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Cellular Signalling

Cellular signaling involves receptors, which are essential for cellular and organ activity. These receptors are drug targets which upon binding elicit a biological response. These receptors show specificity, meaning that they have selective binding. Thy can only bind to ligands that show similar chemical bonding, considering both the composition and 3D orientation. However, no drug is entirely specific. They can partially bind to other receptors which is the cause of side effects. This is what’s called as affinity. It is how strongly a molecule binds to its target.

As mentioned before, a ligand is a molecule that binds to a receptor so it can perform its biological response. There exist different types of ligands. Agonists are ligands that binds to a receptor and triggers its biological response. Antagonists are ligands that binds to a receptor to prevent an agonist to bind to the receptor and blocks the biological response. There are 2 types of antagonists. The competitive antagonists bind to the same site as the agonist would, blocking that binding site, whereas the non-competitive antagonist binds to a different site and changes the conformation of the original site, preventing the agonist from binding. Antagonists can also be reversible or irreversible. Reversible means that they bind and detaches regularly from the receptor. This implies that an antagonist can be surmountable if there is a higher concentration of agonist present. However, irreversible means that the antagonists permanently bind to the receptor, forever blocking the effect of it. There exists another type of ligand called the inverse agonist. This ligand binds to receptors that have constitutive activation, meaning that they spontaneously are in they’re active state and produce a response without a ligand. Inverse agonists will reduce the biological response of this receptor, producing negation efficacy. Efficacy refers to the strength of the response. For agonists, there is also two types of them. Full agonists have efficacy, meaning that they will allow a strong response, whereas partial agonists have low efficacy, meaning that they will elicit a weaker response.

For cell-cell communication, there exists many types. Contact-dependent involve juxtracrine communication. The cells directly interact with each other. Paracrine signalling refers to the interaction with nearby cells through substances. Synaptic signalling refers to the signalling in neurons done by neurotransmitters. Endocrine signalling refers to the release of hormones in the blood which will binds to far away cells.

 


 

Extracellular Matrix

The matrix of the extracellular matrix (ECM) is composed of several molecules, with three main superfamilies being most prominent: glycosaminoglycans (GAGs), fibrous proteins, and non-collagen glycoproteins. GAGs promote water retention and often adhere to proteoglycans, contributing to the hydration of the ECM. Fibrous proteins, such as elastin and collagen, provide structural stability, strength, and organization. There are three types of collagen: fibrillar, fibril-associated, and network-forming collagen. Non-collagen glycoproteins include molecules such as fibronectin, laminin, growth factors, and tenascins, which play essential roles in the ECM's functionality.

Integrins, transmembrane heterodimers, act as adhesion molecules and receptors within the ECM. They mediate interactions between cells and the ECM through bidirectional signaling, guided by FAK (focal adhesion kinase) signaling, which influences cell motility, survival, proliferation, and differentiation. Upon binding to an RGD (arginine-glycine-aspartate) sequence on a molecule, integrins interact with actin filaments inside the cell, relaying signals to stabilize cell position, modulate migration, and promote growth and survival through focal adhesion formation.

The ECM engages in various signaling interactions. Chemical signaling involves ECM components interacting with signal molecules that regulate tissue development and patterning. Mechanical signaling occurs in response to mechanical forces like shear stress. Mechanotransduction converts mechanical forces into biochemical signals, adjusting ECM strength based on rigidity. In bidirectional signaling, cells remodel the ECM by pulling on it, affecting its structure and transmitting signals both ways.

ECM composition varies by tissue type. Soft tissues have a softer ECM with less collagen, while hard tissues, like bone, have abundant collagen and a stiff ECM. The quantity of ECM determines tissue stiffness, giving bones rigidity through mineralized collagen fibrils, arteries flexibility through elastic fibers, and blood fluidity due to the high water content in plasma.

The ECM is maintained by cells that produce and excrete proteins, balancing ECM regeneration and degradation. Cells also degrade the ECM for tissue repair and remodeling, using enzymes that break down ECM proteins into biologically active fragments (matricryptic peptides), which recruit progenitor cells and influence cell migration and differentiation.

Scientists mimic the ECM using hydrogels to create ex-vivo scaffolds. These synthetic hydrogels replicate the physical and biochemical properties of natural ECM proteins like collagen, laminin, and fibronectin.

