Chapter 7: The connective tissues. ‘The connective tissues are the second group of tissues derived from mesenchyme. They have two kinds of tissue elements ~ cefls and intercellular ground substance. ‘The last one presented by fibers and amorphous substance. Classification of connective tissues. 1. Fibrous connective tissues. A) Loose irregular connective tissue B) Dense connective tissue: D Regular: tendons, ligaments. > Irregular: reticular layer of dermis. 2. Connective tissues with special properties. A) Adipose tissue B) Reticular tissne C) Mucous tissue, D) Pigment tissue. ‘The fibrous connective tissues have a typical feature - they have a lot of fibers in ground substance. It is clear from the table above that fibrous tissues are of two types: foose and dense. In the loose connective tissue the amorphous substance dominates over fibers. In the dense connective tissues, the concentration of fibers is much higher than amorphous substance. In the loose connective tissue we can find a wide range of cell types. However, in the dense connective tissue the cellular composition is homogenous. The tissues can be regular and irregular. If the fibers are positioned in a special order (parallel to each other), it is regular tissues (like in ligament). If the fibers are disordered in 2 tissue, it is irregular connective tissue. The irregular connective tissue is subjected to mechanical ioad in different directions. That is why fibers are located to resist the load in any direction. In the regular connective tissues the mechanical load has one direction. So, fibers chose the best position to cope it. Connective tissues with special properties (adipose, reticular, pigment, mucous) perform special functions and in majority of cases they have a limited distribution in the organism. Loose irregular connective tissue (LICT). LICT has the largest distribution in an organism among connective tissues. It accompanies the smallest vessels making a stroma of parenchymal organs. It is included to the coats of layered organs. The functions of the LICT are the following: defense, support, feeding, regulating, homeostasis maintsining, shape formation of organs, patticipation in regeneration and inflammation. Structure. As all tissues of internal environment the LICT has cells and intercellular ground substance, Cells of LICT. LICT contains sefferal cellular differons. 1. Differon of fibroblasts (stem cells —> semistem cells + undifferentiated fibroblasts —> differentiated fibroblasts —> fibrocytes, fibroclasts, myofibroblasts). 2. Differon of macrohages (blood stem cell —+ ...+ blood monacytes —+ macrophages of LICT). 3. Differon of plasma cells (blood stem cell —> ...-» B-lymphocytes of blood —> B- lymphocytes of LICT —» plasmablasts —» proplasmacyte — plasma cell), 4. Differon of mast cells (Blood stem cell —> ...—» basophilic leucocyte of blood — mast cell of LICT). Differon of lipocytes (undifferentiated fibroblasts — lipocytes).Lecture#? ‘The connective tissues. 6. Differon of pigmentocytes (precursor cells —+ pigmentocytes). 7. Adventitial cells. 8 Perycytes. 9. Leucocytes All cells of LICT can be ciassified into two groups. 1. Local cells. his group includes all differons exept leucocytes. 2. Foreign cells. They are the leucocytes. All cells of LICT have three sources of development. 1. Cells from mechanocytes line. There are adventitial cells, perycyies, cells of fibroblast differon and lipocytes. They develop from special mechanocytes stem cell of red bone marrow origin. It is different from blood stem cell 2. Cells derived from blood stem cell, There are macrophages, mast cells, plasma cells and leukocytes, Both stem cells have mesenchymal origin, ‘They undergo divergent differentiation 3. Cells af nenroectodemal origin. Such cells are developed from neural crest. They are pigmemtocytes. FIBROBLASTS (FB). During embryogenesis, the fibroblasts are differentiated directly from mesenchymal cells. in postnatal ontogenesis, they are developed from the mechanocytes stem cell. It is located in red bone marrow. Arriving to connective tissue it is transformed to adventitial cell ‘The fibroblast population is the most numerous population in connective tissue. Their function is to produce intercellular ground substance containing fibers and amorphous substance. ‘The differon of fibroblasts contains the following cells as: 1. Immature FBs - they are cells with high NCR, moderate organelles number, large oval basophilic nucteus containing 1-2 nucleoli. They have ability to produce ground substance, but they still able to division. Consequently, their functions are synthesis of ground substance and enlargement of populution size. 2. Mature Fis — they have iow NCR, light euchromatin nucleus with 1-2 nucleoli, In light microscope, the cytoplasm of the cells is lightly basophilic. There are two regions: the first is intemal, around the nucleus with more basophilic cytoplasm (endoplasim), the second is extemal with less basophilic cytoplasm (ectoplasm). The endoplasn contains well developed organelles of protein synthesis: routh EPR, Golgi complex, mitochonria, lysosomes. In the ectoplasn there are well developed components of cytoskeleton providing formation of proceses and active movement of FB. Eetoplasm may be not distinguishable from ground substance while staining. It looks like it gradually transforms into ground substance. Functions of specialized FB are: > Active synthesis of interceliular ground substance components, > Regular influence on fiunctioning of other cells of the connective tissue by constant releasing of wide range of mediators, > Degradation of collagen, which was produced by fibroblasts themselves. It is performed both inside and outside of the cell. The collagenase enzyme is needed for this. The degradation of produced collagen is important process for controlling ground substance state, for tissue homeostasis maiutaining. By destroying and modifying collagen the fibroblasts can modify scar connective tissue, 3. Fibrogytes. Aging FBs transform to fibrocytes. They are inactive cells of spindle shape with dense hyperbasophilic nucleus and with narrow band of cytoplasm. It is poor in organelles. Functions, Fibrocyies are terminal point of fibroblasts development. And finally they die by apoptosis. But contemporary researches shows that fibrocytes can participate in some degree in renewing of ground substance components, and 0 in tissue homeostasis maintaining. During reparation after injury, the cells can transform to the cells with active protein synthesis similar to fibroblasts 4. Myofibroblasts. They are the fibroblasts which have well developed contractive elements. They resemble smooth myocytes. But they are not surrounded by basement membrane and they have 2Lecture#7? ‘The connective tissues. more developed EPR and Golgi complex. In light microscope myofibroblasts are same as smooth myocytes. Functions. > They actively participate in wound repair. Because of high contractive ability they can get wound sides closer. causing wound contraction. At the same time the cells actively produce components of ground substance. It is clear that myofibroblast work facilitates wound healing, > They can be transformed to smooth myocytes, and by this they can participate in regeneration of the smooth muscular tissue, In the scar they are transformed to fibrobiasts and than to fibrocytes. 5. Fibroctasts - they are fibroblasts with well developed lysosomes, Functionally, they are similar to macrophages. Their function is a degradation of the ground substance while it exceeds normal level, for example in the uterus after delivery, in the scars after regeneration. As it was Pointed above, the differentiated fibroblasts have ability to destroy ground substance too. That is why some authors consider fibroclasts as fibroblasts with dominating degradation function. Hence, the fibroblasts and fibroclasts are functional antagonists regulating the volume of ground substance and homeostasis LICT. MACROPHAGES. It is second largest differon of LICT after fibroblasts. They are developed from the descents of blood stem cell - blood monocytes. The blood monocytes migrate to a tissue and they are subject (0 (ransfonnaiion. ‘The transformation results in cell enlargement, fonuing bean-shaped nucleus, accumulation of lysosomes and other organelles: mitochondria, EPR. Golgi complex becomes hypertrophic. ‘The number of pinocytosis vesicles increases. The macrophage membrane expresses large number of microvillia and folds. It increases the square of the cell surface. The macrophages become more motile. The processes of pinocytosis and phagocytsis are activated, ‘The activity of lysosomic enzymes and enzymes of metabolic exchange is increased. ‘There are two functional states of macrophages in the connective tissue: resting macrophages and roaming macrophages. These two ceil types are different in morphology. The following data about macrophages can be revealed y light microscopy. They have bean- shaped nucleus and sharp borders of cytoplasm. Vacuoles are visible in the cytoplasm. ‘The resting macrophages are haruly recognizable from Gbroblasts. They have flattened shape, small densely packed nucleus and cytoplasm with limited number of the organelles. Inactive (resting) macrophages are usually bound o collagen fibers. Roaming (active) macrophages are very motile, ‘That is why the shape of the cells is very iegular. The surface of the cells is rough with numerous pseudopodia ‘The electronic inicroscopy can add some data about macrophages structure. The roaming macrophages have many lysosomes, mitochondria, smooth and rough EPR, glycogen inclusions, phagocytated articles. ‘There a lot of receptors for immune system mediators, hormones and etc on the surface of « macrophage, These receptors enable the cells to interact with many other cells and intercellular ground substance and to perform migration, Functions: DPhagocytosis: it includes recognition, absorption and degradation of bacteria and other antigens, aging celis, components of ground substance and ete. > Antigen presenting: it includes transformation of an antigen to fughly immune form and expressing it on the surface receptors for other cells of immune system. Perhaps, the macrophage induce immune reactions, Contemporary findings allow putting antigen- presenting cells to separate group from roaming macrophages. It is belived that roaming macrophages perform only non-immune phagocytosis, D Secretion: the macrophages produces and secretes the wide range of biologically active substances, such as jaterferon (antiviral substance), antibacterial substances (Iysicim, active forms of oxygen and s0 on). > Participation in antitumor, anticancer immunity. > Regulation of tissue homeostasis. They eliminate aged components of the tissues. They participate in metabolic exchange, especially in lipid exchange. They regulate the state of the ground substance and activity of other cella in a tissue. 3Lecture#!? ‘The connective tissues, D Regulation of regeneration: secretion of substances stimulating regeneration in the macrophage phase of inflammatory process. MAST CELLS. (tissue basophils). It is third largest differon of LICT. The source of development for them is similar with macrophages. It is blood stem cell. Mast cells have similar origin with blood basophils. They have very similar structure and functions, but they aren’t absolutely identical. It is believed that population of mast cells in LICT can be replenished by divisions of young max cells. ‘The tissue basophils in LICT usually located near blood vessels and norves. They have a size about 10 to 30 mom. The shape of the cells may be different: spindle, ovil, irregular. The nuclei are round with heterochromatin dominstion. They are obscured by granules and they are hardly distinguishable in light microscope. ‘There ure moderate developed organelles of general purpose and components of «yioskeleton in the cytoplasm. There are also lipid inclusions. The typical feature of them is ~ large quantity of metachromatic granules (the are stained to the color different fran the color of stain in the solution}. The granules of mast cells differ fiom the same granules of blood basophiles by larger quantity and by variable shape and ultrastructure. There are granules with dense homogenous and gral struciure and crystal-like gramules, as well, The metachromasia of granules is because of presence of heparin. It decreases blood clotting rate and decrease permeability of capiliaries. The granules have histamine too (in rodents the serotonine is also present). These substances cam change the tate of LICT ground substance and increase permeability of microvessels. The granules also contain some enzymes and chemoattractive substances fur eosinophiis aid neuiropliiv. The secretion of granules by mast cells is called degranulation, Jt con be either slow and insignificant (nomally) or rapid and massive (during allergic reactions). Functions: > Regulation of tissne homeostasis — regulation af permeability of blood vessels, blood clotting, tissue supplewent. i is performed due to slow degranulation. > Syuthesis of ground substance components (heparin, chondroitinsulphates, hyaluronic acid, glicoproteins). D Regulatory fimneiion. It is in regulation of working of other cells of LICT and in controlling of ground substance state by mediators release. > Panicipaiion in immune reactions. The madiaion: of the mast cells regulate immune reactions and intensity of immune response. The cells perform phagocytosis of antigen-antibody complex. They absorb the excess of histamine from tisme, They participate in allergic reactions. DSlimuiation of tisswe regeneration. The colls take part in histo- and organogenesis. For exumple, the mast celle stimulate hair growth wnd development. PLASMA CELLS. They develop from B-lymphocytes of blood through the following stages: B-lymphocyte —plesmoblast -~proplasmacyte —plasma cell (or plasmocyte). During the development the NCR iu the cells is decreased, the organelles of protein synthesis are accumulated in the cytoplasm. B-lyraphocytes and plasma cells together are permanent inhabitants of LICT. ‘They are particularly numerous in the lamina propria of mucous coats in many interna! organs. Prasioa cells have a size about 7-16 mem {it was observed larger plasma cells up to 20 mem) and oval shape, The round nucleus is shifted toward one pole. The chromatin in the nuclens Jooks like a wheel wlth spokes. The cytoplasm of the cell is strongly basophitic and often reveals a pale area in the region whors the contricles and Golgi apparatus located. With help of the electronic microscope it can be revealed weil developed EPR. The cisterns of EPR are flattened, They ale located close to each other in parallel order. The mitochondria and Goldgi complex are well developed too. Piasma ceils do not make secretory granules for secretion. Although, sometimes the Russel’s bodies can ive iovealed. They are dense spherical inclusions containing carbohydrates and proteins including sntibodies. In eledronic microscope the Russe!’s bodies are homogenous material located in widen parts of rough EPR. It is believed that Russel’s bodies appear while disturbances of immmunegiobutin synthesis and secretion. 