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Welcome to Indian Dental Academy ...

Welcome to Indian Dental Academy
The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and offering a wide range of dental certified courses in different formats.

Indian dental academy has a unique training program & curriculum that provides students with exceptional clinical skills and enabling them to return to their office with high level confidence and start treating patients

State of the art comprehensive training-Faculty of world wide repute &Very affordable.

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  • 目前已有許多的證據顯示,ECM的更新(turnover)在組織的重組上是一個關鍵的步驟,其中會伴隨許多生理或是病理的反應。最近的研究指出,這些機制包含了很多分解細胞外間質的相關酵素(ECM-degrading enzymes),其中最大的族群即是」間質金屬結合蛋白脢」(Matrix Metalloproteinase,MMP)。MMP是一個酵素族群,其中至少包含了十五個種類,其中大多是需要鋅的生呔類酵素(Zn-dependent endopeptidase)。 <br />

Biomaterials /certified fixed orthodontic courses by Indian dental academy Presentation Transcript

  • 1. THE EXTRACELULAR MATRIX, CELL-BIOMATERIAL INTERACTIONS INDIAN DENTAL ACADEMY Leader in continuing dental education www.indiandentalacademy.com www.indiandentalacademy.com
  • 2. 1. TISSUE RESPONSE TO INJURY A. Inflammation and Repair B. Cell Regenerative Capacity C. Extracellular Matrix Remodeling 2. CELL/TISSUE-BIOMATERIALS INTERACTIONS 3. TECHNIQUES FOR ANALYSIS OF CELLS AND TISSUES A. Light Microscopy a. b. c. d. e. f. B. C. D. E. Tissue Sample Fixation Dehydration&Embedding Sectioning Staining Special Staining Electron Microscopy Three-Dlmenslonal Interpretation Artifacts Artifacts Identification, Genotyping, and Functional Assessment of Cells, Including Synthetic Products,In Cells or Tissue Sections www.indiandentalacademy.com
  • 3. 1.A.Inflammation and Repair Inflammation and repair follow cell and tissue injury induced by various exogenous and endogenous stimuli. Inflammation is a protective response that eliminates (i.e., dilutes, destroys, or isolates) the cause of the injury (e.g., microbes or toxins) and disposes of both the necrotic cells and tissues that occur as a result of the injury. www.indiandentalacademy.com
  • 4. 炎滲液 Pathways of reparative responses after acute inflammatory injury . www.indiandentalacademy.com
  • 5. The inflammatory response initiates the process that heals and reconstitutes the normal tissue. During the reparative phase, the injured tissue is replaced by native parenchymal cells, or by filling up the defect with fibroblastic scar tissue, or both. The outcome depends: Restoration of normal structure and function (1) tissue injury is transient or short-lived (2) tissue destruction is small (3) the tissue is capable of regeneration Scarring results the injury is extensive or occurs in tissues that do not regenerate. www.indiandentalacademy.com
  • 6. ( A ) Initial contact of cell with solid substrate . ( B ) Formation of bonds between cell surface receptors and cell adhesion ligands . ( C ) Cytoskeletal reorganization with progressive spreading of the cell on the substrate for increased attachment strength . www.indiandentalacademy.com
  • 7. An abscess 膿腫 is the outcome when an infection cannot be eliminated, the body "controls" the infection by creating a wall around it. Inflammation processes: @ Acute inflammation @ Chronic inflammation @ Scarring @ Acute inflammation The immediate and early response to injury, of relatively short duration, is characterized by fluid and plasma protein exudation into the tissue, and by accumulation of neutrophils. www.indiandentalacademy.com
  • 8. @ Chronic inflammation • • • This phase is manifested with concurrent tissue destruction, and can evolve into repair involving fibrosis and new blood vessel proliferation. A special type of inflammation characterized by activated macrophages and often multinucleated giant cells is called a granuloma. The pattern occurs where the inciting agent is not removable, including the foreign body reaction, a characteristic inflammatory reaction to the implantation of a biomaterial. www.indiandentalacademy.com
