Connective tissue properI. FunctionII.III.CompositionDerivationIV. IntercellularmaterialV. CellsVI. Types ofconnective tissueVII.Specializedfunctions
Connective tissue cells• Fibroblasts• Macrophages• Mast cells• Plasma cells• Adipose cells• Leukocytes Lymphocyte Monocyte Neutrophil Eosinophil BasophilMononuclear, agranulocytespolymorphonuclear, granulocytes
Connective Tissue Function• Support• Organism defense• Protection- barrier to pathogens• Nutrient distributionblood
Composition• CellsFixedMobile• FibersType I collagen fibers- largeReticular fibers-thinElastic fibers- very fine• Ground substance (Extracellularmatrix, ECM)
Modular representation of ECM protein components(Human)LamininN-terminalEGF EGF EGF B-domain EGF EGF EGF EGF EGF EGF EGF EGF EGF B-domain EGF EGF EGF EGFCoiled-coilG-domainG-domainG-domainG-domainG-domainLamininN-terminalEGF EGF EGF EGF EGF EGF EGF EGF EGF EGF EGF EGF EGF Myosin-likeLaminin β11765 aaLaminin α13058 aaFNIFNIFNIFNIFNIFNIFNIIFNIIFNIFN FNI IFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFibronectin2451 aaHeptadrepeatsEGFEGF EGF EGF EGFFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFibrinogenlikeTenascin C2179 aavWFCollagentriple helix repeatsC-ter.Collagen I, α11441 aaC1qCollagen X680 aaTSPNCollagentriple helix repeatsCollagen IX, α1898 aaSOHemopexin repeatsVitronectin478 aaEGFTenascinEGF-likevWFvWF type C-domain(cysteine-rich)FNIFNIIFNIIIFibronectintype I, II and IIITSPNTrombospondin-like domainEGFLamininEGF-likeSOSomatomedinB-like repeatsC-ter.Collagen C-terminaldomain
ECM protein components• Laminin- huge, linking glycoprotein• Fibronectin-large linking glycoprotein• Collagen type 1-large protein, but formseven larger fibers after polymerization andprecipitation in ECM.
Derivation• All connective tissue cells are derived fromtwo types of stem cellsHematopoietic stem cellsMesenchymal cells
Intercellular material• Ground substanceProteoglycans•Glycosaminoglycan (GAG)- 80-90%•Protein core- 10-20%Glycoproteins•Fibronectin•Laminin•Chondronectin
Figure 5—2. Section ofrat skin. A connectivetissue layer (dermis)shows several fibroblasts(F), which are theelongated cells. H&Estain. Mediummagnification.
Issue Date: originally appeared in Veterinary Therapeutics Winter 2007 (Vol 8, No 4)Effect of Adipose-Derived Mesenchymal Stem and Regenerative Cells on Lamenessin Dogs with Chronic Osteoarthritis of the Coxofemoral Joints: A Randomized,Double-Blinded, Multicenter, Controlled Trial*Linda L. Black, James Gaynor, Dean Gahring, Cheryl Adams, Dennis Aron, Susan Harman, Daniel A. Gingerich, Robert J. HarmanCLINICAL RELEVANCEAutologous stem cell therapy in the field of regenerative veterinary medicine involves harvesting tissue, such as fat, from the patient, isolating the stem andregenerative cells, and administering the cells back to the patient. Autologous adipose-derived stem cell therapy has been commercially available since 2003,and the current study evaluated such therapy in dogs with chronic osteoarthritis of the hip. Dogs treated with adipose-derived stem cell therapy had significantlyimproved scores for lameness and the compiled scores for lameness, pain, and range of motion compared with control dogs. This is the first randomized,blinded, placebo-controlled clinical trial reporting on the effectiveness of stem cell therapy in dogs.*This study was sponsored by Vet-Stem, Inc., Poway, California. Correspondence should be directed to Dr. Black (LBlack@vet-stem.com).
Glycosaminoglycans• Linear chains of repeating disaccharideunits• One of the disaccharides is glucosamineor galactosamine• The other is glucuronic or iduronic acid, orgalactose.