4Lecture#7 ‘The connective tissues. Functions: The only function of plasma cell is to produce immunoglobulines (antibodies) inactivating antigens, By this they participate in humoral immunity reactions. Plasma cells are the only source of the antibodies in the body. ADIPOCYTES (LIPOCYTES). They are distributed everywhere. But the quantity of the cells may vary in wide range. ‘The source of development for lipocytes is undifferentiated fibroblasts, They slat to accumulrte lipid incnsions fusing to one large lipid droplet. The mechanism of fibroblast selection for differentiation to lipocytes is unknown. It is stated thet while fasting after depletion of lipid inclusions the lipocytes can retum to fibroblasts appearance. ‘There are whit» and brown lipocytes. The white lipocytes are located near blood capillaries. ‘They have a ring shave and big size. The nucleus is strongly basophilic, It lies eccentrically. The cytoplasm resembles narvow band around the lipid droplet. The lipid droplet located centrally is stained by sudan. ‘The electronic microscoping reveals a few organelles in the cytoplasm. The functions of white iipocytes are storage of fet and water (much water is produced while fat oxidation), trophic and temperature pattern regulating, ‘The brown lipocytes have smaller size and polygonal shape. The round nucleus is located centrally. The cytoplaam has numerous lipid droplets. The EPR and Golgi complex are moderately developed. There are many mitochondria. They have well developed enstae. They surround lipid droplets. The enzyme complexes of mitochondria contain iron ions. i gives brown color to the cells, ‘The main function of the brown lipocytes is generating heat. Here, there is uncoupling of oxidations and phosporilation. The oxidation does not result in ATP formation. All enrgy taken from oxidation ix sprexd in a form of heat. ‘The termogenin protein is responsible for the uncoupling. The fimction of fipid storage is secondary PIGMENTOCYTES (PIGMENT CELLS). All pigment ceils are derived from neural crest. ‘They have a fot of pigment inclusions (melanin inclusions). The melanin has increased ability to absorb ultraviolet radiation, The pigemntocytes of LICT are unable to produce melanin. They get it from melanin producine “lls - melanocytes located in epithelium. ‘That is why pigmentocytes have lees developed organeiles of protein eynthesis comparing with melanocytes, ADVENTICIAL CELLS. They are undifferentiated cells with high nucleo-cytoplasmic ratio, weakly basophilic cviopiasm with few organelles. They are located newr capillaries. That is why they have second name ~ peryvascular cells, They are considered as ster cells for fibroblasts and lipocytes. The precursore of adventicial cell migrate to LICT from red bone marrow. There are self- maintaing population of mecbnocytes stem cells PERYCYTES. ‘They are the cells snmounding microcirculatory s network. Some authors consider them as precursors for fibroblasts. The perycytes will be particular described in the chapther “Cardio-vasentar system”, LEUKOCYTES. All types f leukocytes migrate to LICT: granulocytes, lymphocytes, monocytes. Th last iv tinsformed to isscrophages. All leukocytes are foreign cells for LICT. Intercellular ground substance. It consists of fibers and amorphous substance. The fibers can be of three types: collagen, elastic and retjeular. The interceliuiar ground substance is produced by cells of LICT. The main cells producing ground substance are fibroblasts. They produce both fibers and amorphous substance. Mast cells also produce some of the components. Partially. the ground substance is filtrated from blood plasma, COLLAGEN FIBERS. They are made of collagen protein, Today, the 19 collagen types have been described, But only five of them are of great importance 3 First type is in the connective tissue of skin, bone, artery wall. % Second type van be found in cartilages. % Third type is focuied in fetal dermis, in the wall of large vessels, in reticular fibers. % Fourth type is included to the structure of basement membranes and eye lens. 5Lecture#7 ‘The connective tissues. 2% Fifth type also participates in basement membrane formation as well as in formation of walle of vessels, main substance of 2 comen, Collagens of 1, 11, TT. and V types are fibrillar because they can form filaments and fibrils, Other collagens are amorphous becanse they have no such ability. VI-XTX types of collagen are incompletely studied Molecules of the collagens are made from three coiled around each other a-chains, The biochemical composition of the chains is also specific. Here is the prevalence of such amino acids as glycine, proline, lysine, hydroxyproline and hydroxylysine, The collagen molecule is made by fibroblasts. Beside them the following cells may produce collagen: osteoblasts, chondroblasts, cementoblasts, dentinabelsts, reticular cells, smooth myocytes, cells of perynervium. ‘The procese of collagen biosynthesis is similar in all listed above cells. The process has two stages: intracellular and extracellular. Intracellular stage First which has to be done is synthesis of polypeptide chain. It takes place in rough EPR. The produced e-chains are accumulated in cisterns of EPR. Here they are subjected to moditication: the hycroriation of some lysine and proline molecules with help of vitamin C and formaion of disulfide bonds. Insufficiency of vitamin C results in formation insufficient amount of hydroxilycine and hydroxiproline. The chains made by this way are unable to coil into threefold spiral. The next is formation of procollagen chains having 3 polypeptide chains Each of them is shifted along others on ‘4 of length. As result of thie the collagen molecules have striations. Then the collagen molecules move to Golgi complex in secretory vesicles. Here they subject to terminal giicosilation. Molecules of procollagen in Golgi complex are packed to excretory vesicles. Tiev they release to the extracellular matrix, Extracellular stage ‘The terminal regions of procollagen molecules are cut off after seretion to tho matrix. It results in formation of tropocollagen. This process takes place only in fibrillar collagens. It enables them to make fibrils, Next step is polymerization of tropocollagen molecules. ‘They are joined “‘side by side” and “end to end”. They muke protofibrilles with diameter 3-5 nm. Five-six protofibrillex make microfibrilles with diameter 10-20 nm. Then, microfibrilles merge with each other making fibrilles with diameter 100 nm, Several fibrilles are united to visible in light microscope fiberes with diameter 1-100 mem. So, the collagen fiber has following levels of organization: polypptice chain —> procollagen molecule —> tropocollagen molecule —> protofibrilles -> microfituilles —> fibrilles -> collagen fibers. ‘The collagen hers of LICT are mainly of T type. They are visible on histological slides as oxyphilic crimped fibers going in many directions along or forming bundles of various width. In polarizing microscope the: collagen bundles have double refraction. In electronic microscope they ook like parallel striated fibers Functions of collagen fibers: 1) support, 2) providing tissue suength; 3) regulatory - ion in morphoconcris, differentiction, regeneration of cells and tissues, regulation of migrotion. secretory snd synthetic activity of cells, participation in cell adhesion, in platelets adhesion and in clot fonnstion; 4) setting the tissue architecture of connective (issue. ELASTIC FIBERS They are less in LICT than collagen fibers. They are made of amorphous elastin and fibrillar Ghriiin, Both are made in rough EPR and then are modified in Golgi complex. Elastin as well as collagen contains many glicine and proline molecules. It also contains two unique amino acids: 4gsmecin od isodermesin, The eletin molegule ee globules. Being eereted to extracellular mfirix they are unitedto the chains and form elastin protofibyilles with width 3am. Later, pratofibritles make central Intex-like network af molecules. tt is so called central component of elastic fiber. Outside of it there is go called fibrillar component of elastic fiber. The elements of it are made of fibrillin, The process of elastic fiber formation can be described as following. At the beginning of the fiber formation fibrillin make microfibrilles (oxytantal fibers) They serve as a framework for further elastin accumulation, Elastin accumulates centrally in the form of amorphons component (or so called elaunin fibers). Accumulated elastin shifts fibrillar component to the periphery.Lecture#7 ‘The connective tissues. The elastic fibers can be visible in light microscope only if special staining techniques have been used (iron hematoxilin, orsein and etc.). They look like thin straight branched lines connecting, to each other and by this making three-imensional network. They are elastic. This means that they can return to primary position after being stretched or compressed. There are immature elastic fibers in LICT beside mature elastic fibers: oxytantal and elaunin fibers. The first type contains only oxytantal fibrilles and nothing else. In elaunin fibers there are both types of fibrilles, but oxytantal fibrilles are located centrally with a small amount of amorphous component. In mature fibers, as it was said above, the fibrillar component is located peripherally. Apart to fibroblasts, the following cells can produce elastic fibers: chondroblasts, chondrocytes and smooth myocytes. Functions of elastic fibers: 1) providing reversible deformability of tissues — elasticity; 2) setting the tissue architecture of connective tissue. RETICULAR FIBERS. They may be reffere as collagen fibers because they are made of collagen type Ill. They are invisible in slides stained by routine hematixilin-eosine. They consist of microfibrilles and there are binding glicoproteins and proteoglicans between them. Perhaps, the reticular fibers can be impregnated by siver salts and they have positive Schiff base reaction. Reticular fibers are located in reticular tissue of hemopoietic and immune organs. However, they ‘can be found in other types of connective tissue. The cells which can produce reticular fibers are fibroblasts, reticular cells, adipocytes, smooth myocytes, cardiomyocytes, neurolemmocytes. ‘The main function of the reticular fibers is support. AMORPHOUS SUBSTANCE. It is the second component of ground substance. It is transparent if it is studied by light microscope. In electronic microscope it has low electronic density. It is made of water (90% of total volume), proteins (glicoproteins, proteoglicans, plasma proteins — albumines, globulins, fibrinogen), lipida, carbohydrates (af first glucosaminoglicans), inorganic substances. Glucoseaminoglican molecules are large. They make three dimensional network. They are hydrophilic and by this they bind a lot of water making gel. Glicoproteins and proteoglicans provide the interaction of the intercellular ground substance with cells, participate in substance transportation, accumulate growth factors and perform some of other functions. There are two functional states of the amorphous substance: sol and gel. Gel is colloid state, where as sol is more liquid state. The changing of functional state is controlled by hyaluronidase enzyme. Functions f amorphous substance are regulatory, metabolic, providing environment for cells being, Dense connective tissues. Comparing to LICT the dense connective tissues have more fibers that cells. The cells are mainly fibrocytes. The fibers may be organized and disordered. That is why two following variants of the dense connective tissues exist: regular and irregular. The regular connective tissue is located in tendons, ligaments fascia, aponevrosis. Sometimes, elastic