometimes eventually ossify (become bone)  Quadriceps tendon Quadriceps tendon striations Patella Downloaded from: Gray's Anatomy (on 25 April 2007 05:54 PM) Figure 113.6 Left patella: anterior aspect. (Photograph by Sarah-Jane Smith.) Sagittal section through the knee joint © 2007 Elsevier Learning Objective 5: Application Describe the component parts and tissues of sesamoid bones, explain their relationships to tendons and the mechanical force(s) predicting where they most commonly occur in the body. Ask yourself: 1. Why does a sesamoid NOT develop at the elbow as opposed to the knee? What would be needed in order for that to occur? 2. From what surrounding tissue would a fractured sesamoid bone obtain its blood supply for healing? 3. If a sesamoid remains cartilaginous, rather than being replaced with bone tissue, will it heal if fractured? Learning Objectives 6. Describe how bones grow, including long bone growth in breadth, length, and at articular surfaces.  ENDOCHONDRAL OSSIFICATION AND GROWTH OF A LONG BONE growth plate Growth = an increase in size Ross, Romrell, and Kaye, 1995. Epiphysis articular cartila (hyaline cartilage) Metaphysis Diaphysis (shaft) PARTS OF AN IMMATURE LONG BO  Growth plate  Produced by interstitial growth of cartilage in a specialized organ: the growth plate. Growth plate chondrocyte columns (isogenous groups) direction of growth Growth Plate Chondrocytes 1. Proliferate by dividing mitotically 2. Enlarge and secrete alkaline phosphatase which calcifies matrix 4. Die to create empty lacunae calcified matrix The remaining calcified cartilage matrix provides a scaffold for incoming osteogenic cells to begin to secrete bone matrix on. marrow calcified cartilage osteocytes osteoblasts bone tissue  Occurs through interstitial growth of articular cartilage.  Occurs through subperiosteal bone deposition, in which new bone tissue is produced by the osteogenic layer of the periosteum.  Bone GROW TH bone tissue growth occurs ONLY through apposition, by the production and deposition of osteoid on a pre-existing surface Note, however, that the growth of a bone organ can also occur through interstitial growth of the growth plate cartilage, the means by which the majority of long bone lengthening occurs. Learning Objective 6: Application Describe how bones grow, including long bone growth in breadth, length, and at articular surfaces. Ask yourself: 1. Which cells are responsible for producing most of the increase in length of a long bone? Breadth? 2. In all cases of endochondral bone formation, what occurs to chondrocytes after they hypertrophy? 3. Which cells produce new bone tissue inside the primary and secondary marrow cavities? 4. How can a remnant of calcified cartilage be differentiated from bone tissue histologically? Learning Objectives 7. Describe the process of bone remodeling, including participant cells and tissues, and explain its role in the formation of mature bone tissue in response to: local mechanical forces within a bone organ; bone growth; and bone healing.  Remodeling = change in form occurs regardless of whether bone originally formed intramembranously or endochondrally is a process by which a growing bone maintains its morphology (shape) restores the shape of a bone as part of fracture healing involves coordinated deposition (production) and resorption (removal) of bone at various sites along bone surfaces and within bones - transforms woven (immature disorganized bone tissue) to mature (lamellar) bone in respon to local mechanical stress experienced by the tissue - alters mature bone architecture in response to local mechanical requirements - is a response to physiological demands for bone mineral reserves part of continual process of bone tissue turnover throughout an individual’s lifetime  Gross shape change of bones during growth oronal section through the ranial vault, showing spatial isplacement, or modeling, hrough growth. Diagram of a long bone, showing spatial displacement, or modelin through growth. From Enlow and Hans, 1996 How do we get from - initially, bone tissue is woven bone very cellular, porous, and to lamellar (mature) its matrix structure, including bone that is either orientation of its collagen fibers rather cancellous or compact? disorganized = woven bone fetal  Bone: Types of Tissue woven (cellular; primary) vs. secondary (=lamellar) - cancellous - compact  WOLFF’S LAW (Wolff’s Law of Transformation) If it is true that functional stresses shape the bone, then it is equally true that a change in the strength and direction of forces will lead to changes in the form and structure of bones. Forces Shaping Bone(s) Weight (mass via gravity)> pressure, i.e., compression Muscle force (and, to a lesser extent, ligaments) > pull, i.e., tension Bone, schematic showing differentiated tissue architecture of lamellar Spongy (=cancellous or (mature) bone trabecular) bone resulting from bone tissue Compact remodeling (=dense or cortical) bone Types of Lamellae * Lamellae= layers of bone * tissue Haversian Systems (also call osteons) Circumferential lamellae Notice lamellae (around circumference of the bone) (layers) are also present in Interstitial lamellae * cancellous bone; (between the Haversian canals they are just of compact bone; these often not as obvious. look like fragments of Haversian Systems – because they are!)  Attachment of Tendon to Bone uscle This tension produces the surface cortical Myotendinous bone tissue which covers junction a bone organ eriosteum one Sharpey’s Fibers  Compact Bone Architecture http://www1.udel.edu/biology/Wags/histopage/ illuspage/icb/icb.htm ian and Volkmann’s Canals convey blood vessels through the compact bone tissue  Haversian Systems of Compact Bone http://www1.udel.edu/biology/Wags/histopage/ illuspage/icb/icb.htm ellae = layers of bone tissue; characteristic of mature bone (aka, lamellar bone) Lacunae house osteocytes, the mature cells of bone erent Views of Compact Bone Tissue m a long bone shaft n ground bone [inorganic] preparations) Longitudinal Section Cross-Section Volkmann’s Canal Haversian Canal Haversian System (osteon) Haversian System Formation X marks the location of a future new Haversian System. This series of schematics shows how a new haversian system is formed In already existing compact bone. Haversian systems are formed in the same manner in the process of transforming woven bone to cortical bone in a given location.  