PolysaccharidesCellulose Chitin GlycogenChondroitin sulfateanionDermatan sulfateKeratan sulfateamineHyaluronateHeparan sulfate5 glycosaminoglycansusuallyattached to a proteinand called a proteoglycanexceptionis hyaluronate
HeparinHeparan sulfate is similar toheparin but differs due toalternating sulfates onsugars
The heparan sulfate chain consists of different domains that vary inthe extent of modification by sulfation and epimerizationEssentials of GlycobiologySecond EditionChapter 16, Figure 8
5 glycosaminoglycan types• Keratan sulfateGalactose-glucosamineBone, cornea
GAGs• Dermatan sulfate Iduronic acid-galactosamine Tendon, skin
GAGs• Heparan sulfate Iduronic acid-glucosamine Basal lamina, nerve tissue
GAGs• Hyaluronic acid Glucuronic acid-glucosamine Cartilage, synovial fluid LARGEST!- no protein core
Glycosaminoglycans consist of repeating disaccharide unitsEssentials of GlycobiologySecond EditionChapter 16, Figure 3Hyaluronic acid is not sulfatedallothers are
The large cartilage CS proteoglycan (aggrecan) forms an aggregate withhyaluronan and link proteinEssentials of GlycobiologySecond EditionChapter 16, Figure 1
GAGs are attached to serines via a linking tetrasaccharide© 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.
Proteoglycan structure• GAGs are linked to a protein core likebristles on a hair brushGAGsProtein core
Figure 5—32. The molecular structure of proteoglycans andglycoproteins.
Aggrecan• 250 kilodaltons- very large!• Protein with chondroitin sulfate andkeratan sulfates attached• Protein has a hyaluronic acid bindingregion• Attaches to hyaluronic acid• 10,000 negative charges per aggrecan• 10% of cartilage weight (cartilage is75% water).
HABR- hyaluronic acid binding site© 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter
Chondrotin sulfate proteoglycans• Aggrecan, cartilage• Versican, fibroblasts• CD44, lymphocytes• Neurocan (nervous system)
Variable number of tandem repeatsRoughley P, Martens D, Rantakokko J, Alini M, Mwale F, Antoniou J. 2006 The involvement of aggrecan polymorphism indegeneration of human intervertebral disc and articular cartilage. Eur Cell Mater. 11:1-7.
VNTR polymorphism inintervertebral disc damage• Variable number of tandem repeats• 19 AA in CS1 domain, number of repeatsfrom 13-33• Normal 28-33• Those with fewer repeats (13) may havefewer CS binding and more cartilage anddisc damage
Roughley P, Martens D, Rantakokko J, Alini M, Mwale F, Antoniou J. 2006 The involvement of aggrecan polymorphism indegeneration of human intervertebral disc and articular cartilage. Eur Cell Mater. 11:1-7.
• “Past studies show that some people with mild to moderate osteoarthritis(OA) taking either glucosamine or chondroitin sulfate reported pain relief ata level similar to that of nonsteroidal anti-inflammatory drugs (NSAIDs) suchas aspirin and ibuprofen. Some research indicates that the supplementsmight also slow cartilage damage in people with OA. Definitive results aboutthe effects of these supplements are expected from an in-depth clinicalstudy currently being conducted by the National Institutes of Health.”• GAIT (The Glucosamine/Chondroitin ArthritisIntervention Trial)
CONCLUSIONS• Glucosamine and chondroitin sulfate alone or incombination did not reduce pain effectively inthe overall group of patients with osteoarthritis ofthe knee. Exploratory analyses suggest that thecombination of glucosamine and chondroitinsulfate may be effective in the subgroup ofpatients with moderate-to-severe knee pain.(ClinicalTrials.gov number, NCT00032890.).Copyright 2006 Massachusetts Medical Society.N Engl J Med. 2006 Feb 23;354(8):795-808.
Syndecans• Integral membrane proteoglycan• Helps bind the cell to collagens Type I, II,and III fibers in extracellular matrix (ECM)• Helps bind cell to fibronectin
Figure 5—33. Schematic diagram of cell-surface synedcan proteoglycan.
© 2000 by W. H. Freeman and Company.