  • 9. @ Scarring Scarring occurs as a composite of three sequential processes: (1) formation of new blood vessels (angiogenesis ) (2) deposition of collagen (fibrosis) (3) maturation and remodeling of the scar (remodeling) The early healing tissue rich in new capillaries and proliferation of fibroblasts is called granulation tissue. www.indiandentalacademy.com
  • 10. Inflammation is also associated with the release of chemical mediators from plasma, cells, or extracellular matrix, which regulate the subsequent vascular and cellular events and may modify their evolution. www.indiandentalacademy.com
  • 11. The chemical mediators of inflammation: vasoactive amines ( 血管活性胺 ) arachidonic acid metabolites ( 花生四烯酸代謝產物 ) 源 自 細 胞 in the cyclooxygenase( 環加氧黴 ) pathway ( the prostaglandins, PG 前列腺素 and the lipoxygenase( 脂質加氧黴 ) pathway ( the leukotrienes, LT 白細胞三烯 platelet-activating factor lysosomal granules of inflammatory cells 源 自 血 漿 nitric oxide Polypeptide growth factors cytokines plasma proteases ( of the coagulation 凝血 , fibrinolytic 纖溶 , kinin 激汰 , and complement 補體四個系統 ) www.indiandentalacademy.com
  • 12. Growth factors may act by endocrine (systemic), paracrine (stimulating adjacent cells) or autocrine (same cell carrying receptors for their own endogenously produced factors) mechanisms. Endocrine 釋放出的 factor 經由血液運送到身體其他較遠的細胞接收 Paracrine 釋放出的 factor 經由擴散由周圍不同類型細胞的 receptor 接收 Autocrine 釋放出的 factor 由同一細胞或周圍同種細胞的 receptor 接收 www.indiandentalacademy.com
  • 13. 1.B.Cell Regenerative Capacity Most types of cell populations can undergo turnover, but the process is highly regulated, Rates of proliferation are different among various cell populations and are frequently divided into three categories: (1) renewing (also called labile) cells have continuous turnover, with proliferation balancing cell loss that accrues by death or physiological depletion; (2) expanding (also called stable) cells, normally having a low rate of death and replication, retain the capacity to divide following stimulation; (3) static (also called permanent) cells not only have no normal proliferation, but have lost their capacity to divide. www.indiandentalacademy.com
  • 14. www.indiandentalacademy.com
  • 15. 1.C.Extracellular Matrix Remodeling The maintenance of the extracellular matrix requires constant collagen remodeling, itself dependent on continued collagen synthesis and collagen catabolism. Turnover of the extracellular matrix is a unique biological problem because of the high collagen content of most extracellular matrix structures and the resistance of these triple helical molecules to the action of most proteases. www.indiandentalacademy.com
  • 16. Connective tissue remodeling, is a highly organized process that involves the selective action of a group of related proteases that collectively can degrade most components of the extracellular matrix. These proteases are known as the matrix metalloproteinases (MMPs 間質金屬結合蛋白脢 ). Subclasses include the interstitial collagenases(MMP-1,MMP-13), stromelysins(MMP3,MMP-10), and gelatinases(MMP-2). MMP 家族是一群含有鋅和鈣離子依賴性的蛋白酶,它們能分解大多數的 ECM , 人類約有二十三種 MMP ,如膠原蛋白酶( collagenases) , MMP-l 和 MMP13 等屬此群,此群主要是切開第一、三和四型 collagen ;另一個家族為 Gelat inases (MMP-2 和 MMP -9) ,此族成員可裂解 laminin 、 f ibr onect in 和 第四型 collagen ,而第四型 collagen 為基底膜最重要的內容物 ; St r omelysills ,如 MMP-3 和 MMP-10 等屬此家族 . www.indiandentalacademy.com
  • 17. Enzymes that degrade collagen are synthesized by macrophages, fibroblasts, and epithelial cells. Collagenases are specific for particular types of collagens, and many cells contain two or more different such enzymes. Particularly important in tissue remodeling are myofibroblasts, a particular phenotype of cells that show both features of smooth muscle cells (contractile proteins such as a-actin) and features of fibroblasts (rough endoplasmic reticulum in which proteins are synthesized). www.indiandentalacademy.com
