Connective Tissue Histology PDF

H::Connec(veTissue Connective Tissue and Extracellular Matrix Connective tissue contains interstitial fluid that provides metabolic support for cells and facilitates the diffusion of nutrients and waste products. Unlike epithelium, muscle, and nerve tissues, connective...

H::Connec(veTissue Connective Tissue and Extracellular Matrix Connective tissue contains interstitial fluid that provides metabolic support for cells and facilitates the diffusion of nutrients and waste products. Unlike epithelium, muscle, and nerve tissues, connective tissue primarily consists of extracellular matrix (ECM), not cells. The ECM is composed of protein fibers (such as collagen and elastic fibers) and ground substance. Ground substance includes anionic, hydrophilic o proteoglycans, o glycosaminoglycans (GAGs), and o multiadhesive glycoproteins (like laminin and fibronectin). § These glycoproteins stabilize the ECM by binding to other matrix components and to integrins in cell membranes. Water in the ground substance enables the exchange of nutrients and metabolic wastes between cells and blood supply. Embryonic Origin of Connective Tissue All connective tissues originate from embryonic mesenchyme, derived mainly from the mesoderm layer of the embryo. Mesenchyme is characterized by viscous ground substance with few collagen fibers. Mesenchymal cells are undifferentiated, with large nuclei, prominentnucleoli, and fine chromatin. (-> high level of synthetic activity) o These cells are often described as “spindle-shaped” due to their scant cytoplasm extending into thin processes. Mesodermal cells migrate in the embryo, surrounding and penetrating developing organs. Embryonic mesenchyme produces not only connective tissue proper but also specialized connective tissues like bone and cartilage. Embryonic mesenchyme also contains stem cells for other tissues such as blood, vascular endothelium, and muscle. Cells of Connective Tissue Fibroblasts are the primary cells in connective tissue proper, originating locally from mesenchymal cells. o They are permanent residents of connective tissue. Other cells like macrophages, plasma cells, and mast cells originate from hematopoietic stem cells in the bone marrow. o These cells circulate in the blood and move into connective tissue to perform their functions.  White blood cells (leukocytes) are considered transient cells in connective tissues, performing various functions temporarily and then undergoing apoptosis. Fibroblasts in Connective Tissue Fibroblasts are the most common cells in connective tissue proper. Fibroblasts produce and maintain most of the tissue's extracellular components. Fibroblasts synthesize and secrete collagen, the body's most abundant protein, and elastin, forming large fibers. Fibroblasts also produce glycosaminoglycans (GAGs), proteoglycans, and multiadhesive glycoproteins for the ground substance. Most of the ECM components secreted by fibroblasts undergo further modification outside the cell before assembling into a matrix. Fibroblast Activity Levels Histologically, there are distinct levels of fibroblast activity. Active fibroblasts differ morphologically from quiescent fibroblasts. The term “fibroblast” is used for the active cell, while “fibrocyte” refers to the quiescent cell. Active fibroblasts have abundant, irregularly branched cytoplasm, rough endoplasmic reticulum (RER), a well-developed Golgi apparatus, a large, ovoid euchromatic nucleus, and a prominent nucleolus. Quiescent fibrocytes are smaller, usually spindle-shaped with fewer processes, have less RER, and a darker, more heterochromatic nucleus. Fibroblast Growth and Differentiation Fibroblasts are influenced by growth factors that affect their growth and differentiation. In adults, fibroblasts rarely divide, but can resume cell cycling and mitotic activity when stimulated by growth factors. This stimulation often occurs in response to a need for more fibroblasts, such as during organ repair. Myofibroblasts, involved in wound healing, have contractile functionsand contain a form of actin found in smooth muscle cells. Adipocytes in Connective Tissue Adipocytes (fat cells) are found in the connective tissue of many organs. These large cells, derived from mesenchyme, specialize in storing lipid as neutral fats or in producing heat. Adipose connective tissue, rich in adipocytes, provides cushioning and insulation to the skin and other organs. Adipocytes have significant metabolic importance. Macrophages