Integrin- fibronectinreceptorHSPG- heparansulfate proteoglycanBeauvais et al. Reproductive Biology and Endocrinology 2004 2:3 doi:10.1186/1477-7827-2-3
Extracellular matrixglycoproteins• Anchor cells to proteoglycans in matrixand to fibers• Fibronectin- general connective tissue• Laminin- mainly basement membrane
Figure 5—34. A: The structures of fibronectin and laminin.
Function domains of fibronectin monomer. Fibronectin bonds tointegrin membrane receptors at ‘RGD’Arg-Gly-Asp sitesEDA, EDB-extra domain A and B- required for normal fiber formationthisoccurs only when fibronectin is attached to cells[Adapted from G. Paolella, M. Barone, and F. Baralle, 1993, in M. Zern and L. Reid, eds., Extracellular Matrix, Marcel Dekker, pp. 3 24.]© 2000 by W. H. Freeman and Company.
disintegrins• RGD containing peptides in snake venomthat block integrin receptor from bindingwith fibrinogen• Prevents blood clotting
Integrin receptor- binds tofibronectinFigure 5—37. Integrin cell-surface matrixreceptor. By binding to a matrix protein andto the actin cytoskeleton (via alpha-actinin)inside the cell, the integrin serves as atransmembrane link. The molecule is aheterodimer, with alpha and beta chains. Thehead portion may protrude some 20 nm fromthe surface of the cell membrane into theextracellular matrix.
INTEGRIN LIGAND* DISTRIBUTIONα 5β 1fibronectin ubiquitousα 6β 1laminin ubiquitousα 7β 1laminin muscleα Lβ 2(LFA-1, see p. Ig superfamily white blood cells1411)counterreceptorsα 2β 3fibrinogen plateletsα 6β 4laminin epithelialhemidesmosomes© 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.
Function of fibronectin• Facilitate cell migration• May be important for determining cellshape• Plays a role in blood clotting.
Laminin- found inbasement membraneBinds to•Collagen type IV•Heparan sulfate•Integrin receptor
Connective tissue fibers• CollagenFormed from collagen protein• Reticular• Elastic- formed from the protein elastin
Collagen• 30% of human body by dry weight• 3 alpha chains in each collagen fiber orfibril• 25 forms of alpha chain• 14 different forms of collagen fibers/fibrils• Lots of hydroxyprolines andhydroxylysines
Procollagen triple helix- three alpha chains.Each alpha chain is transcribed by a differentgene.
Figure 5—21. Collagensynthesis.-alpha chains madeseparately-procollagen secreted-tropocollagen formedfrom cleavage ofprocollagen
© 2000 by W. H. Freeman and Company.
The side-by-sideinteractions ofcollagen helicesare stabilized byan aldol cross-linkbetween two lysine(or hydroxylysine)side chains. Theextracellular enzymelysyl oxidasecatalyzes formationof the aldehydegroups.Lysyl oxidase
Figure 5—19. Schematic drawing of an aggregate of collagen molecules(tropocollagen), fibrils, fibers, and bundles. There is a stepwise overlappingarrangement of rodlike tropocollagen subunits, each measuring 280 nm (1).Collagen type III usually does not form bundles.
J F Lazartigue Treatmentcream shampoo withcollagenThe hydrolyzed marine collagen (2%)consolidates the hair, restoring its elasticity andstrengthening its protective sheath. Its highlynourishing formula acts like a bandage on dry,brittle ends as it rehydrates the scalp.
Collagen types• Collagen type I - 90% of all collagen. Formsbone, tendons, skin. Made by fibroblasts,osteoblasts, odontoblasts.• Collagen type II – found in cartilage. Madeby chondroblasts. Forms fibrils, not fibers.• Collagen type III – component of reticularfibers in lymphoid organs.• Collagen type IV – made by endothelial andepithelial cells
Figure 5—17. Electron micrograph of human collagen fibrils in cross and longitudinal sections. Each fibrilconsists of regular alternating dark and light bands that are further divided by cross-striations. Ground substancecompletely surrounds the fibrils. x100,000.
Figure 5—46.Longitudinal section ofdense regular connectivetissue from a tendon.
