medigraphic.com
Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán

2006, Número 5

<< Anterior Siguiente >>

Rev Invest Clin 2006; 58 (5)


Células troncales mesenquimales: historia, biología y aplicación clínica

Flores-Figueroa E, Montesinos JJ, Mayani H

Idioma: Español
Referencias bibliográficas: 100
Paginas: 498-511
Archivo PDF: 279.38 Kb.


RESUMEN

En los últimos años, el tema de las c élulas troncales o células madre ha llamado la atención a varios sectores de la sociedad por diversas razones. Desde el punto de vista básico, constituyen un inmejorable modelo para estudiar los mecanismos de diferenciación y autorrenovación celular. Sin embargo, es sin duda, su potencial en la terapia celular y la medicina regenerativa lo que ha disparado la generación de estudios y conocimientos sobre este tema. Las células troncales mesenquimales pertenecen al selecto grupo de células troncales de tejido adulto. Poseen un gran potencial de diferenciación a diversos tejidos mesenquimales, como hueso, cartílago, estroma y tejido graso. Reportes recientes, tanto in vitro como in vivo han demostrado una mayor plasticidad celular, ya que son capaces de originar células endoteliales, musculares e incluso células neuronales. Es por esto que el estudio de las células troncales mesenquimales ha cobrado un gran interés y ha generado una gran cantidad de información. En este trabajo de revisión se presenta una visión global sobre ellas, abarcando su historia, métodos de obtención, características biológicas, fuentes de obtención y aplicación clínica.


REFERENCIAS (EN ESTE ARTÍCULO)

  1. Mayani H. A glance into somatic stem cell biology: basic principles, new concepts, and clinical relevance. Arch Med Res 2003; 34: 3-15.

  2. Lisker R. Ethical and legal issues in therapeutic cloning and the study of stem cells. Arch Med Res 2003; 34: 607-11.

  3. Prockop D. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997; 276: 71-4.

  4. Pittenger MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 2004; 95: 9-20.

  5. Liechty KW, MacKenzie TC, Shaaban AF, Radu A, Moseley AB, Dean R, Marshak DR, Flake AW. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 2000; 6: 1282-6.

  6. Bianco P, Gehron Robey O. Marrow stromal stem cells. J Clin Invest 2000; 105: 1663-8.

  7. Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GP. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 1968; 6: 230-47.

  8. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guineapig bone marrow and spleen cells. Cell Tissue Kinet 1970; 3: 393-403.

  9. Friedenstein AJ, Ivanov-Smolenski AA, Chajlakjan RK, Gorskaya UF, Kuralesova AI, Latzinik NW, Gerasimow UW. Origin of bone marrow stromal mechanocytes in radiochimeras and heterotopic transplants. Exp Hematol 1978; 65: 440-4.

  10. Owen M, Friedenstein AJ. Stromal stem cells: marrow-derived osteogenic precursors. Ciba Foundation Symposium 1988; 136: 42-60.

  11. Piersma AH, Ploemacher RE, Brockbank KG. Transplantation of bone marrow fibroblastoid stromal cells in mice via the intravenous route. Br J Haematol 1983; 54: 285-90.

  12. Ashton BA, Phil B, Allen TD, Howlett CR, Eaglesom CC, Hattori A, Owen M. Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo. Clin Orthop Relat Res 1980; 151: 294-307.

  13. Bab I, Ashton BA, Gazit D, Marx G, Williamson MC, Owen ME. Kinetics and differentiation of marrow stromal cells in diffusion chambers in vivo. J Cell Sci 1986; 84: 139-51.

  14. Haynesworth SE, Goshima H, Golberg VM, Caplan I. Characterization of cells with osteogenic potential from human marrow. Bone 1992; 13: 81-8.

  15. Kuznetsov S, Krebsbach PH, Satomura K, Kerr J, Riminucci M, Benayahu D, Robey PG. Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo. J Bone Miner Res 1997; 12: 1335-47.

  16. Friedenstein AJ, Deriglasova UF, Kulagina NN, Panasuk AF, Rudakowa SF, Luria EA, Ruadkow IA. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 1974; 2: 83-92.