and the Mononuclear Phagocyte System Macrophages have a strong phagocytic ability, essential for turnover of protein fibers and removal of apoptotic cells, tissue debris, and particulate material. Macrophages are particularly abundant at sites of inflammation. The size and shape of macrophages vary with their functional activity, typically measuring between 10 and 30 μm in diameter. Macrophages often have an eccentrically located, oval or kidney*-shaped nucleus. Macrophages are found in the connective tissue of most organs and are sometimes called “histiocytes” by pathologists. Under a transmission electron microscope (TEM), macrophages display an irregular surface with pleats and protrusions, indicating their active pinocytotic and phagocytic activities. Macrophages usually have well- developed Golgi complexes and many lysosomes. Macrophages and Mononuclear Phagocyte System Macrophages originate from monocytes, precursor cells circulating in the blood. Monocytes cross the epithelial wall of small venules to enter connective tissue, where they differentiate and mature into macrophages. During early embryonic development, monocytes formed in the yolksac circulate and become resident in developing organs, forming the mononuclear phagocyte system. This system includes macrophage-like cells with specialized functions in various organs, often with specific names. Macrophages are long-living, remaining relatively inactive in tissues for months or years. During inflammation and tissue repair, macrophages become activated and increase in number in the connective tissue stroma. The increase in number of macrophages is due to both proliferation and the recruitment of additional monocytes from the bone marrow. Transformation from Monocytes to Macrophages The transformation of monocytes into macrophages in connective tissue involves increases in cell size and protein synthesis. There is also an increase in the number of Golgi complexes and lysosomes in these transforming cells. Besides debris removal, macrophages secrete growth factors crucial for tissue repair. Macrophages also play a role in the uptake, processing, and presentation of antigens for the activation of (T) lymphocytes, a process integral to the immune system. Mast Cells in Connective Tissue Mast cells are oval or irregularly shaped cells in connective tissue, measuring between 7 and 20 μm in diameter. They are filled with basophilic secretory granules that can obscure the central nucleus. Mast cell granules are electron dense, vary in size from 0.3 to 2.0 μm, and contain high levels of acidic radicals in their sulfated glycosaminoglycans (GAGs). Mast cell granules exhibit metachromasia, meaning they change the colorof some basic dyes (like toluidine blue) from blue to purple or red. This staining feature is similar to basophils but mast cells are agranulocytes. It is common among very acidic substances like GAGs such as heparin. Due to the nature of their granules, mast cells may be difficult to identifyin slides prepared with common fixatives. Mast Cell Functions: Bioactive Substance Release Mast cells are crucial for localized release of bioactive substances in local inflammatory response, innate immunity, and tissue repair. Mast cells release molecules from their secretory granules, including heparin, histamine, serine proteases, eosinophil and neutrophil chemotactic factors, cytokines, and phospholipid precursors (prostaglandins, leukotrienes). Specific Substances Released by Mast Cells Heparin is a sulfated glycosaminoglycan (GAG) acting as a local anticoagulant. Histamine promotes increased vascular permeability and smooth muscle contraction. Serine proteases are enzymes that activate various mediators of inflammation. Eosinophil and neutrophil chemotactic factors attract those specific types of leukocytes. Cytokines are polypeptides directing the activities of leukocytes and other immune system cells. Phospholipid precursors are converted to prostaglandins, leukotrienes, and other lipid mediators of the inflammatory response. Mast Cell Distribution and Function Mast cells are found in the connective tissue of many organs, especially: o near small blood vessels in skin and mesenteries (perivascularmast cells), and o in the tissue lining digestive and respiratory tracts (mucosal mast cells). The granule content of perivascular and mucosal mast cells differs slightly.  