Haversian System Formation Several osteoclasts excavate a cylindrical ped tunnel through existing bone tissue. Osteoprogenitor cells, following ely behind, differentiate into eoblasts and begin to lay down e matrix. They become entrapped, osteocytes, in a circular layer of bone that s the perimeter of the tunnel. Another, then another, wave of eogenic cells follows, producing cessive layers of bone until the tunnel omes quite narrow. A central canal versian canal) remains ransmit blood vessels. The entire final unit – is an osteon – or Haversian System. lindrical Tunnel is formed by the Cutting Cone of Osteoclasts first layer (lamella) of the new Haversian system is formed by oblasts, narrowing the central canal ional lamellae are formed by successive waves of osteoblasts, ing the Haversian system from the outside in he completed new Haversian System  Haversian systems of different ages seen in normal, healthy bone New Haversian Young Haversian system system Old Haversian system tecture of cancellous (spongy, or trabecular) bone tissue ular canals are unnecessary in spongy bone tissue due http://www1.udel.edu/biology/Wags/histopage/ s many interconnecting spaces permitting easy passage of vessels. illuspage/icb/icb.htm Spatial Displacement of a bone spicule depository resorptive side side Direction of drift  Excessive Osteoclastic Resorption in Cancellous Bone Close-up of a horizontal strut showing evidence Low magnification of the interior of a vertebral body of osteoclastic resorption: surface scalloping in which excessive resorption is destroying vertical of multiple Howship’s (osteocytic) lacunae. struts in the cancellous bone. Learning Objective 7: Application Describe the process of bone remodeling, including participant cells and tissues, and explain its role in the formation of mature bone tissue in response to: local mechanical forces within a bone organ; bone growth; and bone healing. Ask yourself: 1. What type of force tends to lead to the deposition of cancellous tissue? Compact? 2. What is a cutting cone, and how does it relate to a Haversian system? 3. How does the blood supply reach the osteocytes of compact bone? Through which vessels in the tissue? Learning Objectives 8. Describe the four stages of bone healing, including the duration of each, the gross and tissue changes characterizing each stage, and the participant cells and intercellular signaling pathways characterizing each stage. Fracture Repair  Fracture Repair – Phase 1: Hematoma/Inflammation Stage Duration: Stage lasts 4 days Initial response Blood vessels rupture leading to hematoma formation Accumulation of blood between fractured ends leads to clotting and formation of hematoma Cell death (necrosis) Damaged osteocytes and marrow cells undergo cell death Activation of inflammation Cellular recruitment Immune cells are recruited Lymphatic vessel expansion Histology and Cell Biology 5th Edition, 2020 Vessels expand within bone Help with fluid balance and trafficking of cells to injury site Debris is cleared  Fracture Repair – Phase 1: Hematoma/Inflammation Stage Cellular response Influx of fibroblasts and progenitor cells Lymphatic vessels help with the recruitment of cells Inflammatory response Recruits immune cells (neutrophils, macrophages, etc) Activation of signaling pathways Cell proliferation – Interleukins and Growth Factors Histology and Cell Biology 5th Edition, 2020 Cell differentiation - BMPs  Fracture Repair – Phase 2: Soft Callus Stage Duration: State lasts 3-4 weeks Fibroblasts Proliferate and contribute to tissue formation Soft callus Cartilage intermediate Helps stabilize the fractured ends Chondroblasts Progenitors differentiate into chondroblasts through Sox 9 MSC Chondroblasts Sox9/5/6 Cell Invasion Histology and Cell Biology 5th Edition, 2020 Vascular endothelial cells invade the soft callus Recruited by VEGF Note: lymphatic vessels support the Blood vessels begin to regrow (re-vascularized) recruitment of cells (chondrocytes, MSCs, and etc)  Fracture Repair – Phase 3: Hard Callus Stage Duration: Begins 3-4 weeks after injury and continues for 2-3 months until union is attained Hard callus formation Soft callus gradually converted into woven bone Bone replaces calcified cartilage Lymphatic vessels Histology and Cell Biology 5th Edition, 2020 Maintain the environment (nutrients and waste)  Fracture Repair – Phase 3: Hard Callus Stage Bone forming periosteum Progenitors differentiate into osteoblasts High levels of osteoblast activity Runx2 upregulation MSC Osteoblasts Runx2 Active bone cellular components Osteoblasts Synthesize ECM with Type I collagen Activate osteoclasts Histology and Cell Biology 5th Edition, 2020 Osteoclasts/chondroclasts Remove cartilage intermediate Leads to final stage: REMODELLING  Fracture Repair – Phase 4: Remodeling Overlaps with hard callus stage May continue for several years Age is a factor: healing will occur much faster in children than adults. Active bone cellular components Osteoclasts remove excess bone Osteoblasts lay down new bone Histology and Cell Biology 5th Edition, 2020 Woven bone of hard callus is gradually converted to mature lamellar bone Learning Objective 8: Application Describe the four stages of bone healing, including the duration of each, the gross and tissue changes characterizing each stage, and the participant cells and intercellular signaling pathways characterizing each stage. Ask yourself: 1. How long does each stage of bone healing last? 2. At what point in bone healing is replacement bone present, but as woven, rather than secondary bone tissue? 3. At what stage of the bone healing process is chondrogenesis most involved? Learning Objectives 9. Using your anatomy atlas and any materials provided in course resources, identify the items named on the session structure list.