  • 18. These cells may also be responsible for the production of tissue forces during remodeling, thereby regulating the evolution of tissue structure according to mechanical requirements. Evidence suggests that growth factors and hormones (autocrme, paracrine, and endocrine) are pivotal in orchestrating both synthesis and degradation of ECM components. www.indiandentalacademy.com
  • 19. Cytokines such as TGF-,8, PDGF, and IL-1 clearly play an important role in the modulation of collagenase and TIMP expression. MMP enzymatic activities are regulated by tissue inhibitors of metalloproteinases (TIMPs), which are especially important during wound repair. Turnover of the extracellular matrix is mediated by an excess of MMP over TIMPs activity. Distortion of the balance between matrix synthesis and turnover may result in altered matrix composition and amounts. www.indiandentalacademy.com
  • 20. 2.CELL/TISSUE-BIOMATERIALS INTERACTIONS Cell interactions with the external environment are mediated by receptors in the cell membrane, which interact with proteins and other ligands that adsorb to the material surface from the surrounding plasma and other fluids (Lauffenburger and Griffith, 2001). . www.indiandentalacademy.com
  • 21. 2.CELL/TISSUE-BIOMATERIALS INTERACTIONS • Cell adhesion triggers multiple functional biochemical signaling pathways within the cell. Most tissue-derived cells require attachment to a solid surface for viability, growth, migration, and differentiation. • Following contact with tissue or blood, a bare surface of a biomaterial is covered rapidly (usually in seconds) with proteins that are adsorbed from the surrounding body fluids www.indiandentalacademy.com
  • 22. 2.CELL/TISSUE-BIOMATERIALS INTERACTIONS • • The chemistry of the underlying substrate (particularly as it affects wettability and surface charge) controls the nature of the adherent protein layer. Cell adhesion to biomaterials is mediated by cytoskeletally associated receptors in the cell membrane, which interact with cell adhesion proteins that adsorb to the material surface from the surrounding plasma and other fluids (Fig. 12) (Saltzman, 2000). www.indiandentalacademy.com
  • 23. Fig 12 ( A ) Initial contact of cell with solid substrate . ( B ) Formation of bonds between cell surface receptors and cell adhesion ligands . ( C ) Cytoskeletal reorganization with progressive spreading of the cell on the substrate for increased attachment strength . www.indiandentalacademy.com
  • 24. Cell binding to the extracellular matrix through specific cellsubstratum contacts is critical to cell-growth control through mechanical forces mediated through associated changes in cell shape and cytoskeletal tension (Ingber, 2002). Focal adhesions are considered to represent the strongest such interactions. They comprise a complex assembly of intraand extracellular proteins, coupled to each other through transmembrane integrins. Cell-surface integrin receptors promote cell attachment to substrates, and especially those covered with the extracellular proteins fibronectin and vibronectin. These receptors transduce biochemical signals to the nucleus by activating the same intracellular signaling pathways that are used by growth factor receptors. www.indiandentalacademy.com
  • 25. (A) Schematic diagram showing the initial pattern design containing differentsized square adhesive islands and Nomarski (DIC) views of the final shapes of bovine adrenal capillary endothelial cells adherent to the fabricated substrate. Importantly, as the bead diameter was decreased to 10 um, cells became more rounded, and the apoptotic index increased to match that in nonadherent cells (Fig. 13). www.indiandentalacademy.com
  • 26. ( B ) Apoptotic index (percentage of cells exhibiting positive TUNEL staining) and DNA synthesis index (percentage of nuclei labeled withs 5bromodeoxyuridine) after 24 hours, plotted as a function of the projected cell area. Data were obtained only from islands that contained single adherent cells; similar results were obtained with circular or square islands and with human or bovine www.indiandentalacademy.com endothelial cells.
