Analysis of the ectopic osteogenic potential of adipose-derived mesenchymal stem cells in mice based on the relative gene expression of osteogenic markers
Osnovni cilj disertacije je da se uporedi i analizira osteogeni potencijal sveže
izolovanih mezenhimskih matičnih ćelija belog masnog tkiva (ADSC) i in vitro
osteoindukovanih ADSC na modelu ektopične implantacije kod Balb/c miševa.
ADSC su nakon izolacije okarakterisane imunocitohemijskom analizom.
Osteogena diferencijacija ADSC ispitana je analizom relativne ekspresije gena osteriksa
(Sp7), osteokalcina (Bglap), alfa 1 lanca kolagena tipa I (Col1a1) i alkalne fosfataze
(Alpl), vizuelizacijom fosfata i analizom ekspresije osteopontina. Miševima su
subkutano implantirani implantati sastavljeni od sveže izolovanih ADSC, mineralnog
koštanog matriksa (BMM) i plazme obogaćene trombocitima (PRP) (SPN grupa),
osteoindukovanih ADSC, BMM i PRP (OPN grupa), PRP i BMM (PN kontrola) i samo
od BMM (N kontrola). Osteogeneza u implantatima je ispitana analizom relativne
ekspresije Sp7, Bglap, Col1a1 i Alpl, histomorfološkom, histomorfometrijskom i
imunohistohemijskom analizom.
Nađena je pozitivna CD29 i negativna CD14 ekspresija kod ćelija, što je
karakteristika ADSC. Nakon petnaestodnevne osteoindukcije ADSC, zabeležena je
najjača ekspresija markera osteogene diferencijacije. Najviša ekspresija gena markera
osteogeneze, prisustvo ćelija nalik osteoblastima i osteoklastima, resorpcija BMM,
visok procenat infiltriranog tkiva i prisustvo tkiva nalik osteoidu, ukazuju na
napredovanje osteogeneze u OPN grupi nakon osme nedelje. U SPN grupi, nađeni su
izraženi znaci inicijacije osteogeneze posle druge nedelje, a od četvrte ekspresija gena i
ostalih markera opada što ukazuje na slabljenje osteogeneze.Rezultati doprinose razumevanju osteogenog potencijala različito pripremljenih
ADSC što treba imati u vidu pri izboru načina i uslova za njihovu primenu u tretmanu
koštanih defekata.The main subject of this doctoral dissertation was to compare and evaluate the
osteogenic potential of freshly isolated, non-treated adipose-derived mesenchymal stem
cells (ADSCs) and the potential of in vitro osteoinduced ADSCs in the ectopic boneforming
model in Balb/c mice.
The ADSCs were characterized after isolation by immunocytochemical analysis.
Osteogenic differentiation of ADSCs was evaluated using relative gene expression
analysis of osterix (Sp7), osteocalcin (Bglap), collagen type I alpha 1 chain (Col1a1)
and alkaline phosphatase (Alpl), along with an assay for detection of phosphate deposits
and an immunoexpression analysis of osteopontin. The implants composed of freshly
isolated ADSCs, bone mineral matrix (BMM) and platelet-rich plasma (PRP) (SPN
group), osteoinduced ADSCs, BMM and PRP (OPN group), BMM and PRP (PN
control) and BMM-only (N control), were implanted subcutaneously in mice. The
implants were analyzed using relative gene expression analysis of Sp7, Bglap, Col1a1
and Alpl, histochemical, histomorfometrical and immunohistochemical analyses.
Positive immunoexpression of CD29 and negative of CD14 was found in the
cells, which is a characteristic of ADSCs. After fifteen days of osteoinduction of
ADSCs, the expression of the markers of osteogenic differentiation was the highest. The
highest level of the bone-related gene expression, the presence of osteoblast-like and
osteoclast-like cells, the resorption of BMM, a high percentage of infiltrated tissue and
the presence of osteoid-like tissue, all suggest the progress of the osteogenesis in OPN
implants after eight weeks. Strongly expressed signs of the initiation of osteogenesis
were found in SPN implants after two weeks, but from the fourth week the expression
of bone-related genes, as well as other analyzed markers decreased.
The obtained results represent a contribution to the understanding of differences
in the osteogenic potential of differently prepared ADSCs which is very important
because of their utilization for the treatment of bone defects