Environmental conditions play crucial roles in ECM behavior. Acidic conditions accelerate ECM degradation and can cause bone demineralization. Basic conditions disrupt ion balance and inhibit matrix remodeling. Salt conditions can lead to electrolyte imbalances, disrupting cell signaling and matrix integrity. Buffer conditions maintain pH homeostasis, preventing acidosis or alkalosis-induced damage.


 

Stem Cells

Stem cells are unique because they have the ability to both proliferate and differentiate. The stem cell pool is maintained through asymmetrical division, where a stem cell divides into two daughter cells: one remains a stem cell, and the other differentiates. Stem cell niches, like Wnt signaling and transcription factors, help maintain self-renewal and allow differentiation. Additionally, the pool can expand through symmetrical division, where two daughter stem cells are produced. These cells can either remain stem cells or differentiate depending on environmental conditions, allowing tissues to adapt to changing circumstances.

A single stem cell can generate many different cell types thanks to transcription factors that cause changes in gene expression and activate signaling cascades. As stem cells differentiate, they move through various potency levels: totipotent (able to form whole organisms), pluripotent (able to form all germ layers), multipotent (able to form multiple cell types), oligopotent (able to form a few related cell types), unipotent (able to form a single cell type), and nullipotent (terminally differentiated cells).

Stem cells differentiate based on signaling molecules, growth factors, environmental factors, and signaling pathways, all of which determine the specific type of cell they become. However, stem cells also have limitations—if their proliferation becomes uncontrollable, they can cause cancer.

Connective tissues vary in their characteristics. Bone is rigid and provides structural strength, rigidity, and compressive strength. Cartilage is flexible and resilient, facilitating movement at joints. Adipose tissue provides shock absorption and energy reserves, while loose and dense tissues attach bone to muscles and form ligaments and tendons.

Bones are held together by hydroxyapatite (HA) and collagen. These components interact through hydrogen bonds (between HA’s OH group and collagen) and ionic bonds (between Ca2+ ions and collagen). Hydroxyapatite crystals have a hexagonal structure, and along with collagen, form a honeycomb pattern within the bone.

There are three types of crystals: ionic crystals (organized lattices formed by ionic bonds), molecular crystals (lattices formed by intermolecular forces between covalently bonded molecules), and atomic crystals (lattices formed by a network of covalent bonds).

Finally, silicates are compounds containing silicon and oxygen, oxide ceramics contain oxygen, and non-oxide ceramics do not contain oxygen.

 


 

Tissue Development

Bone composition of a child is composed of 4 parts: diaphysis (long part), metaphysis (right above the growth plate), growth plate (also called physis) which is cartilaginous, epiphysis (just below the growth plate) formed of transient cartilage (or hyaline cartilage). In an adult bone, only 2 parts can be seen: diaphysis and metaphysis. Here, the epiphysis has completely ossified, and the hyaline cartilage is much thinner.

Bone formation can be done in 4 different ways: embryological and fetal formation (the skeleton of an embryo is completely cartilage, and it ossifies at birth), bone growth, bone remodelling, and fracture healing. Bone formation, in all these ways, can be done in two forms:

1.      Intramembranous ossification (which takes place mainly in the skull and the clavicle). Here, there is a direct formation of bone from the mesenchymal cells. This forms flat bones, responsible for growth in axial skeleton and long bone diameter. Mesenchymal cells in the mesenchyme (a region of the brain) of the fetus will create an ossification center. These cells will differentiate in osteoblasts. Osteoblasts will secrete extracellular matrix, also called osteoid. As the osteoblast secrete its ECM, the matrix starts mineralizing, entrapping the osteoblasts which will differentiate into mature osteocytes. On the outer layer of the bone, the mesenchymal cells will start to condense and form a periosteum (membrane of blood vessels and nerves that wraps around most of your bones). These mesenchymal cells will then be replaced by osteoblasts. Simultaneously, the trabeculae (thin columns and plates of bone that create a spongy structure in a spongy bone -found in the epiphysis) forms. Blood vessels invade the holes found throughout the trabeculae. Finally, the outer layer, also called the periosteum, becomes compact bone.