These locations suggest mast cells function as sentinels for detecting microorganism invasion. Mast Cells in Allergic Reactions The release of chemical mediators from mast cells promotes allergic reactions, known as immediate hypersensitivity reactions, occurring within minutes after antigen exposure in a previously sensitized individual. A dramatic example of immediate hypersensitivity reaction is anaphylactic shock, a potentially fatal condition. Anaphylaxis: Sequence of Events Anaphylaxis starts with the first exposure to an allergen (e.g., bee venom), leading to the production of IgE antibodies by antibody-producing cells. These IgE antibodies bind avidly to receptors on mast cells. Upon a second exposure to the allergen, it reacts with the IgE on mastcells, triggering the rapid release of histamine, leukotrienes, chemokines, and heparin. This release leads to the sudden onset of the allergic reaction. Degranulation of mast cells also occurs due to the action of complement molecules involved in immunologic reactions. Mast Cells: Origin and Differentiation Mast cells originate from progenitor cells in the bone marrow. These cells circulate in the blood, cross the wall of small vessels (venules), and enter connective tissues where they differentiate to become fully functional mature mast cells. While similar to basophilic leukocytes in many aspects, mast cells have a different lineage, especially in humans. Plasma Cells: Characteristics and Lifespan Plasma cells are lymphocyte-derived, antibody-producing cells. Plasma cells are large, ovoid cells with basophilic cytoplasm, rich in rough endoplasmic reticulum (RER), and a large Golgi apparatus near the nucleus. The nucleus of plasma cells is usually spherical but eccentrically placed, with regions of heterochromatin and euchromatin. Plasma cells are found in most connective tissues, with an average lifespan of 10-20 days. Leukocytes in Connective Tissue and Inflammation Leukocytes (other white blood cells, besides* the aforementioned macrophages and plasma cells) are part of a population of wandering cells in connective tissue. Derived from circulating blood cells, they leave the blood by migratingbetween endothelial cells of venules to enter connective tissue, a process that increases during inflammation. Inflammation is a defensive response to injury or foreign substances, characterized by increased blood flow, vascular permeability, leukocyte entry and migration, and macrophage activation for phagocytosis. Inflammation begins with the release of chemical mediators from cells, the extracellular matrix (ECM), and blood plasma proteins. Leukocyte Lifespan and Function in Immune Response Most leukocytes function in connective tissue for only a few hours or days before undergoing apoptosis there. However, some lymphocytes and phagocytic antigen-presenting cellsleave the interstitial fluid of connective tissue, entering the blood or lymph and moving to selected lymphoid organs as part of the immune response. Fibrous Components of Connective Tissue Connective tissue contains fibrous components, which are elongated structures formed from proteins that polymerize after secretion from fibroblasts. The three main types of fibers are collagen, reticular, and elastic fibers. Collagen and reticular* fibers are formed by proteins of the collagenfamily, while elastic fibers are mainly composed of elastin. These fibers are distributed unequally among different connective tissues, with the predominant fiber type determining most specific tissue properties. Collagen in Connective Tissue Collagens are proteins evolved for forming extracellular fibers, sheets, and networks, which are extremely strong and resistant to shearing and tearing forces. Collagen is a key element in all connective tissues, as well as in epithelial basement membranes and the external laminae of muscle and nervecells. Collagen: Abundance and Types Collagen is the most abundant protein in the human body, accounting for 30% of its dry weight. It is a major product of fibroblasts but is also secreted by several other cell types.  