  17. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 1976; 4: 267-74.

  18. Haynesworth SE, Baber MA, Caplan AI. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone 1992; 13: 69-80.

  19. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JE, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science 1999. 284: 143-7.

  20. Colter DC, Class R, DiGiolamo CM, Prockop DJ. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA 2000; 97: 3113-3218.

  21. Sekiya I, Larson BL, Smith JR, Pochampally R, Cui JG, Prockop DJ. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells 2002; 20: 530-41.

  22. Flores-Figueroa E, Gutiérrez-Espíndola G, Guerrero-Rivera S, Pizzuto-Chavez J, Mayani H. Hemopoietic progenitor cells from patients with myelodysplastic syndromes: in vitro growth and long-term proliferation. Leuk Res 1999; 23: 385-94.

  23. Flores-Figueroa E, Arana-Trejo RM, Gutiérrez-Espíndola G, Pérez-Cabrera A, Mayani H. Mesenchymal stem cells in myelodysplastic syndromes: phenotypic and cytogenetic characterization. Leuk Res 2005; 29: 215-24.

  24. Majumdar MK, Keane-Moore M, Buyaner D, Hardy WB, Moorman MA, McIntosh KR, Mosca JD. Characterization and functionality of cell surface molecules on human mesenchymal stem cells. J Biomed Sci 2003; 10: 228-41.

  25. Shih-Chieh Hung, Nien-Jung C, Shie-Liang H, Hung L, Hsiao-Li M, Wai-Hee L. Isolation and characterization of size-sieved stem cells from human bone marrow. Stem Cells 2002; 20: 249-58.

  26. Smith JR, Pochampally R, Perry A, Hsu SCh, Prockop DJ. Isolation of a highly clonogenic and multipotential subfraction of adult stem cells from bone marrow stroma. Stem Cells 2004; 22: 823-31.

  27. Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 1991; 78: 55-62.

  28. Quirici N, Soligo D, Bossalasco P, Servida F, Lumini C, Deliliers GL. Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 2002; 30: 783-91.

  29. Pittenger MF, Mackay AM, Bewck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 248: 143-7.

  30. Ashton BA, Phil BSc, Allen TD, Howlett CR, Eaglesom CC, Hattori A, Owen M. Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo. Clin Orthop Relat Res 1980; 151: 294-307.

  31. Mets T, Verdonk G. Variations in the stromal cell population of human bone marrow during aging. Mech Ageing Dev 1981; 15: 41-9.

  32. Colter DC, Class R, DiGiolamo CM, Prockop DJ. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA 2000; 97: 3113-3218.

  33. Prockop DJ, Sekiya I, Colter DC. Isolation and characterization of rapidly self-renewing stem cells from cultures of human marrow stromal cells. Cytotherapy 2001; 3: 393-6.

  34. Zohar R, Sodek J, McCulloch AG. Characterization of stromal progenitor cells enriched by flow cytometry. Blood 1997; 90: 3471-81.

  35. Caplan AI. Mesenchymal stem cells. J Orthop Res 1991; 9: 641-50.

  36. Herbertson A, Aubin JE. Cell sorting enriches osteogenic populations in rat bone marrow stromal cell cultures. Bone 1997; 21: 491-500.

  37. Javanzon EH, Beggs KJ, Flake AW. Mesenchymal stem cells:paradoxes of passaging. Exp Hematol 2004; 32: 414-25.

  38. Joyner CJ, Bennett A, Triffitt JT. Identification and enrichment of human osteoprogenitor cells by using differentiation stagespecific monoclonal antibodies. Bone 1997; 21: 1-6.

  39. Conget P, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 1999; 181: 67-73.

  40. Guo Z, Yang J, Liu X, LiX, Hou C, Tang PH, Mao N. Biological features of mesenchymal stem cells from human bone marrow. Chin Med J 2001; 114: 950-3.