  • 27. ( C ) Fluorescence micrograph of an endothelial cell spread over a substrate containing a regular array of small circular ECM islands separated by nonadhesive regions created with a microcontact printing technique. Yellow rings and crescents indicate colocalization of vinculin (green) and F-actin (red) within focal adhesions that form only on the regulatory spaced circular ECM islands . www.indiandentalacademy.com
  • 28. The more cells spread, the higher their rate of proliferation. (Fig. 13) (Chen et al., 1997). Cells spread to the limits of the islands containing a fibronectin substrate; cells on circular islands were circular while cells on square islands became square in shape and had 90°corners. When the spreading of the cells was restricted by small adhesive islands (10-30 μm), proliferation was arrested, whereas larger islands (80μm) permitted proliferation. (Fig 13 A) www.indiandentalacademy.com
  • 29. This confirmed that the ability to proliferate depended directly on the degree to which the cells were allowed to distend physically, and not on the actual surface area of substrate binding. Thus, cell distortion is a critical determinant of cell behavior. Interactions of cells with ECM differ from those with soluble regulatory factors owing to the reciprocal interactions between the ECM and the cell's actin cytoskeleton (Ingber, 2003). www.indiandentalacademy.com
  • 30. Thus, the properties of the nature and configuration of the surface-bound ECM on a substrate and the properties of the substrate itself can regulate cell-biomaterials interactions. The key concept is that a biomaterial surface can contain specific chemical and structural information that controls tissue formation, in a manner analogous to cell-cell communication and patterning during embryological development. www.indiandentalacademy.com
  • 31. Employ biomaterials with surfaces designed to stimulate highly precise reactions with proteins and cells at the molecular level. The binding domains of the extracellular matrix (ECM) environment can be mimicked by a multifunctional celladhesive surface created by specific proteins, peptides, and other biomolecules immobilized onto a material. www.indiandentalacademy.com
  • 32. The prototypical binding site present in the adhesive proteins fibronectin and vitronectin is the three amino acid sequence arginine-glycine-aspartic acid (RGD) which binds to a specific type of integrin receptors on the cell surface (see Fig. 3C) www.indiandentalacademy.com
  • 33. This sequence supports the adhesion and spreading of human endothelial cells but not smooth muscle cells, fibroblasts, or blood platelets (Hubbell, 1999). Moreover, cellular responses induced can vary with the surface density of RGD peptides immobilized (Koo et al., 2002). www.indiandentalacademy.com
  • 34. Through selection of ligands, surfaces can be designed 1. to reduce protein and cell adhesion, 2. to prevent coagulation, 3. to encourage endothelial cell attachment and retention, 4. to promote capillary infiltration, and 5. to prevent excessive smooth-muscle proliferation and collagen production. This manipulation of cell-integrin interactions with engineered ligands on synthetic biomaterials could improve function in existing applications such as the healing of vascular grafts. A particularly exciting and active area is the use of chemically patterned surfaces to control cell behavior by creating adhesive and nonadhesive regions and perhaps even chemical gradients. www.indiandentalacademy.com
  • 35. By varying the size and chemistry of the various regions, and thereby the type, architecture, directional migration, and function of cells, a sort of two-dimensional organ can be grown. This strategy has been used to "engineer" constructs of hepatic tissue in which hepatocytes and endothelial cells self-sort to form endothelium-lined liver cell plates (Kim et al., 1998). www.indiandentalacademy.com
  • 36. Different levels of type 1 collagen coating on a culture dish result in different organization of endothelial cells and hepatocytes . High collagen levels cause both cell types to spread across the substratum (left) . www.indiandentalacademy.com
  • 37. hepatocytes endothelial cells On intermediate collagen levels, endothelial cells form a layer on the substratum whereas hepatocytes form a layer on top of the endothelial cells (center) . www.indiandentalacademy.com
  • 38. hepatocyte Low levels of collagen result in an inner layer of hepatocyte aggregate surrounded by endothelial cells (right) . www.indiandentalacademy.com