Collagens are distinguishable by their molecular compositions, morphologic characteristics, distribution, functions, and pathologies. There are 28 collagens in vertebrates, numbered in the order of their identification, with the most important ones listed in Table 5–3. They can be categorized by the structures formed by their α-chains subunits Fibrillar Collagens Fibrillar collagens*, notably types I, II, and III, have polypeptide subunitsthat aggregate to form large fibrils visible in electron or light microscopy. Collagen type I is the most abundant and widely distributed, forming large, eosinophilic bundles typically called collagen fibers. Fibrillar collagens densely fill connective tissue, forming structures such as tendons, organ capsules, and the dermis. Network or Sheet-Forming Collagens Network or sheet-forming* collagens, such as type IV collagen, have subunits produced by epithelial cells. They are major structural proteins of external laminae and all epithelial basal laminae. Linking/Anchoring Collagens Linking/anchoring collagens are short collagens that link fibrillar collagens to each other and to other components of the extracellular matrix (ECM). Type VII collagen (linking) binds to type IV collagen (sheet) of the basal lamina and anchors it to the underlying reticular lamina (of fibrilar type 3 collagen) in basement membranes. Collagen Synthesis Process Collagen synthesis primarily occurs in fibroblasts. The initial procollagen α chains are polypeptides made in the rough endoplasmic reticulum (RER). o Different α chains of various lengths and sequences are synthesized from collagen genes. In the ER, three α chains are selected, aligned, and stabilized by disulfide bonds at their carboxyl terminals and folded into a triple helix. The triple helix undergoes exocytosis, is cleaved to a rodlike procollagen molecule, which is the basic subunit for assembling fibers or sheets. o These subunits can be homotrimeric (all three chains identical) or heterotrimeric (two or all three chains having different sequences). Different combinations of procollagen α chains produce various types of collagen with different structures and functional properties. Posttranslational Processing of Collagen Collagen undergoes numerous posttranslational processing stepsbefore its final assembly in the extracellular matrix (ECM). These steps have been most extensively studied for type I collagen, which constitutes 90% of the body's collagen. Other fibrillar and sheetlike collagens are formed through processes similar to those of collagen type I and are stabilized by linking or anchoring collagens. The complexity of collagen biosynthesis means there are many points where the process can be disrupted by defective enzymes or disease processes. 1. Procollagen α Chain Production and Modification 1. Synthesis on Polyribosomes: Procollagen α chains are synthesized on polyribosomes* of the rough endoplasmic reticulum (RER) and translocated into its cisternae, featuring long central domains rich in prolines and lysines. 2. Hydroxylation of Residues: Prolyl & Lysyl Hydroxylase enzymes in the ER cisternae add hydroxyl (-OH) groups to some prolines and lysines, requiring O2, Fe2+, and ascorbic acid (vitamin C) as cofactors. 3. Glycosylation Process*: Involves attaching galactosyl and glucosyl sugars to specific hydroxylysyl residues, varying across collagen types. *via Glycosyltransferases 2. Collagen Formation and Transport 1. Triple Helix Assembly: The C-terminal regions of selected α chains (α1, α2) are stabilized by cysteine disulfide bonds, which facilitate proper alignment of the polypeptides, enabling the central domains to properly fold into a triple helix structure. 2. Transport and Packaging: Procollagen, with globular terminal sequences, is transported through the Golgi apparatus and packaged in secretory vesicles. 3. Collagen - Extracellular Processing and Fibril Formation 1. Exocytosis to Extracellular Space: Microtubules and microfilaments assist in transporting vesicles to the cell surface for procollagen exocytosis. 2. Procollagen to Collagen Conversion: Procollagen peptidases remove terminal globular peptides, once in the extracellular space, transforming procollagen into insoluble collagen molecules that aggregate into fibrils. 3. Fibril Stabilization and Reinforcement: Proteoglycans and other collagens (e.g., types 5 and 12) associate with new collagen fibrils, stabilizing them and promoting larger fiber formation.  