2.      Endochondral ossification (takes place in long bones). In here, the cartilage templates are replaced by bone. During fetal development, mesenchymal cells cluster together and form a scaffold for bone. These cells differentiate in chondroblasts. These chondroblasts secrete an extracellular matrix which will form a hyaline cartilage model. Surrounding mesenchymal cells adhere and form the perichondrium (protection layer). The chondroblasts become encased in ECM and differentiate into chondrocytes. These chondrocytes start to divide causing interstitial (length) and appositional (diameter) growth. The central chondrocytes will start to hypertrophy and causes the surrounding ECM to calcify. Nutrients cannot travel through the calcify matrix which causes the death of the chondrocytes. This leaves open spaces where blood vessels will start invading. They will invade the diaphysis (where the primary ossification center is), but also the epiphysis (where the secondary ossification center is). This allows the cells in the perichondrium to differentiate into osteoblasts, creating the periosteum. Osteoblasts gradually replace the calcify cartilage and begin the secretion of osteoid (bone ECM). This simultaneously happens in the epiphysis as well. In the primary ossification center (the one in the middle of the diaphysis) osteoclasts will breakdown some of the central bone and create a medullar cavity where bone marrow will eventually start forming.

 

Intramembranous ossification:

1.       Mesenchymal cell aggregation: Mesenchymal cells in the fetal mesenchyme cluster to form an ossification center.

2.      Differentiation into osteoblasts: Mesenchymal cells differentiate into osteoblasts.

3.       Secretion of extracellular matrix (osteoid): Osteoblasts secrete extracellular matrix (osteoid), which starts mineralizing.

4.      Osteocytes formation: Osteoblasts become trapped in the mineralized matrix and mature into osteocytes.

5.      Periosteum formation: Mesenchymal cells condense on the bone surface and form the periosteum, a membrane of blood vessels and nerves.

6.      Osteoblast replacement: The mesenchymal cells in the periosteum are replaced by osteoblasts.

7.      Trabeculae formation: Trabeculae, or spongy bone, forms with gaps where blood vessels invade.

8.      Formation of compact bone: The outer layer of the bone (periosteum) becomes compact bone.

 

Endochondral ossification:

1.      Mesenchymal cell clustering: Mesenchymal cells cluster to form a scaffold for bone.

2.      Differentiation into chondroblasts: Mesenchymal cells differentiate into chondroblasts.

3.      Formation of hyaline cartilage model: Chondroblasts secrete extracellular matrix, forming a hyaline cartilage template.

4.      Perichondrium formation: Surrounding mesenchymal cells adhere to form the perichondrium, a protective layer.

5.      Chondrocyte differentiation: Chondroblasts get encased in ECM and differentiate into chondrocytes.

6.      Cartilage growth: Chondrocytes divide, causing interstitial (length) and appositional (diameter) growth.

7.      Hypertrophy and matrix calcification: Central chondrocytes enlarge, and the surrounding ECM calcifies.

8.      Chondrocyte death and space formation: The calcified matrix prevents nutrient flow, causing chondrocyte death and forming spaces.

9.      Blood vessel invasion: Blood vessels invade the diaphysis (primary ossification center) and the epiphysis (secondary ossification center).

10. Periosteum and osteoblast formation: Cells in the perichondrium differentiate into osteoblasts, forming the periosteum.

11.  Bone formation: Osteoblasts replace the calcified cartilage and secrete osteoid bone ECM), forming bone in both the diaphysis and epiphysis.

12. Medullary cavity formation: Osteoclasts break down the central bone in the diaphysis, forming the medullary cavity where bone marrow will develop.

 

Multiple signaling molecules are involved in regulating the growth plate activity. Oestrogen accelerates the frowth plate ossification by stimulating vascular and bone cell invasion. PthRP maintains endochondral growth plate at a constant width. IHH regulates proliferation and hypertrophy. Growth hormones stimulate chondrogenesis and longitudinal bone growth. Thyroid hormones stimulate clonal expension of chondrocytes progenitor cells and inhibits the subsequent cell proliferation while promoting hypertrophic chondrocyte differentiation.

*Know the structures of oestrogen, PthRP, IHH and Vitamin C

 

 


 

Cell Cycle

The cell cycle is composed of 2 main phases: the mitosis, where the cell divides, and the interphase, where the cell grows and duplicate its DNA. In the interphase, there are 3 subphases. The gap phases 1 and 2, and the S phase. During phase G1, the cells grow and the proteins and organelles are produced. In the S phase, the cells duplicate their DNA. In the G2 phase, the cell continues to grow.  There exists another phase called the quiescent state (G0) where most cells reside. In this state, the cells are not actively dividing but they are still doing their function.