  41. Shih-Chie Hung, Henrich Cheng, Chien-Yuan Pan, May J Tsai, Lung-Sen Kao, Hsiao-Li Ma. In vitro differentiation of sizesieved stem cells into electrically active neural cells. Stem Cells 2002; 20: 522-9.

  42. Majumdar MK, Keane-Moore M, Buyaner D, Hardy WB, Moorman MA, McIntosh KR, Mosca JD. Characterization and functionality of cell surface molecules on human mesenchymal stem cells. J Biomed Sci 2003; 10: 228-41.

  43. Gronthos S, Graves SE, Ohta S, Simmons PJ. The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors. Blood 1994; 84: 4164-73.

  44. Bensidhoum M, Chapel A, Francois S, Demarquay C, Mazurier C, Fouillard L, et al. Homing of in vitro expanded Stro-1-or Stro-1+ human mesenchymal stem cells into the NOD/SCID mouse and their role in supporting human CD34 cell engraftment. Blood 2004; 103: 3313-9.

  45. Galmiche MC, Koteliansky VE, Brière J, Hervè P, Charbord P. Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway. Blood 1993; 82: 66-76.

  46. Zhu GR, Zhou XY, Lu H, Zhou JW, Li AP, Xu W, et al. Human bone marrow mesenchymal stem cells express multiple hematopoietic growth factors. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2003; 11: 115-19.

  47. Majumdar MK, Thiede MA, Haynesworth SE, Bruder SP, Gerson SL. Human marrow-derived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support longterm hematopoiesis when differentiated toward stromal and osteogenic lineages. J Hematother Stem Cell Res 2000; 9: 841-8.

  48. Heil M, Ziegelhoeffer T, Mees B, Schaper W. A different outlook on the role of bone marrow stem cells in vascular growth. Bone marrow delivers software not hardware. Circ Res 2004; 94: 573-4.

  49. Herzog EL, Chai Li, Krause DS. Plasticity of marrow-derived stem cells. Blood 2003; 102: 3483-93.

  50. Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 1995; 18: 1417-26.

  51. Pereira RF, Halford KW, O’Hara MD, Leeper DB, Sokolov BP, Pollard MD et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci USA 1995; 92: 4857-61.

  52. Sandhu JS, Clark BR, Boynton EL, Atkins H, Messner H, Keating A, Hozumi N. Human hematopoiesis in SCID mice implanted with human adult cancellous bone. Blood 1996; 88: 1973-82.

  53. Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 2000; 164: 247-56.

  54. Deng W, Obrocka M, Fischer I, Prockop DJ. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun 2001; 282: 148-52.

  55. Zhao LR, Duan WM, Reyes M, Keene CD, Verfaillie CM, Low WC. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. Exp Neurol 2002; 174: 11-20.

  56. Oswald J, Boxberger S, Jørgensen B, Feldmann S, Ehninger S, Bornhäuser M, Werner C. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells 2004; 22: 377-84.

  57. Asahara T, Murohara T, Suillivan A, et al. Isolation of putative progenitor endotelial cells for angiogenesis. Science 1997; 275964-7.

  58. Reyes M, Lund T, Lenvik T, Aguilar D, Koodle L, Verfaille CM. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001; 98: 2615-25.

  59. Tremain A, Korkko J, Ibberson D, Kopen GC, Digirolamo C, Phinney DG. MicroSAGE Analysis of 2,353 expressed genes in a single cell-derived colony of undifferentiated human mesenchymal stem cells reveals mRNAs of multiple cell lineages. Stem Cells 2001; 19: 408-18.

  60. Seshi B, Kumar S, King D. Multilineage gene expression in human bone marrow stromal cells as evidenced by single-cell microarray analysis. Blood Cells Mol Dis 2003; 31: 268-85.

  61. Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, Deans RJ, Krause DS, Keating A. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy 2005; 7: 393-5.

  62. Zvaifler NJ, Marinova-Mutafchieva L, Adams G, Edwards CJ, Moss J, Burguer JA, Maini RN. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res 2000; 2: 477-88.

  63. Huss R. Isolation of primary and immortalized CD34- hematopoietic and mesenchymal stem cells from various sources. Stem Cells 2000; 18: 1-9.