4. Covalent Cross-Link Formation: Lysyl oxidase catalyzes the formation of covalent cross-links between collagen molecules, reinforcing fibrillar structure. Type I Collagen Fibrils Type I collagen fibrils have diameters ranging from 20 to 90 nm and can extend several micrometers in length. Adjacent rodlike collagen subunits in the fibrils are staggered by 67 nm, creating small gaps (lacunar regions) between their ends. o This structure results in a characteristic feature visible by electron microscopy (EM): transverse striations with a regular periodicity. Type I collagen fibrils assemble to form large, extremely strong collagen fibers, which may be bundled further by linking collagens and proteoglycans. Type II collagen (found in cartilage) forms fibrils but does not form fibersor bundles. Sheet-forming collagen type IV subunits assemble into a latticelike network in epithelial basal laminae. Collagen Appearance in ECM and Microscopy When collagen fills the extracellular matrix (ECM), as in tendons or the sclera of the eye, bundles of collagen appear white. The regular orientation of subunits makes collagen fibers birefringent under polarizing microscopy. In routine light microscopy, collagen fibers are acidophilic, staining pink with eosin, blue with Mallory trichrome stain, and red with Sirius red. Read maybe once: Due to their long and tortuous nature, collagen bundles' length and diameter are better studied in spread preparations rather than sections. o A small mesentery is often used for this purpose; when spread on a slide, it is thin enough to allow light passage and can be stained and examined under a microscope. Collagen Turnover and Renewal Collagen turnover and renewal in connective tissue is generally a slow but ongoing process. In organs like tendons and ligaments, collagen is very stable, while in others, such as the periodontal ligament surrounding teeth, the turnover rate is high. For renewal, collagen must first be degraded. Degradation is initiated by collagenases, which are part of the matrix metalloproteinases (MMPs) class. MMPs clip collagen fibrils or sheets, making them susceptible to further degradation by nonspecific proteases.  Various MMP are secreted by macrophages and play a crucial role in remodeling the ECM during tissue repair. Reticular Fibers in Connective Tissue Composition: o They consist mainly of collagen type III, forming an extensive network (Reticular) of thin fibers (diameter 0.5-2 μm) for supporting various cells. o Reticular fibers contain up to 10% carbohydrate, compared to 1% in most other collagen fibers. Function / Locations: o Reticular fibers are found in delicate connective tissue of many organs, particularly in the immune system. o Produced by fibroblasts, reticular fibers occur in the reticular lamina of basement membranes and typically surround adipocytes, smooth muscle, nerve fibers, and small blood vessels. o Reticular fibers provide supportive stroma for parenchymal secretory cells and microvasculature in the liver and endocrine glands. o Hemopoietic tissue (bone marrow), as well as the spleen, and lymph nodes are also characterized by abundant reticular fibers, supporting proliferating and phagocytic cells. Staining: o Reticular fibers are typically not visible in hematoxylin and eosin (H&E)* preparations but stain black with silver salts, making them argyrophilic (silver-loving). o Reticular fibers are also periodic acid–Schiff (PAS) positive, reflecting their high content of sugar chains bound to type III collagen α chains. *Extra info on staining: Hematoxylin = stains acidic (or basophilic) structures - blue/purple Eosin = stains basic (or acidophilic) structures - pink/red Elastic Fibers in Connective Tissue Elastic fibers are thinner than type I collagen fibers and form sparsenetworks interspersed with collagen bundles in many organs. These organs are typically those subject to regular stretching or bending, such as the stroma of the lungs. Elastic fibers have rubberlike properties, allowing tissues to stretch or distend and return to their original shape. In the walls of large blood vessels, especially arteries, elastin forms fenestrated sheets known as elastic lamellae. Staining: o Elastic fibers and lamellae are not strongly acidophilic and stain poorly with H&E.  o They stain more darkly than collagen with stains like orcein and aldehyde fuchsin. Composition and Formation of Elastic Fibers Elastic fibers and lamellae are composed of fibrillin (350 kDa) and elastin (60 kDa). Fibrillin forms a network of microfibrils embedded in a larger mass of cross- linked elastin. These proteins are secreted by fibroblasts, and by smooth muscle cellsin vascular walls. The formation of elastic fibers occurs in a stepwise manner: o Microfibrils with diameters of 10 nm form from fibrillin and various glycoproteins, serving as scaffolding for elastin deposition. o Elastin accumulates around the microfibrils and eventually makes up most of the elastic fiber, imparting the rubberlike property to these fibers. Elastic