Reticular fibers• Very thin and delicate• Abundant in lymphoid tissue• First to appear in many tissues, buteventually give way to collagen fibers
Adrenal cortex stained for reticular fibers
Elastic fibers• Oxytalan fibers- glycoprotein containingmicrofibrils or microfilaments, 8-10 nmdiameter.• Elaunin fibers- gradual deposition ofelastin on microfilaments• Elastic fibers- heavy deposition ofelastin
Figure 5—28. Electron micrographs of developing elastic fibers. A: In early stages of formation,developing fibers consist of numerous small glycoprotein microfibrils. B: With further development,amorphous aggregates of elastin are found among the microfibrils. C: The amorphous elastinaccumulates, ultimately occupying the center of an elastic fiber delineated by microfibrils. Note thecollagen fibrils, seen in cross section. (Courtesy of GS Montes.)
Unique amino acids in elastin• Isodesmosine and Desmosine- four lysineside chains cross linked to each otherDesmosinelysinehttp://webpages.dcu.ie/~mcdonnes/be201/desmosine.gif
Fibrillin• Microfilament glycoprotein important inelastic fibers.• Marfan’s syndrome- defect in fibrillinsynthesis and elastic fibers.• Connective tissue disorder affectingskeleton, elastic blood vessels• Sever cardiovascular problems- arteriescannot stretch.
Marfan’s syndrome• SkeletalToes, fingers, legs, and arms are disproportionately longDouble-jointedness, or "loose joints"Flat feetConcave or convex breastboneCurvature of spine (Scoliosis)Very tall or taller than most other members in the family• Ocular (Sight and Eyes)Dislocated lenses in one or both eyesGlaucomaCataractsNearsightedness• Cardiovascular (Heart and Blood Vessels)• Nervous• Pulmonary• SkinDilation or tearing of the AortaHeart murmur caused by leakage in the valve between the left chambers of the heart• Severe cases can cause palpitations, shortness of breath and fatigue.Dura (spinal fluid in a membrane surrounding the brain and spinal cord) is weakened and stretches with age• This can cause mild discomfort in the lower back, as well as compression of the vertebrae• Compression can cause abdominal pain and pain, weakness, and numbness in the legsLungs are less elasticSleep apnea or snoringStretch marks can occur without weight change or pregnancyAbdominal or groin hernias can occurSupportMarfan.com
Model of elastic fiber formation in the extracellular space.Rock M J et al. J. Biol. Chem. 2004;279:23748-23758©2004 by American Society for Biochemistry and Molecular Biology
Hematopoietic stem cellsMesenchymal stem cells
Fibroblasts• Most common cell type in connectivetissue• Synthesize collagen, reticular, and elasticfibers• Synthesize glycosaminoglycans andglycoproteins
Figure 5—4.Active (left) andquiescent (right)fibroblasts.
Figure 5—5. Electronmicrograph revealingportions of severalflattened fibroblasts indense connective tissue.
Macrophages• 10-30 um in diameter• Shape can vary tremendously• Phagocytosis and digestion ofBacteriaForeign bodiesBlood cellsDead cells• opsonization
Network of macrophage cells• Table 5-2• Macrophage-connective tissue• Microglia- brain• Kupffer cell- liver• Osteoclast- bone• Monocyte - macrophage precursor inblood
FAB, fragmentantigen bindingFigure 14—2. Two light chains andtwo heavy chains form an antibodymolecule.
Opsonization© 2001 by Garland Publishing
© 2001 by Garland Publishing
Other macrophage functions• Ingest bacteria, pass information on tolymphocytes• Secrete important enzymes such ascollagenase, elastase• Produce cytokines such as Tumor necrosis factor Interleukins GM-CSF (granulocyte macrophage-colony simulatingfactor)
superoxideSuperoxide dismutaseRespiratory burstmyeloperoxidasehypochlorous acid
Cl 2+ H 2OHClO + HCl
Respiratory burst• hydrogen peroxide (H 2 O 2 )• the superoxide anion (O 2- )• nitric oxide (NO)• Hypochlorous acid (HOCl)• all are directly toxic to bacteria
Chronic granulomatous disease• An immunodeficiency disease in which multiplegranulomas form as a result of defectiveelimination of bacteria by phagocytic cells.• Caused by defects in NADPH oxidaseresponsible for producing superoxide radicalslethal to bacteria.• Granulomas form when an intracellularpathogen or its constituents cannot bedestroyed, causing a local inflammation.