The cell division is essential for proliferation (mitosis), reproduction (meiosis), growth and development and repair of tissues. To progress through the cell cycle, cyclin-dependent kinases (Cdks) play a crucial role. Indeed, they drive the cell cycle transitions. They are activated by cyclins that bind to the Cdks. Specific cyclins activate different part of the cell cycle. Cyclin E takes care of the transition to the S-phase. Cyclin A takes care of the progression through the G2 phase. Cyclin B takes care of the transition to the M-phase. Cyclin D takes care of the progression through all the cell cycle. Cyclin D is significant when the cells re-enter the mitotic cycle from the G0 state.

This cell cycle is heavily regulated. There are 2 checkpoints at the end of the growth phases to male sure that the environment has enough nutrient and that there is no damage of the cell to continue with the cycle. There are also many other types of regulators such as Cdk inhibitors, protein kinase, ubiquitin ligases and mitogens.

Mitogens (external growth factors) are especially important for cells to re-enter the cell cycle. Indeed, they stimulate mitosis by activating signaling pathways which promote cyclin D expression and enables progression through the G1 checkpoint. There are 2 types of mitogens. The mitogen activated protein kinase (MAPK) are enzymes that modify proteins and activate the gene expression pathway. The carbohydrate-binding protein don’t modify proteins and just activate the mitotic signal.

For a healthy life of an organism, cells need to divide but also to die. Cell death is therefore extremely important. There are 2 ways to achieve cell death: active and passive cell death. The active cell death is a programmed death call apoptosis. In this type, the cells die for the benefit of the organisms. They break down in a clean way without affecting the nearby cells. Apoptosis can happen through two pathways:
1. Intrinsic pathway: this pathway responds to internal damage and involves the mitochondria releasing signals to trigger death. Upon damage, the mitochondria will receive apoptotic stimuli. This organelle will release cytochrome c which will bind to inactive Apaf1 adaptor protein. This will allow the Apaf1 to expose the caspase activation and recruitment domains (CARD) and the oligomerization domain. The Apaf1 will then oligomerize and become a heptamer. Inactive caspase-9 monomers be recruited by the heptamer to form an apoptosome where the capsapase-9 will be activated by dimerization. The activation of executioner caspases will be done by activated caspase-9 dimers. They will then cause apoptosis.
Bcl2 can also cause an intracellular pathway. Bcl2 governs the release of cytochrome c and other proteins in the cytosol by a process called MOMP (mitochondrial outer membrane permeabilization). There are 3 families of Bcl2: anti-apoptotic proteins (Bcl2 and BclXL) which inhibits apoptosis, pro-apoptotic effectors (Bak and Bax) which activates apoptosis, pro-apoptotic BH3-only proteins which either inhibit anti-apoptotic proteins (Bad) or activate oligomerization directly (Bim and Bid).
2. Extrinsic pathway: responds to external signals (death signals) coming from external molecules. Killer lymphocytes have a Fas ligand on their plasma membrane. This ligand binds to the inactive Fas death receptor on the target cell plasma membrane and activates it. This will release the FADD adaptor protein from the death domain (receptor’s domain inside the cell) and expose its death effector domain (DED). This will bind to an inactive caspase-8 monomer and activate it by dimerization. A DISC (death-induced signaling complex) assembly happens simultaneously. Capsase-8 will be cross-cleaved leading to a subunit rearrangement. This will release mature activated caspase-8 dimers into the cytosol. These proteins will activate by cleavage of executioner caspases 3 and 7 which will trigger apoptosis.
Passive cell-death, also called necrosis, is an injured-induced cell death that can lead to inflammation and is generally harmful. The cell will burst and lose its nucleus.

 

Intrinsic Pathway

  Apoptotic Stimuli: Internal cell damage triggers apoptotic signals in the mitochondria.

  Cytochrome c Release: Mitochondria release cytochrome c into the cytosol.

  Apaf1 Activation:

  • Cytochrome c binds to inactive Apaf1.

  • Apaf1 exposes its CARD (caspase activation and recruitment domain) and oligomerization domain.

  Apaf1 Oligomerization:

  • Apaf1 oligomerizes to form a heptamer.