Properties of Fibers and Lamellae The elastic properties of fibers and lamellae arise from the structure of elastin subunits and their unique cross-links. Elastin molecules contain mainly: o lysine-rich regions (for cross-linking) interspersed with... o hydrophobic domains, rich in glysine* and proline, desmosine and isodesmosine, which form extensible random-coils. Formation: o During deposition of elastin on fibrillin microfibrils, lysyloxidase converts the amino groups of lysines to aldehydes. o Four oxidized lysines on neighboring elastin molecules condense covalently to form a desmosine ring, cross-linking the polypeptides. Characteristics: o Elastic fibers, firmly bound by many desmosine rings but maintaining the rubberlike properties of their hydrophobic domains, can stretch reversibly when force is applied. o Elastin is resistant to digestion by most proteases but can be hydrolyzed by pancreatic elastase. Ground Substance of ECM The ground substance of the extracellular matrix (ECM) is a highly hydrated, transparent, complex mixture of glycosaminoglycans (GAGs), proteoglycans, and multiadhesive glycoproteins. It fills the space between cells and fibers in connective tissue, facilitating the diffusion of small molecules. Due to its viscosity, ground substance acts as both a lubricant and a barrier against invaders. The physical properties of ground substance significantly influence cellular activities.  When prepared for histologic analysis, componentse of ground substanceaggregate as fine, poorly resolved material, appearing in TEM preparations as electron-dense filaments or granules. Glycosaminoglycans (GAGs) in ECM Glycosaminoglycans (GAGs), also known as mucopolysaccharides, are long polymers composed of repeating disaccharide units. These disaccharides usually consist of a hexosamine (glucosamine or galactosamine) and an uronic (aka hexuronic) acid (glucuronate or iduronate). The largest and most ubiquitous GAG is hyaluronan (also known as hyaluronic acid or hyaluronate). Hyaluronan has a molecular weight ranging from hundreds to thousands of kDa and is a very long polymer of the disaccharide glucosamine- glucuronate. Glycosaminoglycans (GAGs) in ECM Unlike other GAGs, hyaluronan is synthesized directly into the ECM by hyaluronan synthase, an enzyme complex in the cell membrane. Hyaluronan forms a viscous, pericellular network that binds a significant amount of water, playing a crucial role in allowing molecular diffusionthrough connective tissue as well as lubrication of various organs and joints. Characteristics of Other Glycosaminoglycans in ECM All other GAGs besides hyaluronan are much smaller, with molecular weights of 10-40 kDa, and are sulfated. They are bound to proteins as parts of proteoglycans and are synthesized in Golgi complexes. The four major GAGs in proteoglycans are dermatan sulfate, chondroitin sulfate, keratan sulfate, and heparan sulfate. Each of these GAGs has different disaccharide units, further modified with carboxyl and sulfate groups, and varying tissue distributions. GAGs’ high negative charge extends their conformation and allows them to sequester/bind cations and water. These water-binding characteristics endow GAGs with space- filling, cushioning, and lubricant functions. Proteoglycans in ECM Proteoglycans are composed of a core protein with various numbers and combinations of sulfated GAGs covalently attached.  They are synthesized on the rough endoplasmic reticulum (RER), mature in the Golgi apparatus (where GAG side chains are added), and are secreted from cells by exocytosis. Proteoglycans differ from glycoproteins in that their attached GAGs often constitute a greater mass than the core polypeptide. After secretion, proteoglycans bind to hyaluronan via link proteins, and their GAG side-chains can associate further with collagen fibers and other ECM components. Diversity of Proteoglycans in ECM Proteoglycans are diverse, partly due to enzymatic variations in Golgi complexes. A region of the ECM may contain several different core proteins, each with one or many sulfated GAGs of various lengths and compositions. Perlecan is a key proteoglycan in all basal laminae. Aggrecan, one of the best-studied proteoglycans, is very large (250 kDa) with a core protein heavily bound with chondroitin and keratan sulfate chains. Aggrecan binds to hyaluronan via a link protein. o Abundant in cartilage, aggrecan–hyaluronan complexes fill the space between collagen fibers and cells, significantly