and collagen regular connective tissues are concerned. In the collagen connective tissue the main fibers are collagen fibers. The elastic tissue has more elastic fibers. The elastic dense regular tissue is found in vocal folds, yelloy’ ligaments of vertebrae. The irregular dense connective tissue is in the reticular layer of skin dermis. As it was said above, the following structures are made of dense regular connective tissue as tendons, ligaments, fascia, aponevrosis. Let describe the structure of tendon. It is made of thick, parallel collagen fibres. Such fibres are separated one from another by single layer of fibrocytes and they are called bundles of first grade. Fibrocytes have many processes. They are lack of protein synthesis organelles. However, they are capable to produce intercellular ground substance. This ability depends on functional load on the tendon. All fibrocytes are connected with eachother by processes making entire three 7Lecture#7 The connective tissues. dimentional network. The cells junctions are communicating junctions (nexuses). Pethaps, the cells are united not only mechanically, but electrically and chemically as well. In it’s turn, the cell membrane of the processes is connected with ground substance with help of the cell adhesion molecules. That is why even slight change of mechanical load on tendon leads to changing of synthetic activity of fibrocytes. Bundles of first grade stay together surrounded by LICT. All together they are called bundles of second grade. Surronding LICT is called endotenonium. It has trophic function because of having vessels. It plays the role in regeneration because it contains stem cells (dense regular connective tissue has no stem cells, because fibrocytes are irreversibly differentiated cells). ‘Several bundles of second grade unite to the bundles of third grade, They are surrounded by thicker layer of LICT called perytenonium. It has functions similar to endotenonium. Some tendons are bundies of third grade. Although, there are tendons, which are the bundles of fourth or even fifth grade. Funetions of dense regular connective tissue are support, transmitting mechanical load from muscle to bone, joints strengthening and etc. Dense irregular connective tissue is located in reticular layer of the skin dermis and it makes capsules of many organs. It has cells and ground substance. The cells are mainly fibrocytes, but some fibroblasts also present. Some mast cells, plasma cells, macrophages may be found, Intercellular ground substance consists of fibers directed irregularly and amorphous substance. There is LICT around vessels in DICT. Function of dense irregular connective tissue is mechanical support. Regeneration ability of dense connective tissues is low. They have no cambial cells. The cells which are able to division are located in inserted LICT layers. They have insignificant volume in compare with dense tissue volume. Connective tissues with special properties Such group of the connective tissues includes reticular, adipose, pigment and mucous tissues. The tissues have general structural principle of connective tissues. Their peculiarities are: 1) well-defined localization in organism (exclusion is white adipose tissue, which is wide spread in organism); 2) performing of special functions; 3) one cellular differon predominates in the tissue; 4) special structure of intercellular ground substance (fibers or amorphous substance). 1. RETICULAR TISSUE. It is located in organs of immune or hemopoietic system It provides environment for hemopoiesis and immunogenesis. It has cells and intercellular ground substance. The cells of reticular tissue are the following: 1) reticular cells (fibroblast-like); 2) macrophages; 3) adventitial cells (undifferentiated). Reticular cells have many processes. Their processes are connected with each other help of communicating junctions (nexuses). They have light nucleus and slightly acidophilic cytoplasm with moderate number of organelles for protein synthesis. The reticular fibers are located near reticular cells. The fibers can even be depressed into cytolemm fold of reticular cells. Function of reticular cells is synthesis of ground substance. Macrophages of reticular tissue have various structure and specialization. They perform phagocytosis, secretion, regulation, antigen-presenting and other functions. Intercellular ground substance includes amorphous substance and reticular fibers. The compositign of ground substance is similar to LICT. The reticular fibers make three-dimensional framework. They are made of collagen type III. Functions of reticular tissue are — nutrients supplement, support, defense, regulation and homeostasis. 2. ADIPOSE TISSUE. It is a sort of LICT where lipocytes dominate. It has cells and ground substance. The composition of ground substance is same as in LICT. The dominating cells are Tipocytes, but there are other cells of LICT. Accordingly to lipocytes type the adipose tissue has ‘two variants: white adipose tissue and brown adipose tissue.Lecturet#7 ‘The connective tissues. White adipose tissue is placed in skin hypodermis, epiploon, between muscular fibers, in walls of internal organs. It has following functions: 1) Storage for nutrients (especially lipids), storage for water (water is produced while lipid oxidation), storage for lipid-soluble vitamins A.D,E,K and for steroid hormones (especially female sexual hormones); 2) Energy supply. Fats breakdown gives a lot of energy; 3) Temperature regulating. Adipose tissue is important in isolation of body from warm dispersion. Also adipose tissue can produce more warm in cold conditions by accelerating fat metabolism; 4) Defense and support functions. Adipose tissue can protect underlying organs from mechanical damage. Also it gives support for organs. While rapid weight loss the organs can loose their original position (for example kidneys); 5) Cosmetic function. Adipose tissue of hypodermis plays an important role in giving our body attractive shape; 6) Endocrine function. Not so far ago, it was revealed that lipocytes are active in producing several hormones such as estrogens and leptin. Leptin is a hormone which regulates food consumption. It suppresses releasing of neuropeptid Y (NP-Y) by hypothalamus. NP-Y is a hormone which stimulates food searching behavior, induces hunger. While fasting, the leptin secretion falls down. ‘At the same time the satiation increases leptin production. Insufficient synthesis of leptin results in obesity. White adipose tissue consists of lobules separated from each other by LICT. There are blood capillaries and nervous fibers in the LICT. There are fibroblasts, macrophages, leukocytes and other cells apart from lipocytes in adipose tissue. Brown adipose tissue is well developed in newboms and in animals that have winther hibernation period. Adults have a few of it. It is generally located around large vessels, kidneys, in mediastenum. It has brown lipocytes and intercellular ground substance. Few whilte lipocyte may present in the tissue. Lipocytes are arranged in lobules separated by thinnest LICT layer. The tissue hhas very well blood supply. Also it has sympathetic innervation. Moreover, the nervous fibers contact almost with each brown lipocyte. Functions: 1) heat regulation (in newborns; in hibernating animals it provides large heat output after waking); 2) storage of lipids. 3. MUCOUS TISSUE. It is an embryonic connective tissue. It is a modified LICT. Here, the intercellular ground substance significantly dominates over cells. Here are few fibers, but a lot of hyaluronic acid. It is located in fetus dermis, umbilical cord, in amnion. The tissue of adult organism, which resembles mucous tissue in structure, is vitreous body of eye. It has cells and intercellular ground substance. The cells are mucous cells. They resemble fibroblasts. They have many processes and produce ground substance components. Ground substance has thin collagen fibers and amorphous substance. The feature of amorphous substance is high concentration of carbohydrates and hyaluronic acid. The hyaluronic acid gives resilience for tissue. In dermis the tissue is gradually substituted by normal LICT. In umbilical cord this tissue protects vessels from compression. 4, PIGEMNT TISSUE. It is sort of connective tissue. It resembles LICT in structure, but it contains many pigmentocytes. It is most developed in iris and tunica vascularis of eye. It is located in skin in some area (nipple areola, around anus, in scrotum), in birthmarks. It has many pigment cells, as well as, fibroblasts, macrophages, leukocytes and etc. Pigment cells are of two types: melanocytes and melanophores. Melanocytes can produce melanin by themselves. Melanophores take melanin from melanocytes and accumulate it. Some authors concem that connective tissue has only melanophores, whereas melanocytes are located in epithelium? Ground substance is similar to LICT. Functions are same to LICT, but due to melanin inclusions it can protect cells and tissues from ultraviolet radiation.Lecture#7 ‘The connective tissues. Participation of LICT and blood in inflammatory process. Blood and LICT have very close functional relationships. It is especially evident on example of defensive reactions of an organism (e.g. inflammation, reparative regeneration, immune reactions and so on). Inflammation as well as immune reactions is general biological defensive-adaptive reaction of an organism. It results from any physical, chemical, mechanical, biological damage. Morphologists can separate the process of inflammation into four overlapping phases: alteration phase, leukocyte phase, macrophage phase and fibroblast phase. In short the morphology of an inflammation can be described as following. Alteration phase is characterized by tissue damage. The inflammatory mediators are released. Mediators can be humoral (derived from blood plasma) and cellular (secreted by various cells). Mast cells degranulate right after the tissue damage. The substances released in degranulation cause increasing of blood vessels permeability and increasing of their diameter. Vasoactive substances are also released by macrophages, basophils, platelets and other cells. It results in diffusion of blood plasma to the tissue and to neutrophil migration to the tissue (leukocyte phase). Neutrophils phagocytate bacteria in inflammation site. During phagocytosis the neutrophils can die making the pus. Dieing cells release chemoattractants which can attract new neutrophils. Massive neutrophils migration to the tissue causes leucocyte billow formation. It separates intact tissues from damaged or dead. At the same time neutrophils release attractive substances for monocytes migration. Monocytes are transformed to macrophages. The leukocyte phase is changed by macrophage phase. At the same time the T- and B-lymphocytes migration takes place. They serve as a basis for immune reactions processing. Later lymphocytes regulate quality and quantity of repairing tissue components. Macrophages phagocytate died cells of tissue, died neutrophils, microorganisms and they release substances activating fibroblasts (fibroblast phase). Precursors of fibroblasts divide and migrate to the inflammation site. They start to produce intercellular substance and to repair damaged tissue. In case of foreign body, the fibroblasts make dense collagen capsule around it. It serves as defensive border for protecting surrounding tissues. 10Chapter 8: The cartilage and bone. Skeletal connective tissues are made accordingly the same principle as all tissues of intemal environment. They made of amorphous substance and fibers. They develop from sklerotome mesenhyme. The major function of them is support, which brings the special features in the structure, The first specific feature is that extracellular matrix excels in volume the cells. The second feature is that extracellular matrix has denser structure that in other connective tissue. It can 4e mineralized (in bones) and no-mineralized (cartilages). Fhe classification of skeletal connective tissues is the following: Cartilages Bone tissues Hyaline Woven bone Elastic Lamellar bone L Fibrocartilage Dentin CARTILAGE A specialized CT to resist compression, and provide modest rigidity with flexibility, by having its cells, chondrocytes, produce a firm resilient matrix of ground substances, and fibres or fibrils. The apid growth of cartilage is used to assist the growth of bones and the repair of fractures. Based on the composition of the matrix, three kinds are distinguished: hyaline, elastic, and fibrocartilage. A HYALINE CARTILAGE 1 Occurs fused with bone or as discrete pieces, looking hyaline/translucent (glass-like) to the unaided eye. Most surfaces, except joint/articular ones, are covered by a nutritive CT perichondriumicapsule with collagen and elastic fibres, fibroblasts and blood vessels. It merges gradually via a chondrogenic zone with the cartilage proper. 1a, Development, Cartilages develop from sklerotome mesenhyme. In cartilage development there are 4 stages: .. Stage of chondrogenic islet formation. In the place of future cartilage, the cells loose their processes, divide by mitosis and form dense accumulation of cartilage cells- chondrogenic islet. Typical extracellular matrix is absent. 2. Stage of primary cartilage tissue (chondroid). Here, mesenhymal cells transform to cartilage cells: the cells obtain rough EPR, Golgi complex, accumulate mitochondria and inclusions. They start to produce extracellular matrix: specific collagen II and glucoseaminoglycans. 