Ex vivo karakteristike mezenhimalnih matičnih ćelija izolovanih iz sinovijalne tečnosti kolenog zgloba pasa različite starosti

Abstract

Cilj ovog rada je bio da se utvrdi: da li ćelije izolovane iz sinovijalne tečnosti (ST) sadrže ćelije koje imaju fenotipske i funkcionalne karakteristike mezenhimalnih matičnih ćelija (MMĆ), kao i da se in vivo ispita njihov hondrogeni potencijal, uporede karakteristike izolovanih matičnih ćelija (MĆ) tokom starenja i okarakterišu zglobne strukture kolenog zgloba pasa kako bi se definisale promene vezane za starenje. Uzorci ST, sinovijalne membrane (SM) i hrskavice sa subhondralnom kosti uzeti su iz kolenog zgloba 28 pasa rase nemački ovčar, muškog pola, koji su prema uzrastu i nivou fizičke aktivnosti svrstani u 4 grupe: mladi radni policijski psi (n = 4) i mladi neradni vlasnički psi (n = 5) starosti 3-5 godina; stari radni policijski psi (n = 13) starosti 10-12 godina i stari neradni vlasnički psi (n = 6) starosti 10-13 godina. Kultura adherentnih ćelija je uspostavljena iz ST 5 mladih i 5 starih radnih pasa. Adherentne ćelije su uzgajane tokom 3 nedelje pre određivanja CD90 i CD34 molekula metodom protočne citometrije. Istovremeno, ispitano je njihovo vreme udvajanja u kulturi (PTD), broj CFU-F i aktivnost markera starenja β-galaktozidaze. Adipogeni, osteogeni i hondrogeni potencijal su ispitani nizom imunocitohemijskih, biohemijskih, imunohistohemijskih i molekularnih metoda. Hondrogeni potencijal reprezentativnog uzorka ćelija je ispitan i metodom ksenotransplantacije umnoženih ćelija imunodeficijentnim miševima. Da bi se stekao sveobuhvatni uvid u promene koštano-zglobnog sistema tokom procesa starenja i da bi se ustanovio mogući značaj fizičke aktivnosti za nastanak ovih promena, pored kliničkog pregleda, izvršeno je radiološko ispitivanje oba kolena zgloba, zgloba kuka i lumbosakralnog dela kičme, makroskopski pregled svih zglobnih struktura kao i histološka i imunohistohemijska (IHH) analiza hrskavice i SM. Sinovijalna tečnost je ispitana sa citološkog i biohemijskog aspekta kako bi se isključilo prisustvo inflamacije. Ukupna debljina hrskavice, broj hondrocita na vertikalnom preseku, njihova gustina i površina određeni su histomorfometrijski. Analiziran je i izgled površine hrskavice, oblik hondrocita, morfologija izogenih grupa, zona koja deli kalcifikovanu od nekalcifikovane hrskavice, subhondralna kost i zastupljenost proteoglikana i glikozamioglikana (PG/GAG). Na SM, procenjivan je broj slojeva ćelija, prisustvo prstolikih nabora i ćelijske infiltracije. Imunohistohemijski je ispitivano prisustvo i distribucija kolagena tipa I, II i X u hrskavici i CD44 molekula u sinovijalnoj membrani...

The aim of this study was to determine: whether synovial fluid (SF) of dogs contains cells that have phenotypic and functional characteristics of mesenchymal stem cells (MSCs), to investigate there in vivo chondrogenic potential, to compare MSCs characteristics during aging and to define the changes associated with aging in stifle joints structures. Samples of SF, synovial membrane (SM) and cartilage with subchondral bone were taken from the 28 German Shepherd dogs, all males, who were assigned to four groups according to their age and physical activity: young working military dogs (n = 4) from 3 to 5 years old (3.8 ± 0.9); young non-working client-owned dogs (n = 5) from 3 to 5 years old (3.8 ± 0.8); aged working military dogs (n = 13) from 10 to 12 years old (10.5 ± 0.7) and aged non-working, client-owned dogs (n = 6), from 10 to 13 years old (11 ± 1.5). Canine SF adherent cells (cSFSc) from 5 young and 5 aged dogs were expanded (37ºC, 5% CO2, humidified atmosphere) for three weeks before their phenotypes were characterized by flowcytometry for the presence of CD90 and CD34. At the same time, population doubling time (PDT), number of CFU-F and SA-β-galactosidase activity (marker of cell ageing) have been determined. Adipogenic, osteogenic and chondrogenic potentials have been tested using imunocytochemical, biochemical, immunohistochemical (IHC) and molecular methods. Chondrogenic potential of representative cell samples was examined using xenotransplantation method in immunodeficient mice. In order to gain a comprehensive insight into the age related changes of musculoskeletal system and to determine the possible role of physical activity for the occurrence of these changes, in addition to clinical examination, radiological examination of both stifle and hip joints and lumbosacral part of the spine, macroscopic examination of all stifle structure as well as histological and IHC analysis of cartilage and SM was performed. SF cytological and biochemical analysis were done in order to exclude the presence of joint inflammation. The whole cartilage thickness, total chondrocytes number per vertical section, chondrocyte density and chondrocyte surface area in each cartilage zone were determined by histomorphometry. It was also analyzed: the appearance of the cartilage surface, chondrocyte shape and isogenous group morphology, tidemark morphology, subchondral bone structure and PG/GAG content. On SM, it was evaluated the number of cell layers, the presence of short and finger-like villi and the presents of cell infiltration...