  Apoptosome Formation:

  • Inactive caspase-9 monomers are recruited by the Apaf1 heptamer.

  • Caspase-9 is activated through dimerization, forming an apoptosome.

  Executioner Caspase Activation:

  • Activated caspase-9 dimers trigger the executioner caspases, leading to apoptosis.

  Bcl2 Family Role:

  • Bcl2 proteins regulate the release of cytochrome c via MOMP (mitochondrial outer membrane permeabilization).

  • Three Bcl2 families:

    • Anti-apoptotic proteins (Bcl2, BclXL) inhibit apoptosis.

    • Pro-apoptotic effectors (Bak, Bax) activate apoptosis.

    • Pro-apoptotic BH3-only proteins (Bad, Bim, Bid) either inhibit anti-apoptotic proteins or activate oligomerization directly.

 

Extrinsic pathway:

  External Death Signal: A killer lymphocyte presents a Fas ligand on its plasma membrane.

  Fas Receptor Activation:

  • The Fas ligand binds to the inactive Fas death receptor on the target cell’s plasma membrane, activating the receptor.

  FADD Protein Release:

  • The activated Fas receptor releases the FADD adaptor protein from its death domain (inside the cell).

  • The FADD protein exposes its death effector domain (DED).

  Caspase-8 Activation:

  • FADD binds to inactive caspase-8 monomers, activating them through dimerization.

  DISC Formation:

  • A DISC (death-induced signaling complex) assembles at the same time.

  Caspase-8 Cleavage:

  • Caspase-8 undergoes cross-cleavage, causing subunit rearrangement.

  • Mature, activated caspase-8 dimers are released into the cytosol.

  Executioner Caspase Activation:

  • Caspase-8 cleaves and activates executioner caspases 3 and 7, which trigger apoptosis.

 

Organisation of Tissues

Tissues have a particular organisation that determines their properties. 3 aspects come into play. First, the localisation and the concentration of the ECM molecules play an important role. Indeed, in cartilage, collagen is localized adjacent to the chondrocytes which makes signaling possible between the ECM and the cell. Therefore, there is a high concentration of ECM molecules. In bone, collagen is localized in the nucleation sites which allows the minerals to deposits withing and around it. The bone, contrary to the cartilage, contains low concentration of ECM molecules. Secondly, the molecular architecture determines the mechanical properties of the tissue. The architecture of cartilage tissue allows it to withstand high pressure while having low friction. They have high GAG concentration which allows more compressive force, and the parallel formation of elastin fibers allows low friction. The bridge like formation of collagen fibers allows a resistance to high pressure. All these gives cartilage the ability to absorb shock and allow pain free movements. Bone on the other hand has a plywood-like structure which allows a distribution of force to bear the load. The perpendicular woven bone layers give strength, and the arcade-like structure of trabecular bone provides lightweight strength and shock absorption. Finally, the molecular modification of ECM molecules, like cross-linking, allows the formation of covalent interactions and makes molecules more resilient.

Multiple diseases can come from a disbalance in the tissue organisation. Osteoporosis is caused by a disbalance between bone formation and bone resorption. The rate of disintegration is higher than the one of formation. This causes the bone to become more fragile and therefore more prone to breakage. Osteogenesis imperfecta is a genetic disease that affect the crosslinking of collagen network leading to weaker and more fragile bone. Osteoarthritis is caused the degradation of the collagen network and a loss of GAGs. This leads to a thinner hyaline cartilage, reducing flexibility of movement, increasing friction and pain.

Glycosaminoglycans have many properties. The most important ones are viscosity, lubrication, low compressibility, structural support, protein interaction and they are negatively charged. This contributes to their functions for cell signalling, development and proliferation, anti-coagulation, shock absorption and water retaining.

There exist 5 types of glycosaminoglycans, each having different functions. Heparan sulphate (heparin) have two conformations (stereochemistry) and they control blood coagulation. Their structure allows binding to leukocytes. Keratan sulphate is the only GAG containing a galactose ring instead of an acid, giving it hydration and signaling agent functions. Chondroitin sulphate is important in cell adhesion, growth, migration and receptor binding due to its high negative charge. Dermatan sulphate plays a role in wound repair and blood coagulation regulation. Hyaluronic acid has strong hydrophilic properties playing a crucial role in water retention.