3. Stage of cartilage differentiation. The following differentiation of cartilage cells occurs. They start to produce sulphated glucoseaminoglycans or chondroitin sulphates. The secreted matrix separates chondrocytes to separate lacunae. The perichondrium is formed. 4, Stage of age changes in cartilage. The age changes mainly affect hyaline cartilage. They are mineralizeg. This can be accompanied by failure of nutrition for internal parts of cartilage. The cells cnlarge anid start to produce substances which can bind calcium. It leads to cartilage cells death, Than, the chondroclasts destroy mineralized regions. 2 Matrix, apparently amorphous with HE staining in LM, contain: + (@ Ground substances rich in soluble collagens and the proteoglycan - aggrecan: a core protein with chondroitin and keratan sulphates side chains, and a link protein for attachment to hyaluronic acid. The aggregated proteoglycans impart basophilic and metachromatic staining properties, and bind water and confer resilience.Lecture#8 The cartilage and bone. + (b) Collagen fibrils visible in EM; and LM after silver impregnation, digestion of the ground substance, or in polarized light. The reinforcing fibrils are oriented in some relation to stresses experienced by the cartilage. 3 Chondrocytes or cartilage cells are large and rounded, each lying in a space - lacuna - enclosed by matrix. Cells often are grouped in nests of 2, 4, or 6 as a result of mitoses and restricted cellular ovement. EM reveals cells to have short stubby processes, fat droplets, glycogen and the GER and Golgi complex appropriate for secretion of the matrix components: proteoglycans, type II collagen [with the homotrimeric molecule «1(II)3], and glycoproteins. Other type of cartilage cells is chondroblast. It is cambial cell which is usually located in perichondrium. The main function is providing appositional cartilage growth. They are synthetically active cells. Chondroclasts are the cells of cartilage which can destroy extracellular matrix and cells, if it is necessary. They are derived from macrophages. They is likely to osteoclasts of bone. They have very well developed lysosomes. + Growth occurs in two ways: * (a) Appositional/perichondral by the recruitment of fresh cells, chondroblasts, from perichondral stem cells, and the addition of new matrix to the surface; + (b) Interstitial by the mitotic division of, and deposition of more matrix around, chondrocytes already established in the cartilage. Growth is vulnerable to X-rays, poor nutrition, and disturbed blood supply, for example, from fractures at the growth plate. 5 Territories Most noticeably in articular cartilage there are: - (i) the chondron - the chondrocyte and the pericellular matrix immediately around it; (ii) proteoglycan-rich territorial matrix outside the chondron; (iii) interterritorial matrix, lying between the territorial matrices. The matrix of the chondron has its own profile of special collagens, proteoglycans, and cartilage glycoproteins, whereas the differences between territorial and interterritorial matrices are more quantitative, and related to collagen fibril thickness and orientation. 6 Nutrition - cartilage is avascular and no blood vessels serve the matrix directly, but cartilage canals may carry vessels through the matrix to non-cartilaginous regions, e.g., secondary ossification centres. Therefore, nutriment and wastes must diffuse through the matrix for the cells to stay alive and perform their slow tumover of the matrix macromolecules. The diffusion may break down and various degenerations then occur, e.g., calcification. This last is prompted, organized and nade use of in the process of endochondral ossification. B ELAS{IC/YELLOW CARTILAGE Is more opaque and flexible than the hyaline kind, but the cells are similar in appearance and distribution; and it occurs as separate pieces with a perichondrium. 2 Matrix is permeated by many elastic fibres that can be selectively stained by stains such as orcein or Verhoefi’s. The matrix is not prone to degeneration and calcification. C FIBROCARTILAGE 1 In the intervertebral (IV) disc, fibrocartilage at first appears to have a rather disorderly matrix with ‘nany thick collagen fibres, amongst which are dispersed only a few chondrocytes in lacunae. 2Lecture#8 The cartilage and bone. However, the fibres are orderly in their alternating orientations and layering, like the burst-resisting “ibres of an old-style bias-ply car tyre. 2 The matrix gives the staining reaction of collagen, mostly type I, except for close around the cells where proteoglycans are abundant. 3 Lacks a perichondrium and is not seen as discrete pieces; rather it is a strong tension-resistant, but flexible transitional tissue located between tendon and bone, bone and bone, hyaline cartilage and hyaline cartilage. 4 In the IV disc, the enclosed central nucleus pulposus is not cartilage, but nevertheless has collagen type II, which diminishes in the innermost layers of the annulus fibrosus as it is replaced by type I. D DISTRIBUTION OF THE THREE CARTILAGE VARIETIES | Hyaline - articular surface of most synovial joints; costal cartilages; nasal and respiratory tract cartilages; basis of most of the fetal skeleton; fracture callus, 2 Elastic - external ear, pharyngotympanic tube, epiglottis, and some laryngeal and bronchiolar cartilages, 3 Fibrocartilage - intervertebral disc's annulus fibrosus (around a nucleus pulposus of notochordal origin, present until late in life); pubic symphysis; femoral ligamentum teres; many tendon insertions into bone; and the articular surface of some joints, e.g., temporomandibular. E REGENERATION OF CARTILAGE. If a cartilage has perichondrium, the regeneration of the cartilage will be good. It will be performed by cellular division of cambial cells of perichondrium and by cell differentiation. The cartilage of joints has no perichondrium, thatwhy it regenerates very slowly. In case of cartilage damage, the cells of surrounding connective tissue may take part in regeneration even by transforming to chondrocytes. Regeneration can be stimulated by injection of chondrocyte suspension from epiphisis of young animals, salycilate and growth factors treatment. BONE Bone is a hard CT with cells, osteocytes, in much matrix, and serves for support, attachment, leverage, protection and mineral storage. | To obtain great strength and rigidity with some elasticity, the matrix is composed of densely nacked collagen fibrils infiltrated with bone mineral as fine crystals of calcium salts resembling iydroxyapatite crystals. Mineral constitutes about 65 per cent of the dry weight of bone. The densely packed collagen fibrils are primarily type I. There are small amounts of distinctive non- collagenous proteins, e.g., calcium-binding osteocalcin and bone sialoproteins (Chapter 5.C.8). 2 Matrix is strong but dense, thus nutritive fluids cannot diffuse freely through it. Osteocytes therefore have to differ from chondrocytes in having many long processes extending through canaliculi (narrow passages) and making contact with one another and, indirectly, with blood vessels. THe cell body lies in a cavity, a /acuna, in the matrix. 3 Throughout life, for mineral homeostasis, and for its special problems of growth, bone is subject to an unending turnover, with selective destruction and replacement - the remodelling process. A CLASSIFICATIONS OF BONE 1 Based on the size of the spaces within the bone, and its trabecular (lattice-like) or dense nature: .- (a) Cancellous/spongy/trabecularLecture##8 The cartilage and bone. (b) Compact/dense 2 Based on the presence or absence of lamellae (layers) and osteons/Haversian systems (a) Woven/primitive .- (b) Lamellar/Haversian Woven bone's matrix has disorderly fibrils, whereas in lamellar bone the fibrils of a lamella share a predominant orientation. Note that a particular bone will have areas of woven and lamellar bone, depending on how far remodelling has involved all regions. 8 HAVERSIAN BONE 1 An Haversian system is roughly cylindrical and arranged around one or two small vessels in a central Haversian canal. 2 Osteocytes and bone lamellae making up the system are disposed in 4-20 concentric rings centred on the canal. 3 A lamella is the territory formed and maintained by the osteocytes lying in a ring when seen in a cross-section. From the orderliness of the fibrils, lamellae can be distinguished in polarized light, but it is only in a smaller unit, the domain, that SEM reveals the fibrils to be aligned in the same direction. 4 Haversian canals branch and join up with others. Their vessels originally entered the bone from the periosteum or marrow via Volkmann's canals, around which osteocytes are not especially ordered D MATURE HUMAN BON! Studied from the outside working inwards has: | Periosteum of dense CT divisible into: + (a) an external fibrous layer of collagen and elastic fibres, fibroblasts, other cells, vessels and nerves; and + (b)an inner cambial layer of bone cells, mostly resting osteoblasts 2 Dense cortical bone. Where wide, e.g., femoral shaft, this layering is often present: © (a) external circumferential/basic lamellae lie outside; + (b) the main thickness with many osteons of various generations (primary, secondary, etc); interstitial lamellae fill the chinks between osteons and are lamellae of earlier osteons that have been spared total ersion; © @ endosteal/internal circumferential lamellae lie to the inside, with their osteocyte bodies lying parallel with the inner surface. In practice, some areas of dense bone remain woven or primary and are not replaced by this classic lamellar architecture, 3 Cancellous medullary bone whose trabeculae are lined by a thin cellular endosteum and have some lamellae, but can be sustained by marrow blood vessels without the need for Haversian canals. 4 Marrow cavities lie between trabeculae, inside the tubular shaft, or in the diploic spaces of flat skull bones, D BONE CELLS 1 OsteoblastLecture#8 The cartilage and bone. I Lies on the surfaces of bone, in a one-cell thick layer, as most of the endosteum and inner periosteum. 2 May be in two states: + (a) active, forming bone matrix, with a large Golgi complex and much GER in a plump cell, appearing in LM to have a pale juxtanuclear vacuole (Golgi) in the basophilic cytoplasm; + (b) resting or bone-maintaining; small cell with a dark nucleus and flattened against the bone. 3 Forms the collagen, glycoproteins, and proteoglycans of the matrix, and controls the deposition of mineral crystals on the fibrils. 2 Osteocyte 1 Osteoblast becomes an osteocyte by forming matrix around itself and becoming buried or immured 2 Young osteocyte thus resembles an active osteoblast; older ones have smaller, flattened bodies. 3 Processes extending from the body down the canaliculi are not visible by LM; but EM shows that osteocytes, like osteoblasts, remain connected by gap junctions. 4 The mature osteocyte is involved in maintaining the matrix of its territory. SEM evidence puts into doubt the proposal that osteocytes can resorb bone by osteolysis, Lacunae empty of osteocytes indicate dead bone. 3 Osteoclast I Large, multinucleated cell, with a pale acidophilic cytoplasm. 2 Lies on the surface of bone, often in an eaten-out hollow - Howship’s lacuna. 3 Cell surface is attached to the bone by podosomes to create a sealed compartment against the bone, in which the moving long cell processes of the ruffled border can agitate the resorbing - bone- destroying - materials. 4 Cytoplasm has vacuoles and lysosomes, since the mechanism of bone resorption is partly an enzymatic digestion, by cathepsins and collagenase, and also from acid made by an osteoclastic proton pump. 5 In dense bone, many osteoclasts act together to erode resorption tunnels, which are later partially ‘illed in with lamellar bone to become osteons. 4 Bone cell dynamics | Skeletal growth, changes of shape, and the physiological responses of bone need changes in the populations of ‘blasts and ‘clasts. These rely on a proliferation of osteoblasts or a precursor, while osteoclasts come from the fusion of blood-derived monocytes, which also partipate indirectly as macrophages in the bone resorption. 2 The osteoprogenitor cell is a small, organelle-poor cell on the surface or lying just behind the osteoblasts. It might be just an inactive osteoblast: that it is more of a stem cell is shown by its occasionally becoming chondroblastic, e.g., in tumours and fracture repair. BONE FORMATION introduction 1 Occurs in only one way - by the appositional or surface-depository action of osteoblasts; soon accompanied by the selective destructive action of osteoclasts in a remodelling process, continuously adapting the growing bone to developing soft tissues and dynamic mechanical forces, whilst meeting metabolic mineral demands. 2 Growth by remodelling is necessary because no interstitial growth is possible (except in growth cartilages). 3 Bone formed in the fetus is woven: only later is it mostly replaced by lamellar bone. 4 Dependent on whether bone is formed de novo in a soft tissue area, or in a site already taken by an established cartilaginous model, two situations of bone formation are noted - intramembranous and endochondral.Lecturet#8 The cartilage and bone. A INTRAMEMBRANOUS OSSIFICATION 1 Seen in the skull vault, facial skeleton, and parts of the clavicle. 