  • 39. A key challenge in tissue engineering is to understand quantitatively cells respond, and to control nonspecific interactions. In addition, special relationships may accrue 產生 for biodegradable polymers, since the polymer disappears as functional tissue regenerates. Thus, polymer degradation may yield a dynamic surface whose chemistry might be unpredictable, but could possibly be manipulated to provide an additional level of control over cell interactions. Moreover, covalently immobilized growth factor, can retain its biological activity, and potentially DNA delivered upon a biomaterial surface can be efficiently taken up by cells and the encoded gene expressed in a wound-healing environment (Swindle etal., 2001; Richardson etal., 2001). www.indiandentalacademy.com
  • 40. Topography has also been studied for its effect on cell behavior, including depth and width of groove, and roughness. Surface texture influences cell behavior, including 1. adhesion and movement attachment, 2. spreading area, 3. proliferation, 4. orientation of cells to the topography, 5. biochemical activity, and 6. neurite (nerve) growth. Moreover, fibroblasts, neurons, and other cells will orient along fibers, ridges, and grooves with potential therapeutic application in nerve regeneration, and texture has been shown to influence macrophage spreading and fibroblast growth. www.indiandentalacademy.com
  • 41. 3.TECHNIQUES FOR ANALYSIS OF CELLS AND TISSUES Gross examination Overall specimen configuration ; many diseases and processes can be diagnosed at this level Light microscopy (LM) Study overall microscopic tissue architecture and cellular structure ; special stains for collagen , mucin , elastin , organisms , etc . are available Transmission electron microscopy (TEM) Study ultrastructure ( fine structure and identify cells and their organelles and environment www.indiandentalacademy.com
  • 42. Scanning electron microscopy ( SEM ) Study topography and structure of surfaces Enzyme histochemistry Demonstrate the presence and location of enzymes in gross or microscopic tissue sections Immunohistochemistry Identify and locate specific molecules, usually proteins, for which a specific antibody is available www.indiandentalacademy.com
  • 43. In situ hybridization Localizes specific DNA or RNA in tissues to assess tissue identity or recognize a cell gene product Microbiologic cultures Diagnose the presence of infectious organisms Morphometric studies Quantitate the amounts, configuration, and distribution of specific structures www.indiandentalacademy.com
  • 44. Chemical, biochemical, and spectroscopic analysis Assess concentration of molecular or elemental constituents Energy –dispersive X -ray analysis ( EDXA ) Perform site-specific elemental analysis on surfaces Autoradiography (at LM or TEM levels) Locate the distribution of radioactive material in sections www.indiandentalacademy.com
  • 45. 3.A.Light Microscopy Key features of tissue processing for examination by light and electron microscopy www.indiandentalacademy.com
  • 46. 3.A.a.Tissue Sample The tissue is obtained by surgical excision (removal), biopsy (sampling), or autopsy (postmortem examination). A sharp instrument is used to remove and dissect the tissue to avoid distortion from crushing. Specimens should be placed in fixatives as soon as possible after removal. www.indiandentalacademy.com
  • 47. Fixation To preserve the structural relationships among cells, their environment, and subcellular structures in tissues, it is necessary to cross-link and fix the tissue in a permanent state. Fixative solutions prevent degradation of the tissue when it is separated from its source of oxygen and nutrition by coagulating proteins. This prevents cellular hydrolytic enzymes, which are released when cells die, from degrading tissue components and spoiling tissues for microscopic analysis. Fixation also immobilizes fats and carbohydrates, reduces or eliminates enzymic and immunological reactivity, and kills microorganisms present in tissues. www.indiandentalacademy.com
  • 48. 4% formaldehyde( 甲醛 ) solution is the routine fixative in pathology for light microscopy. For TEM and SEM, glutaraldehyde( 戊二醛 ) preserves structural elements better than formalin. Adequate fixation in formalin and/or glutaraldehyde requires tissue samples less than 1.0 and 0.1 cm, respectively. For adequate fixation, the volume of fixative into which a tissue sample is placed should generally be at least 5 to 10 times the tissue volume. www.indiandentalacademy.com