  64. Huss R, Lange C, Weissinger EM, Kolb H, Thalmeier K. Evidence of peripheral blood-derived, plastic-adherent CD34-/low hematopoietic stem cell clones with mesenchymal stem cell characteristics. Stem Cells 2000; 18: 252-60.

  65. Conrad C, Gottgens B, Kinston S, Ellwart J, Huss R. GATA transcription in a small rhodamine 123lowCD34+ subpopulation of a pheripheral blood-derived CD34-CD105+ mesenchymal cell line. Exp Hematol 2002; 887-95.

  66. Campagnoli C, Roberts IAG, Kumar S, Bennett PR, Bellantuono LL, Fisk NM. Identification of mesenchymal stem/progenitor cell in human first-trimester fetal blood, liver and bone marrow. Blood 2001; 98: 2396-2402.

  67. Fernández M, Simon V, Herrera G, Cao C, Del Favero H, Minguell JJ. Detection of stromal cells in peripheral blood progenitor cell collections from breast cancer patients. Bone Marrow Transplant 1997; 20: 265-71.

  68. Purton LE, Mielcarek M, Torok-Storb B. Monocytes are likely candidate ‘stromal’ cell in G-CSF-mobilized peripheral blood. Bone Marrow Transplant 1998; 21: 1075-6.

  69. Purton LE, Lee MY, Torok-Storb B. Normal human peripheral blood mononuclear cells mobilized with granulocyte colony stimulating factor have increased osteoclastogenic potential compared to nonmobilized blood. Blood 1996; 87: 1802-8.

  70. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Hematol 2000; 109: 235-42.

  71. Goodwin HS, Bicknese AR, Chien SN, Bogucki BD, Oliver DA, Quinn CO, Wall DA. Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant 2001; 7: 581-8.

  72. Gang EJ, Hong SH, Jeong JA, Hwang SH, Kim SW, Yang IH, Ahn C, Han H, Kim H. In vitro mesengenic potential of human umbilical cord blood-derived mesenchymal stem cells. Biochem Biophys Res Commun 2004; 321: 102-8.

  73. Bieback K, Kern S, Kluter H, Eichler H. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 2004; 22: 625-34.

  74. Lee OK, Kuo TK, Chen W, Lee KD, Hsieh S, Chen T. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 2004; 103: 1669-75.

  75. Wang JF, Wang LJ, Wu YF, Xiang Y, Xie CG, Jia BB, et al. Mesenchymal stem/progenitor cells in human umbilical cord blood as support for ex vivo expansion of CD34+ hematopoietic stem cells and for chondrogenic differentiation. Hematol 2004; 89: 837-44.

  76. Mareschi K, Biasin E, Piacibello W, Agglietta M, Madon E, Fagioli F. Isolation of human mesenchymal stem cells: bone marrow versus umbilical cord blood. Haematologica 2001; 86: 1099-1100.

  77. Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of posnatal human mesenchymal stem cells: candidate MSC-Like cells from umbilical cord. Stem Cells 2003; 21: 105-10.

  78. Panepucci RA, Siufi JLC, Silva WA, Proto-Siquiera R, Neder L, Orellana M, et al. Comparison of gene expression of umbilical cord vein and bone marrow-derived mesenchymal stem cells. Stem Cells 2004; 22: 1263-78.

  79. Bianco P, Riminucci M, Gronthos S, Robey PG. Bone Marrow Stromal Stem Cells: nature, biology, and potential applications. Stem Cells 2001. 19: 180-92.

  80. in’tAnker PS, Scherjon SA, Kleijburg-van der Keur C, Noort WA, Claas FHJ, Willemze R, et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 2003; 102: 1548-9.

  81. in’tAnker PS, Scherjon SA, Kleijburg-van der Keur C, Groot- Swings MJS, Claas FHJ, Willemze R, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004; 22: 1338-45.

  82. Hu Y, Liao L, Wang Q, Ma L, Ma G, Jiang X, Zhao RC. Isolation and identification of mesenchymal stem cells from human fetal pancreas. J Lab Clin Med 2003; 141: 342-9.