Alpha 1 antitrypsin-relatedemphysema• A1AT is deficient in some individuals• Normally blocks neutrophil elastase in lungs• If A1AT is lacking, elastase digests lung elasticfibers• Lungs lose elasticity• Emphysema can also occur due to irritants intobacco smoke and over-stimulation ofmacrophages
Cytokines and enzymes involved inthe inflammation response• CSF, colony stimulating factors• ILs, interleukins• TNF, tumor necrosis factor• MIF, macrophage inhibitory factor• INF, interferoncytokines• MMP, matrix metalloproteases• Collagenase• elastase
Mast cells• Round, oval 20-30 um diameter• Basophilic granules in cytoplasm• Metachromasia- mast cells stain purple withmethylene blue. Due to heparin content inthe cell.
Mast cells secretes• SecretesHeparin - anticoagulentHistamine - increases capillary permeabilityEosinophil chemotactic factor of anaphylaxis• ECF-A (old)• eotaxin 1 and eotaxin 2 (new)-97 aminoacidsSlow reacting substance of anaphylaxis• SRS-A (old)• Leukotrienes (new)
Heparin is a glycosaminoglycan© 2002 by W. H. Freeman and Company.
© 1999 by American Society for NeurochemistryPublished by Lippincott Williams and Wilkins.
Figure 5—10.Section of rattongue. Severalmast cells in theconnective tissuesurround musclecells and bloodvessels. PT stain.Mediummagnification.
Immediate Hypersensitivity Reaction• Can occur within seconds• Massive release of histamine from mastcells• Reduces mean arterial blood pressurebloodenters peripheral capillaries• Caused by IgE secretion from plasmacells• Epinephrine can reverse HIS by causingvasoconstriction.
Figure 5—12. Mast-cell secretion. 1: IgE molecules are bound to the surface receptors. 2: After a second exposure to an antigen (eg, beevenom), IgE molecules bound to surface receptors are cross-linked by the antigen. This activates adenylate cyclase and results in thephosphorylation of certain proteins. 3: At the same time, Ca2+ enters the cell. 4: These events lead to intracellular fusion of specific granulesand exocytosis of their contents. 5: In addition, phospholipases act on membrane phospholipids to produce leukotrienes. The process ofextrusion does not damage the cell, which remains viable and synthesizes new granules. ECF-A, eosinophil chemotactic factor of anaphylaxis.
Tryptase and protease-activatedreceptor-2• Tryptase, serine protease highly enrichedin mast cells. Plasma levels are indicatorof anaphylactic shock.• PAR-2, receptor for tryptase
Plasma cell• Eccentric nucleus• Dark staining cytoplasm, lots of RER• 10-20 um diameter• Don’t leave the connective tissue• Produce antibodies
Figure 5—14.Ultrastructure of a plasmacell. The cell contains awell-developed roughendoplasmic reticulum,with dilated cisternaecontainingimmunoglobulins(antibodies). In plasmacells, the secreted proteinsdo not aggregate intosecretory granules. Nu,nucleolus. (Redrawn andreproduced, withpermission, from Ham AW:Histology, 6th ed.Lippincott, 1969.)
Leukocytes(Mononuclear cells;agranulocytes)• Monocytes 25 um Macrophage precursor• Lymphocytes T cells, cell mediated immunity B cells, humoral immunity. Differentiate into plasmacells. Small, 10 um Uniform shaped nucleus
Leukocytes(polymorphonuclear cells;granulocytes)• All have multiple lobed nuclei• Neutrophils Microphagocytosis• Eosinophil Destroys antigen antibody complexes• Basophil Function very similar to mast cells Only source of histamine in blood
VI. Types of connective tissue
Figure 5—41. Section of ratskin in the process of repair ofa lesion. The subepithelialconnective tissue (dermis) isloose connective tissueformed soon after the lesionoccurs. In this area, the cells,most of which are fibroblasts,are abundant. The deepestpart of the dermis consists ofdense irregular connectivetissue, which contains manyrandomly oriented thickcollagen fibers, scarce groundsubstance, and few cells. H&Estain. Medium magnification.