Histology for TE 2024 PDF

Histological evaluation Essential tool in Tissue engineering study PhD. Nguyễn Thị Ngọc Mỹ 1 HCMC, 27th Sep 2024 Contents Histology - Histological evaluation Microscopy Sample preparation techniques Tissue clas...

Histological evaluation Essential tool in Tissue engineering study PhD. Nguyễn Thị Ngọc Mỹ 1 HCMC, 27th Sep 2024 Contents Histology - Histological evaluation Microscopy Sample preparation techniques Tissue classification 2 Histology – Histological evaluation Histology is the study of the tissues of the body and how these tissues are arranged to constitute organs. Greek root: ‘histo’ means “tissue” or “web” Histology involves all aspects of tissue biology, with the focus on how cells’ structure and arrangement optimize functions specific to each organ. [1,2]. Stedman's Medical Dictionaries (2005) Junqueira's Basic Histology: Text and Atlas, 13rd Edition (2013) The McGraw-Hill Companies 3  Liver – Macroscopic observation Left lateral view Right lateral view Ventral view Lobes of Liver (*) Gall bladder (mouse only) * 4 Ruberte, J., Carretero, A., & Navarro, M. (2017). Morphological mouse phenotyping: anatomy, histology and imaging. Elsevier.  Liver tissue – Microscopic observation Hematoxyline and Eosin Staining Fluorescent staining Normal mice Mice fed high-fat diet (Lipid –Bodipy stain (green), Cell Nuclei–Propidium iodide stain (Red)) 5 Ruberte, J., Carretero, A., & Navarro, M. (2017). Morphological mouse phenotyping: anatomy, histology and imaging. Elsevier. Histology – Histological evaluation Histology is the study of the tissues of the body and how these tissues are arranged to constitute organs. Histological evaluation The examination of tissue specimens under a microscope. “Either for research or diagnostic purposes, for both qualitative and quantitative measure”. Bone defects after 1 month transplantation Original bone (without treatment or damage). control groups PLGA group TiO2/PLGA+P-O2 Definitionof histologic examination - NCI Dictionary of Cancer Terms - National Cancer Institute Monitoring and Evaluation of Biomaterials and their Performance In Vivo | ScienceDirect 6 Nanobiomaterials in hard tissue engineering - ScienceDirect Microscopy Resolution: is a measure of the ability to distinguish 2 points as two point Naked eyes About 100-200 µm Light microscope About 0.2 µm Electron microscope About 0.2 µm – 0.2 nm Tools, Techniques and Assessment in Biology: A Course Guide for Students and Teachers 7 (John Adds, Erica Larkcom, 1999) Stereo microscope Light microscopes (KHV soi nổi) Fat Bright field microscope (KHV vùng sáng) Ovary 1 (Buồng trứng) Phase-Contrast Microscope 1 Oviduct (Ống dẫn (KHV phản pha) trứng) Uterus (Tử cung) (1) Follcles (Nang trứng) 8  LIGHT MICROSCOPE Types Principles Bright-field Visualization by microscopy ordinary light that passes through the stained specimen Phase- Lens system that contrast produces visible microscopy. images from transparent objects and, importantly, can be used with living, cultures cells For observation of unstained cells and tissue specimen 9  FLUORESENCE MICROSCOPE Types Principles Fluorescence The specimen is microscopy usually irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum. Fluorescent compounds (fluorophore) are used as fluorescent stains. Intracellular c-Rel staining Cell nuclei staining (DAPI) Merged image 10  Electron microscope Types Principles Scanning Electron beam electron scans over the microscope surface of the (SEM) specimen Transmission Electron beam electron transmits through microscope the specimen to (TEM) form an image 11 Sample preparation techniques The most common procedure used in histologic research is the preparation of tissue samples that can be studied with the microscope. 1. Embedding 2. Sectioning 3. Staining 12 Sample preparation techniques 1. Embedding technique Embedding is the process in which the tissues or the specimens are enclosed in a mass of the embedding medium using a mold. Small pieces The tissue is transferred Alcohol is The tissue is The paraffin- of tissue are through a series of removed in then placed infiltrated tissue placed in increasingly concentrated toluene or in melted is placed in a solutions of alcohol solutions, ending in other agents paraffin small mold with chemicals to 100%, which removes all in which both until it melted paraffin preserve water alcohol and becomes and allowed to tissue in a paraffin are completely harden. “life-like” soluble infiltrated state and with paraffin. inactivating degradative enzymes 13 Sample preparation techniques 2. Tissue sectioning The paraffin block containing tissue is placed on a microtome and sliced by the steel blade into extremely thin sections (about 1 – 10 µm) The specimen should be thin so that: - the light can pass through it - only a single layer of cells is present ➔ so that tissue composition can be clearly distinguished. 14  Tissue section In thin tissue sections, the 3D structures of tissue appear to have only two dimensions. Two-dimensional (2D) appearance will vary depending on the direction of section. 15 Sample preparation techniques 3. Staining In order to reveal structural detail using microscopy, some form of staining is required. Un-stained skin tissue section Hematoxyline and Eosin staining 16 Sample preparation techniques 3. Staining The routine stain used universally as a starting point in providing essential structural information is the hematoxylin and eosin (H&E) stain. EOSIN: HEMATOXYLINE: - Acidic dye - Basic dye For staining basic For staining acidic components components Cytoplasm and ECM Cell nuclei are stained components in blue shades of pink 17  Decellularized pericardium Native Pericardium Hematoxyline and Eosin staining (x200) Nguyen MTN, Tran HLB. Effect of Modified Bovine Pericardium on Human Gingival Fibroblasts in vitro. Cells Tissues Organs. 