contributing to the physical properties of cartilage. Other proteoglycans include: o Decorin, with few GAG side chains binding to type I collagen fibrils (-> collagen fibril formation), and o Syndecan, with an integral membrane core protein that attaches ECMto cell membranes. Embryonic Mesenchyme and Proteoglycans Embryonic mesenchyme is characterized by high levels of hyaluronan and water, leading to wide spacing between cells and a matrix conducive to cell migrations and growth. In both developing and mature connective tissues, core proteins and GAGs (particularly heparan sulfate) of many proteoglycans bind and sequester various growth factors and signaling proteins. During the early phase of tissue repair, the degradation of these proteoglycans releases stored growth factors, which then stimulate new cell growth and ECM synthesis. Multiadhesive Glycoproteins in Ground Substance Multiadhesive glycoproteins make up the third major class of macromolecules in the ground substance of the ECM. Multiadhesive Glycoproteins feature multiple binding sites for cell surface integrins and other matrix macromolecules. These glycoproteins are large molecules with branched oligosaccharide chains, facilitating the adhesion of cells to their substrate.  An example is laminin, a large (200-400 kDa) trimeric glycoprotein with binding sites for integrins, type IV collagen, and specific proteoglycans, aiding in cell adhesion. Laminin is abundant in all basal and external laminae, playing a crucial role in the assembly and maintenance of these structures. Fibronectin and Integrins in ECM Fibronectin is a 235-270 kDa dimer, predominantly synthesized by fibroblasts. Fibronectin has binding sites for collagens and certain GAGs, forming insoluble fibrillar networks throughout connective tissue. Fibronectin provides specific binding sites for integrins, playing a crucial role in cell adhesion and cellular migration through the ECM. Integrins are integral membrane proteins that act as membrane receptorsfor specific sequences on ECM glycoproteins like laminin, fibronectin, some collagens, and others. Integrins bind their ECM ligands with relatively low affinity, enabling cells to explore their environment while maintaining attachment. Integrins are heterodimers consisting of two transmembrane polypeptides: the α and β chains. The diversity in integrin α and β chains allows cells to bind different specific ECM ligands. Focal Adhesions in Mesenchymal Cells Integrin- microfilament complexes in fibroblasts and mesenchymal cellsform focal ad hesions. o Focal adhesions can be observed via transmission electron microscopy (TEM) or immunocytochemistry. Focal adhesions are typically present at the ends of actin filaments, bundled by α-actinin. Cytoplasmic stress fibers and focal adhesion kinases in these adhesions allow cells to respond to pulling forces or other physical properties of the ECM's by altering cellular activities. Interstitial Fluid in Connective Tissue Interstitial fluid in connective tissue, also known as ground substance water, resembles blood plasma in ion composition. It contains plasma proteins of low molecular weight from capillaries. Despite being a small proportion of connective tissue proteins, up to one- third of the body’s plasma proteins are in connective tissue interstitial fluid due to its large volume and distribution. Capillary Functions and Pressures in Connective Tissue Capillaries in connective tissue deliver nutrients to cells and remove metabolic waste to the liver and kidneys. Interstitial fluid acts as a solvent for nutrients and waste products. Two main forces influence water movement in capillaries: o Hydrostatic pressure from the heart's pumping forces water out of capillaries. o Colloid osmotic pressure, mainly due to plasma proteins like albumin, draws water back into capillaries. Colloid osmotic pressure, caused by blood proteins that cannot pass through capillary walls, counters the water outflow caused by hydrostatic. Osmotic pressures from ions and low-molecular-weight compounds are nearly equal inside and outside capillaries, thus having a negligible net effect. Lymphatic Capillaries in Connective Tissue Water reabsorption into capillaries is often less than the amount forced out. Excess fluid in connective tissue drains into lymphatic capillaries. Lymphatic capillaries return this excess fluid to the blood. These capillaries originate in connective tissue as delicate endothelialtubes. Types of Connective Tissue Connective tissue