2 In one or more ossification centres for a given bone, mesenchymal cells become osteoblasts and start to lay skeletal claim to territory by forming branching trabeculae/struts of bone, The initial thin struts may be called spicules. 3 This trabecular bone becomes denser by widening of the trabeculae, and is then remodelled extemally and internally, e.g., in the skull vault to two denser plates, tables, with spongy bone - diploe - between them. 4 The remodelling plates expand from their centres, but during growth remain separated by CT sutures for better adjustment to the enlarging brain, eyes, nasal cavities, etc. Skull bones grow by complex interactions and remodelling patterns that must cope also with, for instance, more teeth in the older child's jaws and the need for articulating cartilages on the mandible . B PREPUBERTAL LONG BONE | Diaphysis is the long tubular shaft containing marrow. The dense bone is the cortex, the marrow constitutes the medulla. 2 Epiphyses lie at each end of the bone. Each has: (a) a cap of hyaline articular cartilage over a «~ (b) cushioning lattice of secondary-ossification-centre bone; (©) this bone on its deeper aspect is fused with an epiphyseal plate/growth disc of hyaline cartilage. 3 Metaphysis is a lattice of bone trabeculae (primary ossification bone) with cross-struts, that joins each end of the shaft to an epiphyseal plate. 4 Endosteum lines all internal bony surfaces. 5S Periosteum ensheaths the bone, except for a small circumferential perichondrium around the epiphyseal plates, and where tendons and ligaments fasten to the bone. The articulating surfaces are bare. 6 Longitudinal growth, while the bone is under the stresses of use, is provided for by the interstitial growth of cartilage in the growth plates. C ENDOCHONDRAL/INTRACARTILAGINOUS OSSIFICATION 1 Mesenchymal cells retracting their processes round up to become chondroblasts, which form a minute hyaline cartilage precursor having roughly the shape of the eventual bone, e.g., the femur. Other mesenchymal cells differentiate and make a perichondrium. 2 In the central, shaft, region of the cartilage: + (a) Cartilage cells hypertrophy. + (b) Matrix around them becomes basophilic, then calcifies. + (©) Perichondral cells close by become osteoblasts; i.e., the perichondrium becomes a periosteum, + (@) Periosteal bone deposition forms a subperiosteal bony collar or ring around the centre of the shaft. + (©) At the same time, through gaps in the bone, osteogenic/vascular buds invade the calcified cartilage matrix to form the primary ossification centre of bone forming in, and on the calcified walls of, spaces eroded in the matrix. 3. Cells and functions of an osteogenic bud are (a) macrophages | (b) chondroclasts |- for selective cartilage erosion (c) endothelial cells? i (a) progenitor cells of osteoblasts and osteoclasts/chondroclasts; (£) marrow cells - to populate intertrabecular spaces; (g) endothelial cells - to form capillaries and sinusoidsLecture#8, The cartilage and bone. 4 Primary ossification zone establishes itself across the width of the shaft and starts extending in both directions towards the epiphyses, resulting in two transverse fronts of ossification across the diaphysis. At each front is the cartilaginous growth plate. 5 Epiphyseal plate. (This only becomes plate-like after secondary ossification has started within the epiphysis.) Starting farthest from the front, the zones are: + (a) Reserve/quiescent zone of hyaline cartilage with small cells and few cell divisions + (b Proliferative zone where chondrocytes multiply and arrange themselves in ordered parallel columns (palisades) of disc-shaped cells. Growth, interstitial, in the long axis of the bone occurs mainly here. + (©) Hypertrophic/maturing zone has the chondrocytes no longer dividing, but enlarging at the expense of their matrix. © @ Calcification zone, where the matrix stains basophilic and is impregnated by crystals of calcium salts. Mineralization may be triggered by the seeding action of caleium-rich matrix vesicles released from chondrocytes. Whether or not these chondrocytes then all die is disputed, + (©) Erosion zone encroaches into the calcification zone, Vascular bud elements destroy some cartilage, but leave longitudinal spicules as a scaffold on which bone is laid down. In HE preparations the calcified cartilage cores are blue, the superficial bone red. 5 Within the cartilage of the young epiphysis, a secondary ossification centre develops, again by processes of cartilage cell hypertrophy, matrix calcification, and its erosion by vascular elements penetrating from the perichondrium. However, orderly columns of chondrocytes and a defined marrow cavity are lacking, 7 The epiphyseal, secondary, ossification centre spreads to occupy much of the epiphysis and forms the bony border to the cartilaginous epiphyseal plate. The cartilage grows (thus lengthening the whole bone) keeping pace with the front of ossification invading it from the metaphyseal side, until puberty. Then resorption and ossification slowly overtake halting chondrocyte proliferation, until the primary ossification front fuses with the secondary epiphyseal bone - epiphyseal fusion/closure. The growth plate is obliterated, but an irregularity in the trabecular bone pattern marks its site. 8 Hyaline cartilage remains as a thin cap over the epiphysis to be the articular surface. 9 Growth in width of the shaft is by a periosteal deposition on the outside surface, coordinated with an osteoclastic resorption on the inner, marrow, aspect. These patterns may be reversed at sites of change in shape or drift. At the same time, shaft bone is remodelled internally to be more lamellar and have the layers. D OSTEOID | The osteoid seam is a very poorly mineralized, narrow zone of organic matrix seen sometimes with LM between the true bone and active osteoblasts. 2 It results from a definite lag between the formation of collagen fibrils and the later deposition of mineral crystals. 3 The presence of osteoid can be determined for sure by methods, e.g., von Kossa's silver, imicroradiography, EM, which are able to show the absence of mineral, but certain stains for decalcified sections are reliable. 4 The seam widens significantly in osteomalacia and rickets when too little Ca°* is available, e.g., in kidney disease. E PHYSIOLOGICAL FACTORS AFFECTING CONNECTIVE TISSUES | HormonesLecture#8 The cartilage and bone. * Growth: for matrix synthesis, particularly in epiphyseal cartilage; lack causes dwarfism, excess gigantism, or in adult-onset, acromegaly. * Parathyroid: acts on osteoblasts to cause osteoclasts to resorb bone, thus raising blood Ca”*; lack of hormone results in death by tetany; large eroded spaces from an excess fill with fibrous CT in ‘osteitis fibrosa’ - hyperparathyroidism. (There is also the paradox that small doses of PTH stimulate bone formation.) * Calcitonin: acts on the ‘clast to block bone resorption and lower blood Ca”, but its role seems to be minor (except in treating Paget's disease - uncoordinated ‘clasts and blasts). + Thyroid: acts indirectly on all cell activities by controlling metabolic rate; lack thus slows growth; an excess favours resorption. + Sex: affect genital tract CT, e.g, endometrial stroma; also the timing of secondary ossifications and epiphyseal closure (premature fusion and dwarfing follow an excess of gonadal hormone in childhood). * Glucocorticoid: excess impairs bone and CT matrix synthesis; used clinically to reduce inflammation, pethaps by reducing prostaglandins + Insulin, Norepinephrine, etc: control fat cell metabolism. * Parathyroid hormone-related protein/PTHrP is a PTH-like peptide released from various tumours that causes a cancer-linked destruction of bone, and hypercalcaemia. (PTHrP acts more in a local - paracrine - manner than the endocrine PTH.) 2 Agents | Vitamin D: in its active form is needed for Ca’* to be absorbed in the gut; low blood Ca®* from a lack of D prevents mineralization of growth cartilage matrix, resulting in rickets, and causes the failure of osteoid to mineralize in osteomalacia; excess D may raise blood Ca” to the point where soft tissue calcifications occur. 2 Prostaglandins: stimulate osteoclastic bone resorption, 3 Peptides: although thought of originally in the contexts of immunity and haemopoiesis, the cytokines (see F below) influence matrix formation and destruction, and the numbers and activities of all connective tissue cells, e.g., macrophages stimulate fibrogenesis, and osteoblasts interact with osteoclasts, ete 3 Diet | Calcium, phosphorus: see 2.1 above for Ca’* deficiency. 2 Vitamin A: excess and deficiency disturb ossification and remodelling in different ways. 3 Vitamin C: deficiency (scurvy) impairs collagen synthesis in all CTs. 4 Copper: needed for making elastin. 5 Toxic elements, e.g. Pb, "Sr, F, may substitute for the natural elements and ions in the mineral crystals of bone and teeth. 4Use Ts respond to more use by making a matrix better able to withstand the greater forces, e.g., osteoblasts build more and wider bone trabeculae; conversely, disuse leads to the few thin and frail trabeculae of osteoporosis.Chapter 9: The muscular tissues. General morphofunetional characteristics. Muscular tissues are united by one function — contraction. Skeletal muscle tissue provides locomotion. Smooth muscular tissue contracts the walls of internal organs and vessels. Cardiac muscle tissue pumps blood along vascular network. Myoneural tissue is responsible for pupil contraction and dilatation. Myoepithelial tissue facilitates excretion of secretory products by glands. Thus, we can assume that there are many various contractive processes in an organism. All of them arte based on myofibril contraction. The myofibrils are special organelles of contraction, which are made of actin and myosin filaments. Processes of contraction, which are based on cytoskeleton protein interactions, are in any cell of an organism, However, in muscles the contraction is the main function. Structural basis for this function is myofibrils. They were developed in phylogenesis on a base of contractive elements of cytoskeleton. Summarizing we can say that second common feature of muscular tissues is a having of myofibrils. Concerning other elements of structure it is possible to say that muscular tissues have more differences than similarities. They have different origin. The source of development for skeletal muscular tissue is myotom of a somit. Cardiac muscular tissue is derived from myoepicardial plate, which is part of visceral layer of splanchnotom. Smooth muscle tissue is originated from mesenhyme, generally splanchnotomic mesenhyme. Myoneura tissue have been developed from neuroectoderm, whereas myoepithelial tissue — from skin ectoderm. The tissue elements of skeletal muscle tissue are symplasts and cells (myosatellite cells). Other muscular tissues are made exclusively from cells: in cardiac muscle tissue they are named as cardiomyocytes, in smooth — smooth myocytes, in myoneural — myoneurocytes, in myoepithelial — myoepitheliocytes. Muscle cells do their work in close collaboration with nervous tissue. By this, the skeletal muscle tissue receives somatic innervation, whereas all others receive autonomic innervation. Regeneration abilities of muscular tissues are also different. Skeletal muscle tissue has cambial elements (myosatellite cells) and it can regenerate satisfactory in proper conditions. It can combine cellular and intracellular regeneration. Smooth muscular tissue and myoepithelial tissue have cambial elements as well and they have good regeneration. In cardiac muscle tissue of adult person the cambial elements are absent. So, the majority cardiomyocytes have no ability for division. It cardiac muscle tissue has been damaged; it is substituted by connective tissue. Regeneration of cardiac muscle tissue can be only on intracellular level. Regeneration potential of myoneural tissue is not well studied. Classification of muscular tissues. There are several approaches in muscular tissues clasifiation, 1. Physiological classification. Here, the muscular tissues are divided into voluntary and involuntary. The voluntary muscular tissue is skeletal muscular tissue. It’s contraction is controlled by conciseness. The contraction of the muscle is rapid. It can stay in contractive state relatively short period. The relaxation is also rapid. The involuntary muscles are all other types of muscular tissues. It is interesting to point out that smooth myocytes contractions are long. They can stay contracted for a long time. The relaxation time is also long. The contractions of cardiac muscle tissue are automatic. The differences in contraction can be explained by peculiarities of innervation and also by presence of condueting cardiomyocytes in cardiac muscle tissue. 2. Morphological classification. It is based on structural phenomenon such as presence of myofibrils striation. Accordingly to this classification all muscular tissues are divided into: = Striated. = Unstriated. Unstriated muscular tissues are smooth muscular tissue, myoneural and myoepithelial muscular tissue. Myofibrils are arranged in such way that the striation is invisible. Striated muscular tissues are skeletal striated ‘muscular tissue and cardiac muscle tissue. Here, the myofibrils are arranged in sarcomeres. They have lighter and darker regions. The interchange of the regions gives striated appearance to the tissues. 