  83. Yen BL, Huang HI, Chien CC, Jui HY, Ko BS, Yao M, Shun CT, Yen ML, Lee MC, Chen YC. Isolation of multipotent cells from human term placenta. Stem Cells 2005; 23: 3-9.

  84. Wang HS, Hung SC, Peng ST, Huang CC, Wei HM, Guo YJ, et al. Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord. Stem Cells 2004; 22: 1330-7.

  85. Dicker A, Le Blanc K, Astrom G, van Harmelen V, Gotherstrom C, Blomqvist L, et al. Functional studies of mesenchymal stem cells derived from adult human adipose tissue. Exp Cell Res 2005; 308: 283-90.

  86. Kern S, Eichler H, Stoeve J, Kluter H, Bieback K. Comparative Analysis of Mesenchymal Stem Cells from Bone Marrow, Umbilical Cord Blood or Adipose Tissue. Stem Cells 2006; Enero 12. Publicación en línea.

  87. Piersma AH, Ploemacher RE, Brockbank KG. Transplantation of bone marrow fibroblastoid stromal cells in mice via the intravenous route. Br J Haematol 1983; 54: 285-90.

  88. Piersma AH, Brockbank KG, Ploemacher RE, Ottenheim CP. Recovery of hemopoietic stromal progenitor cells after lethal total-body irradiation and bone marrow transplantation in mice. Transplantation 1985; 40: 198-201.

  89. Pereira RF, O’Hara MD, Laptev AV, Halford KW, Pollard MD, Class R, et al. Marrow stromal cells as a source of progenitor cells of nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta. Proc Natl Acad Sci USA 1998; 95: 1142-7.

  90. Azizi SA, Stokes D, Augelli BJ, DiGirolamo C, Prockop DJ. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats-similarities to astrocyte grafos. Proc Natl Acad Sci USA 1998; 95: 3908-13.

  91. Allers C, Sierralta WD, Neubauer S, Rivera F, Minguell JJ, Conget PA. Dynamic of distribution of human bone marrowderived mesenchymal stem cells after transplantation into adult unconditioned mice. Transplantation 2004; 78: 503-8.

  92. Lazarus H, Haynesworth S, Gerson S, Rosenthal N, Caplan A. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): Implications for therapeutic use. Bone Marrow Transplant 1995; 16: 557-64.

  93. Almeida-Porada G, Porada CD, Tran N, Zanjani ED. Cotransplantation of human stroma cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points alter transplantation. Blood 2000; 95: 3620-7.

  94. Maitra B, Szekely E, Gjini K, Laughlin MJ, Dennis J, Haynesworth SE, Koc ON. Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant 2004; 33: 597-604.

  95. Kinnaird T, Stabile E, Brunett MS, Lee CW, Barr S, Fuchs S, Epstein SE. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res 2004; 94: 678-85.

  96. Murphy JM, Kavalkovitch KW, Fink D, Barry FP. Regeneration of meniscal tissue and protection of articular cartilage by injection of mesenchymal stem cells. Osteoarthritis Cartilage 2000; 8(Suppl B): S25.

  97. Horwitz E. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999; 5: 309-13.

  98. Mauney JR, Volloch V, Kaplan DL. Role of adult mesenchymal stem cells in bone tissue engineering applications: current status and future prospects. Tissue Eng 2005; 11: 787-802.

  99. Freyman T, Polin G, Osman H, Crary J, Lu M, Cheng L, Palasis M, Wilensky RL. A quantitative, randomized study evaluating three methods of mesenchymal stem cell delivery following myocardial infarction. Eur Heart J. En prensa.

  100. Berry MF, Engler AJ, Woo YJ, Pirolli TJ, Bish LT, Jayasankar V, Morine KJ, Gardner TJ, Discher DE, Sweeney HL. Mesenchymal Stem Cell Injection After Myocardial Infarction Improves Myocardial Compliance. Am J Physiol Heart Circ Physiol. En prensa.




2020     |     www.medigraphic.com