2018;206(6):296-307. doi: 10.1159/000501807. Epub 2019 Jul 29. 18 PMID: 31357195. Histology Stains - Embryology (unsw.edu.au) 19 Sample preparation techniques 3. Staining Immunostaining Defined as the use of specific antibodies to detect a single target protein (or so-called antigen). 20 Immunofluorescence staining Tiêu bản não phôi chuột được nhuộm với kháng thể kháng Annexin V (Abcam). Kháng thể thứ cấp được đánh dấu huỳnh quang Alexa Fluor® 594 phát màu đỏ. Nhân tế bào được hiển thị bằng cách nhuộm DAPI phát màu xanh. Immunofluorescence using fluorophore-conjugated antibodies. Intracellular c-Rel staining Cell nuclei staining (DAPI) Merged image 21  Immunohistochemistry staining A section of small intestine treated with an antibody against the enzyme lysozyme. The secondary antibody labeled with peroxidase was then applied and the localized brown color produced histochemically with the peroxidase substrate 3,3′-diamino-azobenzidine (DAB). The method demonstrates lysozyme-containing structures in macrophages. Nuclei were counterstained with hematoxylin. (X100) 22  Human Treated Dentin and DPSCs for Dentin-like tissue formation hDPSC adhesion on hTDS surface after 3 days Dentin-like tissues in vivo (8 weeks) Immunohistochemical staining 4 weeks 6 weeks 8 weeks Newly-formed tissues and expression of dentin matrix DSPP: Dentin sialophosphoprotein Tran, Ha Le Bao, and Vu Nguyen Doan. "Human dental pulp stem cells cultured onto dentin derived 23 scaffold can regenerate dentin-like tissue in vivo." Cell and tissue banking 16.4 (2015): 559-568. TISSUE CLASSIFICATION Epithelial tissue Connective tissue Muscle tissue Nervous tissue 24 Epithelial tissue Epithelial tissues are composed of closely aggregated polyhedral cells with strong adhesion to one another and attached to a thin layer of ECM as the basement membrane. TYPES OF EPITHELIA Covering or Lining Epithelia Secretory Epithelia & Glands The principal functions of epithelial tissues include the following: Covering, lining, and protecting surfaces (eg, epidermis) Absorption (eg, the intestinal lining) Secretion (eg, parenchymal cells of glands) 25 Layers of skin Classification of Epithelial tissue Covering or lining Epithelia Secretory Epithelia Blood vessels - The endothelium Simple squamous epithelium Connective tissue Connective tissues provide a matrix that supports and connects tissues and cells together in organs The major constituents of connective tissue are the cells and extracellular matrix (ECM) Differences in composition and amount of the cells and ECM together are responsible for the structural, functional, and diversity of connective tissue. 30 Connective tissue Provide a matrix that supports and physically connects other tissues and cells together in organs Contains blood vessels 3 main components: cells, protein fibers, and ground substance Connective tissue - Protein fibers Collagen Collagen is the most abundant protein in the human body, representing 30% of its dry weight. Major product of fibroblasts Collagen type I, the most abundant and widely distributed collagen, forms large, eosinophilic bundles usually called collagen fibers. Hematoxylin and Eosin staining SEM imaging 32 Connective tissue - Protein fibers Elastin Present with collagen bundles in organs with bending or stretching properties Secreted from fibroblasts (and smooth muscle cells in vascular walls) 33 Connective tissue – Ground substance Three main groups: Glycosaminoglycans (GAGs): long polysaccharides consisting of repeating disaccharide units Bind large amounts of water, which causes the polyanions to swell and occupy a large space in the tissue Hyaluronic acid (HA or hyaluronan) Proteoglycans = a core protein + GAGs One of best known proteoglycans, aggrecan Glycoproteins = protein + oligosaccharide ➔ For cell adherence (have multiple binding sites for cell surface receptors (integrins) and for other matrix macromolecules) Laminin, Fibronectin 34 Connective tissue – Cells Fibroblasts Connective tissue – Cells Blood vessels Endothelial cells Connective tissue – Cells Adipocytes Muscle tissue Muscle tissues are composed of cells that optimize for contractility. Three types of muscle tissue: Skeletal muscle contains bundles of very long, multinucleated cells with cross-striations. Their contraction is quick, forceful, and usually under voluntary control. Cardiac muscle also has cross-striations and is composed of elongated, often branched cells bound to one another at structures called intercalated discs that are unique to cardiac muscle. Contraction is involuntary, vigorous, and rhythmic. Smooth muscle consists of collections of fusiform cells that lack striations and have slow, involuntary contractions. 38 Muscle fibers in the longitudinal section Muscle fibers in cross section 40  Lumen Endothelial cells Elastic lamina (composed of elastin fibers) Nervous tissue Nervous tissue is found in the brain, spinal cord (brain and spinal cord belong to CNS), and nerves (PNS). It is responsible for coordinating and controlling many body activities Two kinds of cell in nervous tissue: nerve cells (or neurons) and glial cells Neurons for processing information and generating responses Glial cells for supporting and protecting neurons Neurons 42  White matter Neuron Gray matter Histological evaluation in TE Papers for discussion: ➔7 and 11. AF-MSC and Chondrocyte spheroids ➔18. Decellularized Blood vessel ➔12 and 18.Fibroblast sheet and decell. 44 45