has graded variations in structure due to different cell, fiber, and extracellular matrix (ECM) combinations. Classifications of connective tissue are based on structural characteristics or major components. Connective Tissue Proper Classification Connective tissue proper is classified as either loose or dense. Classification depends on the amount of collagen present. o Less collagen: Loose connective tissue o More collagen: Dense connective tissue Loose Connective Tissue Characteristics Also known as areolar tissue. Contains cells, fibers, and ground substance in roughly equalproportions. Fibroblasts are the most common cells, but other connective tissue cells, nerves, and small blood vessels are present. Collagen fibers are predominant, with elastic and reticular fibers also present. Loose Connective Tissue has a moderate amount of ground substance, giving it a delicate, flexible consistency that is not highly stress-resistant.  Loose Connective Tissue widespread, underlining epithelial layers and filling spaces in muscles and nerves. Dense Connective Tissue Composition Dense connective tissue has similar components to loose connective tissue. It contains fewer cells, predominantly also fibroblasts. There is a clear predominance of bundled type I collagen fibers over ground substance. The abundance of collagen provides protection and structural strength to organs. Dense Irregular Connective Tissue Dense irregular connective tissue has randomly interwoven collagen bundles. The collagen network is three-dimensional, offering resistance to multi- directional stress. Examples include the deep/reticular dermis of skin and capsules surrounding organs. Dense irregular tissue often grades into loose connective tissue, making clear distinctions challenging. Dense Regular Connective Tissue Dense regular connective tissue primarily consists of parallel- aligned type I collagen bundles and fibroblasts. It is designed for resistance to prolonged or repeated stresses from the same direction. Dense Regular Connective Tissue Examples Dense regular connective tissue is exemplified in tendons, aponeuroses, and ligaments. Tendons are strong, flexible cords connecting muscles to bones. Aponeuroses are sheetlike tendons. Ligaments are bands or sheets that stabilize components of the skeletal system. Consists almost entirely of densely packed parallel collagen fibers with little ground substance and few blood vessels. Appears white in the fresh state due to densely packed collagen. Fibrocytes in Dense Regular Connective Tissue Fibrocytes have elongated nuclei and lie parallel to collagen fibers. Their cytoplasmic folds envelop collagen bundles and they maintain the tissue matrix. In tendons, referred to as “tendinocytes”.  Cytoplasm is sparse and has similar acidophilia to collagen, making it hard to distinguish in H&E-stained preparations. In aponeuroses, collagen bundles form multiple layers at 90° angles to each other. Some ligaments, like elastic ligaments along the vertebral column, contain many parallel elastic fibers besides collagen. Tendons and Ligaments Outer Surface Tendons and ligaments have an outer layer of dense irregular connective tissue. This layer is continuous with the outer layers of adjacent muscles and bones. Collagen bundle size varies in different tendons and ligaments. Regular connective tissue structures have poor vascularization, leading to slow repair. Reticular Tissue Characteristics Reticular tissue is characterized by abundant type III collagen fibers, also known as reticulin. Reticular fibers form a delicate network supporting various cell types. Reticular cells, modified fibroblasts, produce these fibers and partially cover them. Glycosylated reticular fibers provide a framework in hemopoietic and some lymphoid organs (bone marrow, lymph nodes, spleen). o This framework facilitates leukocyte and lymph passage and creates specialized microenvironments. o Macrophages and dendritic cells are dispersed in reticular tissues for cell monitoring and debris removal. Mucoid Connective Tissue Mucoid (or mucous) connective tissue is a key component of the fetal umbilical cord, known as Wharton’s jelly. Contains an abundant ground substance primarily composed of hyaluronan. It is gelatinous, with sparse collagen fibers and scattered fibroblasts. Includes mesenchymal stem cells, with potential applications in regenerative medicine. Mucoid tissue is similar to tissue in the vitreous chambers of eyes and pulp cavities of young teeth.