3, Histogenetic classification of muscular tissues. It considers a source of developmet for the tissues. The classification can be presented in the table below. MUSCULAR TISSUES. Mesodrem origin ‘Mesenchyme origin | Ectoderm originMyotom ‘Coelom ‘Smooth muscles Myoepithelial ‘Myoneural Skeletal Cardiac Smooth muscular tissue. HISTOGENESIS. The source of development for smooth muscular tissue is splanchnotom mesenchyme. The cells of splanchnotom mesenchyme migrate and surround organs, which are to have smooth muscular tissue. The beginning of differentiation of smooth muscular tissue is characterized by elongation of mesenchyme cells and by transformation of them to spindle-shaped or star-shaped cells. The organelles of protein synthesis appear in cytoplasm of the cell. They start production of specific proteins, which are typical for smooth muscle cells (myofibrils proteins). Then, the proteins are used for myofibrils construction. When myofibrils have been accumulated, the cells can response on irritation by contraction. Some of the cells keep staying in undifferentiated position, Later, they can be used for regeneration. STRUCTURE. In adult organism the smooth muscular tissue is included to walls of internal organs, into coats of alimentary tube, into blood vessels, into bronchi and so on. The smooth muscular tissue of vessels can differ from smooth muscular tissue of other localization. Stuctural-functional unit of the tissue is smooth myocyte (sometimes, intercellular substance made by myocytes is mentioned as second elementary unit). The smooth myocyte is the cells of spindle shape. The length may vary from 20 to 500 mom, like in a uterus (there they also have special star-like shape). Nuclei are of oval or band shape with dense chromatin and with 1-2 nucleoli. The smooth myocyte is surrounded by cytolemm, Outside of eytolemm there is basement membrane with reticular fibers. There are all organelles of general purpose in myocyte cytoplasm. They are generally located near the nucleus. They are presented by rough EPR, Golgi complex, mitochondria and etc. There are many vesicles located under the cytolemm (eaveoli). Such vesicular apparatus is needed for Ca** storage. Such ions are needed for contraction. The apparatus serves as analogous of sarcoplasmic reticulum and T-tubules, as well. There are elements of reduced sarcoplasmic reticulum as small vesicles and cisterns in smooth myocyte. Caveoli may have connections with them. Membranes of caveoli and sarcoplasmic reticulum have calcium ion channels in membrane. There are glycogen inclusions in the cytoplasm. On a periphery of myocyte there are dense bodies made of a-actinin protein. They are analogues of Z-lines of sarcomere, There are two variants of dense bodies: 1) attached to internal surface of cytolemm with help of adhesive proteins. They look like folds of internal surface of cytolemm. 2) freely lying in cytoplasm. They are arranged as chain, Actine and intermediate filaments are attached to the dense bodies. The last makes three dimentional network in cytoplasm. ‘The important component of cytoplasm of smooth myocytes is contractive filaments or myofillaments. They make myofibrils, Such filaments are arranged along longitudinal axis of myocyte. Such arrangement is not strict, so myocyte is not look as striated, Thin actin filaments at one end are attached to dense bodies. They have no troponin and tropomyosin in compare with skeletal muscles. They interact with thick myosin fibers making so called contractive unites. The myosin filaments of smooth muscles are different from those of skeletal muscles. They are less stabile. Even some investiganots consider that smooth muscle myosin is depolimerizied when there is no contraction (in resting state), That is why there is no fibrils in smooth myocyte, there are no sarcomeres, and that is why the striations are absent. Formation of myosin occurs right after initiation of contraction. This process is activated by calcium ions as well as process of contraction. Calcium ions are injected from caveoles. sarcoplasmic reticulum and mitochondria, The units, which have been made, are under an angle to myocyte length. Mechanism of smooth myocyte contraction is generally similar to contraction mechanism of skeletal muscular tissue, which will be discussed later. The ions of calcium participate in the mechanism as triggers. They are released by sarcoplasmic reticulum, caveoli and mitochndria, Incoming nervous impulse activates calcium influx. Calcium makes complex with calmodulin protein. The complex *Ca-calmodulin” activates enzyme kinase of light myosin chains. It does phosphorylation for light myosin chains. This gives ability to interact with actin active centers. They interact with active centers of actin, While this interaction the myosin molecule change conformation as if it pulled itself forward. As result of recurring interaction the myosin molecules crawl along actin. This makes dense bodies close to each other. It results in contraction of smooth myocyte. Intermediate desmin filaments resist excessive deformation of smooth myocyte during contraction. ATP is needed for contraction, The hydrolysis of ATP is very slow here. It causes long time of contraction. 2‘There is an enzyme which terminates the contraction. It is myosin phosphatase. It splits phosphate residue from light chains of myosin (dephosphorilation). But the feature of smooth muscles is that not all myosin bridges are destroyed after dephosphorilation. Some of myosin heads have being connected with actin filaments. It helps in tonus keeping in smooth myocytes without excessive energy wasting. Smooth myocytes does not work alone. They form myocyte complexes. Nerve endings have no touch to each smooth myocyte. They connect with one in a complex. The complex consists of 10-12 myocytes. Myocytes in the complex are connected with each other with help of gap junctions (nexuses) and desmosomes. There is no basement membrane in the nexus region, The nexuses help in exitation transmitting from one myocyte to another. So, the excitation becomes spread on whole complex. ‘There are several functionally different myocytes in the complex. 1. Contractive myocytes are specialized in contraction. 2. Secretory myocytes produce and secrete intercellular matrix. 3. Pace-maker myocytes can induce action potential and transmit it to neighbor myocytes. 4. Cambial (undifferentiated) myocytes serve as source of cells for regeneration. Having similar origin, the smooth myocytes are genetically close to fibroblasts and to other similar cells producing ground substance. They are able to produce ground substance of muscular tissue, Sometimes it is considered as second tissue element of smooth muscular tissue. Regeneration of smooth muscular tissue is performed not only with help of undifferentiated smooth myocytes, but also adventitial cells can be employed. In reparative regeneration some myofibroblasts can participate in regeneration of smooth muscular tissue due to close genetic origin. The intracellular regeneration of smooth myocytes is also possible. It is based on hypertrophy and hyperplasia of organelles. Myoepithelial tissue. Tissue element of the tissue is myoepitheliocyte or basket cell. The source of development for the tissue is skin ectoderm. It’s localization is — terminal portions and some excretory ducts of sweat, mammary, lachrymal and salivary glands. The cells are differentiated from ectoderm together with secretory cells. Myoepitheliocytes are closely adjusted to secretory cells. Structure. Myoepitheliocytes have star-like shape. They have many processes by which they capture terminal portion of a gland. There are actin filaments in processes. The synthesis of myosin filaments is activated by calcium ions and it takes place right before contraction. Ca” influx is induced by nervous impulse. Myofibrils have no striation, Contraction of processes results in compression of terminal portion of the gland and in evacuation of secretion products. There is basement membrane outside of myocyte. Regeneration. There are some cells with signs of undifferentiated cells amoung differentiated myocytes. The regeneration takes place due to their division and differentiation to mature myoepitheliocytes. Some other findings show that regeneration is performed with help of cambial cells of striated epithelia. Cells of epithelium can be differentiated to secretory cells and myoepitheliocytes as well. Myoneural tissue. This type of muscular tissue is in the following muscles: sphincter and dilatator of pupil. The source of development for the tissue is neuroectoderm. The structural-functional element of myoneural tissue is myomeurocyte or myopigementocyte. They ate spindle shaped cells with single nucleus. They have myofibrils such as actin and myosin, which are made as in smooth muscular tissue. The thick myosin fibrils are made right before contraction. It is initiated by calcium ions. There are many mitochondria and pigment granules in the cell. There are nexuses and desmosomes between cells. Innervation of the tissue is performed by autonomic nervous system, Regeneration of the tissue isn’t fully investigated. Endocrine myocytes are referred as modified myocytes. They are located in muscular coat of afferent and efferent artery in renal glomerulus (juxtaglomerular cells). They produce hormone — rennin, They are characterized by well developed apparatus of protein synthesis and reduced apparatus of contraction. It is important to note that there is significant contractive ability in myofibroblasts. Skeletal muscular tissue. Funetions: 1) Locomotion. It is the main tissue of skeletal muscles. Apart to skeletal muscles it is a part of tongue, walls of oral cavity, esophagus, larynx, anus of rectum, eye moving muscles. 2) Thermoregulation. Contraction of skeletal muscular tissue results in formation of heat. It helps to warm up the body in cold conditions.Tissue elements of skeletal muscle tissue are symplasts (striated muscular fibers) and cells (myosatellite cells) HISTOGENESIS OF SKELETAL MUSCULAR TISSUE. The source of development for skeletal muscular tissue is myotom of somites. The majority of myotom cells becomes mitotic myoblasts (Gi-myoblasts), which actively proliferate. Part of Gi-myoblasts are segregated from main pool and later they become myosatellite cells. Other myoblasts have the differentiating mitosis. After differentiation they become postmitotic myoblasts (Go-myoblasts). They are single nucleated, spindle shaped cells with ability to produce some specific proteins. They migrate from myotom to the destination place — place of definite muscle location. They migrate together with myosatellite cell. While migration these cells save their undifferentiated state. This stage is called myoblastic stage (I stage of myogenesis). II stage of myogenesis is - myosymplastie stage. Here, myolasts are arranged in the chains. They fuse with each other. It results in myosymplst formation. Active protein synthesis takes place in cytoplasm. Synthesized proteins are combined making myofibrils. The myofibrils are located on a periphery of the cell Nuclei are located centrally. There are no more divisions of nuclei after myoblasts fusion, The myosymplast ‘grows in length by involvement of new myoblasts in fusion, and in width by producing of contractive elements. III stage ~ stage of muscular tubules. Here is enlargement of myofibril number. They still lie on the periphery. Nuclei are located centrally too. Muscular tubule can be split along their length. This is the way of increasing their number. Growing in length, they continue involving of new myoblasts in fusion. IV stage — stage of mature muscular fiber. Here, the volume of myofibrils in the cell becomes so significant that they shift nuclei to the periphery. Smooth EPR becomes well developed. The fiber accumulates mitochondria, Well developed in myoblasts, rough EPR subjects to reduction, STRUCTURE OF MUSCULAR FIBER. The muscular fibers are the functional and structural unit of skeletal muscular tissue. They have length up to 20-30 em and width about 100 mem. They have two parts: 1) myosymplast and 2) myosatellite cells. Symplast is covered by sarcolemm and contains many (up to several Thousands) nuclei, Sarcolemm consists of thick basement membrane and of plasmalemm of muscular fiber. There are spaces between plasmalemm and basement membrane where satellite cells are. In light microscope these cells are undistinguishable from connective tissue cells. In electronic microscope it is possible to find their own plasmalemm and weakly developed organelles Myosatellite cells are cambial cells of skeletal muscular tissue. They are very important in regeneration. Protoplasm of the muscular fiber is known as sarcoplasm. It has many organelles of general purpose (excluding centrioles): mitochondria, lysosomes and ete. Golgi complex is developed relatively weak. Smooth EPR is well developed. It is known as sorcoplasmic reticulum. Rough EPR is weakly developed. There are inclusions of glycogen and lipids which are needed for energy supplement. There are myoglobin pigment inclusions. Myoglobin is ferrum-containing enzyme as hemoglobin is. It has higher affinity to oxygen. That is why it can facilitate oxygen transmition from blood to the muscular fiber. Myoglobin is especially abundant in the red muscular fibers providing their color. In some regions of musculer fiber there are projections of plasmalemm inside of muscular fiber in a form of tubules, They pass across the fiber. They are called T-tubules (from Latin transverses ~ located across). T-tubules