  • 49. 3.A.c Dehydration and Embedding In order to support the specimen during sectioning, specimen water must be replaced by paraffin wax or other embedding medium, such as glycol methacrylate ( 乙二醇 甲基丙烯酸酯 ). This is done through several steps, beginning with dehydration of the specimen through increasing concentrations of ethanol. However, since alcohol is not miscible with paraffin (the final embedding medium), xylol (an organic solvent) is used as an intermediate solution. Following dehydration, the specimen is soaked in molten paraffin and placed in a mold larger than the specimen, so that tissue spaces originally containing water, as well as a surrounding cube, are filled with wax. The mold is cooled, and the resultant solid block containing the specimen can then be easily handled. www.indiandentalacademy.com
  • 50. Sectioning Tissue specimens are sectioned on a microtome. The shavings are picked up on glass slides. Sections for light microscopic analysis must be thin enough to both transmit light and avoid superimposition of various tissue components. If thinner sections are required for TEM analysis, a harder supporting (embedding) medium (usually epoxy plastic) and a correspondingly harder knife (usually diamond) are used. www.indiandentalacademy.com
  • 51. the conventional paraffin technique requires overnight processing, frozen sections can be used to render an immediate diagnosis In this method, the specimen itself is frozen, so that the solidified internal water acts as a support medium, and sections are then cut in a cryostat ( 冷凍切片機 ). Although frozen sections are extremely useful for immediate tissue examination, the quality of the appearance is inferior to that obtained by conventional fixation and embedding methods. www.indiandentalacademy.com
  • 52. Staining Tissue components have no intrinsic contrast and are of fairly uniform optical density. Therefore, in order for tissue to be visible by light microscopy, tissue elements must be distinguished by selective adsorption of dyes (Luna, 1968). Since most stains are aqueous solutions of dyes, staining requires that the paraffin in the tissue section be removed and replaced by water (rehydration). www.indiandentalacademy.com
  • 53. The stain used routinely in histology involves sequential incubation in the dyes hematoxylin ( 蘇木素 ) and eosin ( 伊紅 ) (H&E). Hematoxylin has an alkaline (basic) pH that stains bluepurple; substances stained with hematoxylin typically have a net negative charge and are said to be "basophilic" (e.g., cell nuclei containing DNA). Substances that stain with eosin, an acidic pigment that colors positively charged tissue components pink-red, are said to be "acidophilic" or "eosinophilic" (e.g., cell cytoplasm, collagen). www.indiandentalacademy.com
  • 54. Electron Microscopy Sections are stained with salts of heavy metals (osmium, lead, and uranium), which react differentially with different structures, creating patterns of electron density that reflect tissue and cellular architecture. www.indiandentalacademy.com
  • 55. Electron Microscopy www.indiandentalacademy.com
  • 56. Three-Dimensional Interpretation www.indiandentalacademy.com
  • 57. Artifacts Artifacts are unwanted or confusing features in tissue sections that result from errors or technical difficulties in obtaining, processing, sectioning, or staining the specimen. The most frequent and important artifacts are autolysis, tissue shrinkage, separation of adjacent structures, precipitates formed by poor buffering or by degradation of fixatives or stains, folds or wrinkles in the tissue sections, knife nicks, or rough handling of the specimen. www.indiandentalacademy.com
  • 58. Identification, Genotyping, and Functional Assessment of Cells, Including Synthetic Products, In Cells or Tissue Sections Cellular apoptosis and proliferation can be quantified (Watanabe et al., 2002). Immuno-histochemical markers allow detection of proteins that are highly expressed in a tissue section. Tissue micro-assays permit the comparative examination of potentially hundreds of individual specimens in a single paraffin block. In addition, laser-assisted micro-dissection techniques permit isolation of individual or a homogenous population of cells on selected cell populations under direct visualization from a routine histological section of complex, heterogeneous tissue (Eitoum etal., 2002). Very exciting new imaging technology, termed molecular imaging, may permit analysis of viable and in vivo tissues (Stephens and Allan, 2003; Weissleder and Ntziachristos, 2003; Webb et al., 2000). www.indiandentalacademy.com
  • 59. www.indiandentalacademy.com Leader in continuing dental education Thanks for your attention… www.indiandentalacademy.com