surround each myofibril. To do so they are highly branched and they have connections between each other. There are L-cisterns (L - longitudinal) of sarcoplasmic reticulum from the both sides of the T-tubule. Reaching the T- tubule the L-cistemns fuse and make terminal cistems (T-cisterns), which are located across. Together with T- tubules the terminal cisterns make triads — itis special membrane system playing an important role in initiation of muscle contraction. There are special contacts between T-tubules and terminal cisterns. Through the contacts calcium ions can be transported. This helps the sarcoplasmic reticulum to accumulate calcium ions. The main pert of the fiber is made of organelles of special purpose ~ myofibrils. There are up to 2000 myofibrils in one fiber. The diameter of myofibril may reach 2 mcm, the length of it is equal to the length of muscular fiber. In light microscope each myofibril looks striated. There are light and dark disks. In polarizing microscope the dark disks have double refraction and that is why they are called anisotropic disks or A-disks. The light disks have no such feature, so they named as isotropic disks or I-disks. In the middle of I-disk there is black line, which is called Z-line. It resembles zigzag in longitudinal section of myofibril, but it looks as four- angular frame in cross section. The nodes of the frame are the place of actin filaments attachment. There is light band H in the middle of A-disk. Centrally in the H-band there is black M-line, The region of the myofibril between two neighboring Z-line is called sarcomer. The sarcomer is a structural- functional unit of the myofibril. It consists of Z-line, ¥ of I-disk, A-disk, of I-disk and second Z-line. Each sarcomer consists of thin actin and thick myosin filaments. Myofillaments are made of contractive proteins. The 4actin filaments consist of actin protein and alos of troponin and tropomyosin proteins. Molecules of actin protein have globular structure. It is so called G-actin. Several molecules of G-actin an join together making a long chain, Such the chain is called F-actin of fibrillar actin Each atin filament has two chains of this type. They are arranged as the double helix. In the grooves of the helix there are molecules of tropomyosin. They are also in the form of double helix. There are troponin molecules attached to the tropomyosin protein on the same distance away from each other. The troponin protein has 3 subunits: T1,C (shortly can be marked as TaT, Tal, TnC).TnT is responsible for the attachment to the tropomyosin molecule. TnC is responsible for binding calcium ions. Tal resists the interactions of myosin heads with active centers on the actine molecule. The intervals between troponins molecules are 40 nm. The diameter of the thin filaments is 5 nm. The thick filaments have diamenter 12 nm and they contain the myosin protein, Each myosin molecule consists of two parts: head and tail. It can be bent in two sites (joint sites). The head of myosin has ATP-ase activity, It can degrade ATP with liberation of energy. This energy is needed for contraction and relaxation as well. The myosin molecules join to bundles and make thick filament. It looks like a brush: the heads of myosin stick out of main rod. This picture is only on peripheral parts of the myosin filament. They are absent in the central part (so called “bold or naked” region). the feature of skeletal muscles. The myosin filaments of smooth muscles have heads sticking out of rod all the way along the myosin filament. Apart to the myosin, there are such proteins as titin, nebulin, myomesin and C-protein in the thick filaments. Molecules of titin look as a spring and they bind ends of the filaments to the Z-lines. These molecules make kind of framework inside of the sarcomer, maintaining the position of thin and thick filaments relatively to each other. They also resist over stretching of filaments. Nebulin connects thin and thich filaments. Myomesin and c-protein make connections between thick filaments in the M-line region. On electronic photos, the myosin heads are visible as cross bridges, if the fiber is contracted, In sarcomere, the thick disc are only in the A band. The thin filaments are generally in I band, but their end slightly enter A band. That part of A disk, which contains both actin and myosin filaments looks darker, whereas that part with only myosin filaments is lighter. The last one is H-line. M-line in the middle of a sarcomere is the place of connection of all myosin filaments. The myomesin and C-protein facilitate this connection. Examining the cross section of a myofibril, it is possible to see that one thick filament is surrounded by six thin filaments. The thin filaments are tightly connected to Z-lines. Z-lines are made of a-actinin, desmin and vimetin proteins MECHANISM OF MUSCULAR CONTRACTION. ‘The generally accepted theory is the model of muscular contraction suggested by H. Huxley or sliding- filaments hypothesis (1954). The summary of it is in the following. The nervous impulse reaches the muscular fiber. It is transmitted on post-synaptic membrane of motor-end plate. It is membrane of muscular fiber. Next the excitation goes to the T-tubules. With help of them it is transmitted deep to the muscular fiber. It involves the closely lying cisterns to the contraction. They release the stored calcium ions to the sarcoplasm. Unlike the smooth muscle, where the main target for calcium is myosin filaments, the calcium in skeletal muscles works on actin filaments. The calcium ions can disclose the active centers on thin filaments for the interaction with actin: the calcium ions migrate to troponin C.(Tn C) molecules and bind them. Troponin Tn I closes the active centers on actin filaments during relaxation phase. Ca binding changes the configuration of troponin and it causes opening the centers. Thus, the héaids of myosin, which have high affinity to actin centers, Tace the opportunity to bind them, Have been bound the active center, the myosin filaments are bent in the joint site. Doing this they pull the rest of the filament. Than they lose their connection to the active centers and relax. After this they are ready to bind next active center. The rate of such actions is 500 times per second, It causes sliding of thick filaments along thin filaments. To relax myosin head the ATP energy is needed. The head of myosin has ATP-ase activity, so it can provide release of energy for contraction. If death has been developed, the production of ATP is rapidly fallen. Therefore, the myosin heads are unable to get off actin filaments. This is the reason of cadaveric rigidity development. The development of cadaveric rigidity depends on agony duration, temperature of environment and other conditions. But it is constant for the definite complex of conditions. Disappearing of cadaveric rigidity is also take place in the definite time intervals as result of autolysis. The cadaveric rigidity can be destroyed artificially. All these facts about epy cadaveric rigidity are used in the forensic medicine to determine the time, which lasts from the death case. In the absence of activating nerve impulses Ca ™* ions are pumped back to cisterns of sarcoplasmic reticulum. The active centers on actin filaments become closed again by troponin. In the electron microscope the contractions is characterized by moving of Z-lines close one to another, decreasing of I-band, disappearance of M line and also by appearing of cross bridges from myosin heads. The number of these cross bridges increases 5during contraction. The following relaxation is characterized by reverse processes. There are functionally antagonistic muscles, such as flexors and extensors. The elongation of extensors during contraction of flexors is passive process. There are no specific relationships between actin and myosin filaments. They slide along each other passively. With help of specific proteins (dystrophin, vinculin, tallin, and others) and adhesive molecules (integrins, fibronectin and others) the myofibrils are connected with the basement membrane and components of intercellular matrix of endomysium. a / REGENERATION OF SKELETAL MUSCULAR TISSUE. oe we G17 Physiological regeneration. In the normal conditions the aging and degradation of muscular fiber parts take place. Reparation of them occurs with help of cellular and intracellular regeneration. Intracellular regeneration is needed to replace aging cellular organelles and other parts of muscular fibers. Cellular regeneration employs division of myosatellite cells, transforming them to myoblasts, incorporation of myoblasts to existing muscular fibers and differentiation of them to the fragment of the fiber. Reparative regeneration. It is also performed on intracellular and cellular level with help of myosatellite cells, There is inflammatory reaction in the place of damaged fiber at the beginning. At the same time some muscular fiber are partially destroyed little bit away from the damage site. Cells of immune system, such as macrophages and neutrophils, actively consume dead tissues and cells. They clean the space for following regeneration. If phagocytes are over activated, they can release inflammatory substances causing additional degradation of muscular fibers. The substances formed during tissue degradation can stimulate regeneration. Regeneration of vessels (angiogenesis) follows regeneration of muscular fiber. The regeneration of muscular fibers is the composition of two closely related processes: 1) formation of growth buds (intracellular egeneration); 2) division and differentiation of myosatellite cells (cellular regeneration). In the first case the formation of growth buds on the ends of damaged muscular fibers takes place. The growth bud is pear shaped enlargement of protoplasm on the end of the fiber. Due to intracellular regeneration (formation of new sarcomers and etc) the buds can grow toward each other. In the second case the activation of myosatellite cell near the wound takes place. Dividing and differentiating myosatellite cells provide development of stages looking similar to phases of myogenesis: 1. Myoblasts stage. There is transformation of satellite cells to myoblasts, which undergo active mitotic divisions. 2. Myosymplast stage. Myoblasts merge with each other making myosymplasts with myofibrils on the periphery and nuclei in the central part. 3. Stage of muscular tubules. 4, Stage of mature muscular fibers. It is important to note that myoblasts can both merge with cach other making muscular tubules and be incorporated to growth buds enhancing their growth. ‘Newly made regions of muscular fibers are thinner that preexisting ones. They are not fully differentiated. Correct regulated physical load facilitate their transformation to normal fibers. CONDITIONS OF GOOD REGENERATION OF SKELETAL MUSCULAR TISSUE. In a case of small injury the regeneration of skeletal muscle is complete. Recently, with help of microsurgery methods it has became possible to achieve complete regeneration of skeletal muscle even in case of massive injury. It allows reimplantation of amputated limbs. The conditions of good regeneration of skeletal muscular tissue are: 1. Closing the wound sides by suing them. 2. Complete removal of dead tissues from regeneration zone. Restraining development of connective tissue. 3. Suing of nerves and vessels with help of microsurgery. 4. Keeping intact basement membrane of muscular fibers. It prevents entering of fibroblasts to damaged muscular fibers. It facilitates orientation of muscular tubules. GROWTH AND ADJUSTMENT TO DIFFERENT LOAD ON SKELETAL MUSCLE. There are significant changes in muscular tissue during ontogenesis. They are connected with it’s growth and adaptation to different environmental conditions. ‘The growth of muscular tissue is performed by two processes: 1) widening of myofibrils and 2) lengthening of myofibrils. The widening of muscular fiber can be due to formation of new myofibrils as well as due to widening of already existing myofibrils. It is also possible that wide already existing myofibrils can be split to smaller ones. At the same time the muscular fiber accumulates sarcoplasma and organelles. 6The lengthening of myofibrils and muscular fiber as whole can be performed in two ways: 1) adjustment of new sarcomeres to the ends of myofibrils, 2) fusion of myosatellite cells with muscular fiber. The new components of sarcoplasma are also produced in sufficient amount. Hypertrophia of skeletal muscular tissue — is original adaptation of muscular tissue, which takes place after a long period of time of increasing muscular load. It is characterized by increasing of anabolic processes over catabolic processes. The hypertrophia is based on the increasing of myofibrils number and size. If training is performed to increase the endurance of muscles, the main increasing takes place in sarcoplasma. It increases in volume. But if training is performed to increase the power of muscles, the main increasing takes place in myofibrilar apparatus. Atrophia of muscles can be observed in such conditions as hypodynamia (low physical activity), denervation (loss of nerve supply to a muscle) and fasting. In case of fasting and hypodynamia the atrophia is some kind of adaptation to extreme conditions. There is the inherited atrophis (or better to say dystrophia). It is result of genetic defects. Some genetic defects are connected with the failure of dystrophin proteins synthesis. ‘These proteins are responsible for binding of myofibrils with sarcolemma and intercellular ground substance of endomysium. The binding provides the normal biology of muscular fibers. The deficiency of dystrophin proteins causes degradation of muscular fibers and growth of adipose and connective tissue instead of muscular fibers. Stimulation of regeneration and hypertrophia of skeletal muscular tissue. There are several ways to stimulate regeneration of muscular tissue. 1) treatment by anabolic hormones (androgens and it’s synthetic analogs, insulin, growth hormone); 2) treatment by vitamins. The vitamins which play important role in protein synthesis are of great value here (B12, pholic acid, potassium orotate). 3) It was shown in the experiment that the regeneration is increased by injection of thoroughly cut muscle to the regeneration zone. 4) implantation of myosattelite cells culture is one of contemporary methods of muscle growth stimulation. 5) the early limited muscular load on muscle is also very important. STUCTURE OF SKELETAL MUSCLE AS AN ORGAN. The muscle consists of many muscular fibers connected by connective tissue. The number of muscular fibers in muscle may vary in wide limits: from several hundreds of thousands to several millions. The connective tissue between muscular fibers is called endomysium, The fibers of endomysium are closely connected with the basement membrane of muscular fiber. Several muscular fibers (10-100) are surrounded by thicker layer of connective tissue, which is called perymysium. Connective tissue contains nerves and vessels, which supply the muscle. The muscle as whole is surrounded by epimysium. It is dense regular connective tissue. From the both ends of the muscle there are tendons. They are tightly attached to the muscle. The sarcolemma of muscular fibers makes many interdigitations, which come and enter between collagen fibers of the tendon. The collagen fibers of the tendon are closely adjusted to the basement membrane of muscular fibers. TYPES OF MUSCULAR FIBERS. There are three types of muscular fibers. I type ~ red muscular fibers. They have small diamenter. The sarcoplasma prevails over myofibrils. There is a lot of myoglobin in sarcoplasma, which provides the red color of the fibers. It is idle (tonic) muscular fiber. They have many mitochondria and they have high activity of redox systems. They have good deposits of nutrients and they can work during a long time, but slowly and with small power of contraction. They contain many satellite cells and have good blood supply. They are found in muscles, which perform long tonic work, such as breast muscles in migrant birds. IIB type - white muscular fibers. They have large diamenter and well developed myofibrils. But sarcoplasma is developed less than in red muscular fibers. That is why it contains less nutrients and mitochondria, The fibers have low activity of oxidative enzymes, whereas activity of glycolytic enzymes is very high. They have good storages of glycogen. They are known as rapid, tetanic fibers. They can make contraction of strong power, but not for a long time. They get tired very fast. They have relatively weak blood supply. The muscle of limbs have to perform strong and rapid movement. That is why they are made of such type of muscular fibers, This fibers are subject to hypertrophy faster and more significant than the red muscular fibers. IB type — Intermediate type of muscular fibers. Structurally and functionally they are in intermediate position between red and white muscular fibers. They use glycogen as well as lipids for nutrition. They have approximately the same level of oxidative and glycolitye enzyme activity. Every human being has very particular ratio of these three types of muscular fibers. That is why people have different abilities for sports and physical training,Cardiac muscle tissue. Developemnt. The source of development of cardiac muscle tissue is myoepicardial plate — the part of visceral splanchnotom in neck region of embryo. The cells of the plate actively transform to myoblasts, which undergo mitosis and differentiation. The myofillaments are produced in cytoplasm of myoblasts. Then they form myofibrils. At the beginning, the myofibrils have no striations and definite orientation in cytoplasm. In the process of further differentiation they acquire longitudinal orientation and they become attached to forming Z- lines by thin myofillaments. Due to increasing order of filaments the myofibrils become striated. The cardiomyocytes are formed. The cytoplasm accumulates organelles: mitochondria, rough EPR, free ribosomes. In the differentiation process the cardiomyocytes keep reproducing themselves. In some cells the division of cytoplasm is absent. It causes formation of cells with two nucleuses. Developing cardiomyocytes have definite orientation in space. They get placed in the chains. In the places where they touch each other they form intercellular junctions — intercalated disks. The cardiomyocytes undergo divergent differentiation, [t results in formation of three types of cardiomyocytes: 1) working or typical; 2) conducting or atypical; and 3) endocrine cardiomyocytes. At the end of differentiation the cardiomyocytes loose their ability to reproduce themselves, There are no cambial cells in mature cardiac muscle tissue. Structure. The cardiac muscle tissue is made of cells ~ cariomyocytes. The cardiomyocytes are only tissue clement of the tissue. They are connected with each other with help of intercalated disks and they make functional muscular fibers or “functional symplast”, which isn’t morphologically true symplast. Functional muscular fibers are branched and they have connection on the side surfaces also. Is results in formation complex three-dimensional network. Cardiomyocytes have elongated shape with a few processes. They have nucleus and cytoplasm, Many cells (more than half in mature heart) have two nucleuses. The nuclei are big, light. Hey are located in the center of cardiomyocyte. The cytoplasm of cardiomyocytes is oxyphilic. It contains many organelles and inclusions. There are striated myofibrils on the periphery of the cell. Their structure is the similar as in the skeletal muscles. In compare with skeletal muscle, the myofibrils can fuse with each other making holistic structure. They also contain contractive proteins which are different to those in skeletal muscles. There are many mitochondria between myofibrils. Sarcoplasmic reticulum (SPR) and T-tubules are developed less than in skeletal muscles. It is due to less influence of nervous system and automatic work of cardiac muscle. SPR and T-tubule make dyads instead of triads in skeletal muscle (T-tubule is connected with only one cistern of SPR). The typical terminal cisterns are absent. SPR is less active in calcium storage. The cardiomyocytes are covered by sarcolemm. It consists of plasma membrane of cardiomyocyte and basement membrane. The basement membrane is tightly connected with intercellular ground substance by collagen and elastic fibers. The basement membrane is absent in the intercalated disks. The intercalated disk is very interesting site. They are connected with componetes of cytoskeleton and with intercellular ground substance through integrins of cytolemm. They provide mechanical, as well as, chemical connection between cardiomyocytes. In light microscope they are visible as black cross strips. In electronic microscope they are visible as zigzag lines. It is possible to identify horizontal and vertical regions and zones. 1. Zone of desmosomes and adhesive belts. They are located in vertical regions of disks. They are responsible for mechanical binding of cardiomyocytes. 2. Zone of nexuses (gap junctions). It is the place of excitement transmittion from one cell to another. They provide chemical communication between cells. They ate located in horizontal regions of disks. 3. Zone of myofibrils attachment. They are located on vertical regions. They serve as the place for myofibrils attachment, The myofibrils are attached to Z-lines located on the inner surface of cytoplasm. It was found the cadherins are in high concentration in intercalated disks. These molecules are responsible for calcium mediated adhesion of cardiomyocytes with each other. Types of cardiomyocytes. In the different parts of the heart the cardiomyocytes have different properties. For example, in atrium they can divide by mitosis, whereas in ventricles they are not able to do it. There are three types of cardiomyocytes which are different in structure and in functions: working, endocrine and conductive. 1. Working cardiomyocytes. They have the structure described above.2. Endocrine cardiomyocytes. They produce sodium-uretic factor (SUF). They are located mainly in right atrium, SUF inereases excretion of sodium ions by kidneys. It also relaxes smooth myocytes of vascular wall and suppresses secretion of hormones causing hypertension. It results in diuresis increasing, arteries dilatation, decreasing of circulating blood volume and generally in decreasing of blood pressure. It is important to note that during embryogenesis all cardiomyocytes have ability for secretion, but ventricle cardiomyocytes reversibly loose this function. 3. Conductive cardiomyocytes. They sharply differ from working cardiomyocytes. They make conductive system of heart. These cells are twice bigger than working cardiomyocytes. They have less myofibrils. There is an increased amount of glycogen in cytoplasm. Due to this the cytoplasm is not stained well. There are many lysosomes. T-tubules are absent. The function of atypical cardiomyocytes is to generate and conduct electric impulses for the working cells. The appropriate functioning of atypical cells results in automatic heart working. However, the heart working is tightly regulated by autonomic nervous system. Thus, sympathetic nervous system increases heart rate and contraction force, whereas parasympathetic nervous system has opposite effect. REGENERATION OF CARDIAC MUSCLE TISSUE. Physiological regeneration. It can be performed on intracellular. It is very intensive due to big load on cardiac muscle. It can be even more during physical work and in pathological conditions (hypertension). In these cases the elements of cardiomyocyte cytoplasm having been worn out rapidly. They have to be substituted by new elements. If the heart faces overload it can develop hypertrophy (increasing in size) and hyperplasy (increasing in number) of organells, including myofibrils. In youth the heart can develop polyploidy of cardiomyocytes. Working hypertrophy is characterized by adequate developing of vascular network of heart. In pathological conditions the vascular network does not develop adequately. Than growing cardiomyocytes face deficiency in blood supply and part of them die due to this. Dead cardiomyocytes are substituted by connective tissue (cardiosklerosis). Reparative regeneration. It takes place after a wounding of heart muscle, a myocardial infarction However, the cardiac muscle has no stem cells. That is why only the intracellular regeneration is present — regeneration of cellular organelles of neighboring cells. They increase in size and take the function of lost cardiac cells. The dead cardiomyocytes are replaced by connective tissue. Not so far ago it was stated that smaller part of the cardiomyocytes die by necrosis during myocardial infarction. These are the cells located directly in the center of infarction site. The cells located on the periphery die mostly by apoptosis little bit after infarction. This process is the main process in cellular damage during infarction. That is why the main attention of the investigators should be concentrated on prevention of apoptotic death of cells. There were observed the possibility of mitotic divisions in the wall of the atrium, The number of such divisions is small and they generally take place in young people Stimulation of cardiac muscle regeneration. 1) Prevention of apoptotic death of cardiomyocytes by prescribing preparations, which can increase circulation in microcirculatory network, decrease blood clotting and viscosity and by this make blood supply of the cells better. 2) Prescription of anabolic drugs. 3) Early setting of gradual physical exercises. In the last years, there were set the experiments in which the regeneration of cardiac muscle tissue was stimulated by transplantation of myosattelite cells of skeletal muscle tissue. It was stated that transplanted cells can make skeletal muscular fibers, which have close contacts and relationships with cardiac muscle cells. The substitution of cardiac muscle by skeletal muscle is preferable in compare with substitution by connective tissue because skeletal muscle tissue has ability for contraction. Therefore, the further development of this method has very good perspectives. Age changes of muscular tissues. 1.Smooth muscular tissue. It was observed the further differentiation of smooth myocytes in the coats of intemal organs during early period of postnatal ontogenesis. This is characterized by the increasing of myocyte complexes due to increasing of myocytes number in complex, as well as, increasing of myocyte size. Owing to this fact, the muscular coat of the internal organs has been expanded up to puberty. Process of aging results in gradual decrease of myocyte complexes due to enhanced apoptotic death of smooth myocytes. It results in decreasing of muscular coat width in internal organs. Sometimes the abnormal