Co-cultures of osteoblasts and osteoclasts are on the rise because they enable a more complex study. Diseases such as osteoporosis are related to a higher age. Thus, cell isolation from adult individuals is necessary. Osteoblasts can be isolated from the rat femur by three methods: explant culture, explant culture with enzymatic pre-treatment, or enzymatic treatment. The isolation methods yield different populations of osteoblasts which, in a co-culture with peripheral blood mononuclear cells, might result in differences in osteoclastogenesis. Therefore, we examined the differences in osteogenic markers, cell proliferation, and the metabolic activity of isolated osteoblast-like cells in a growth and differentiation medium. We then evaluated the effect of the isolated populations of osteoblast-like cells on osteoclastogenesis in a subsequent co-culture by evaluating osteoclast markers, counting formed osteoclast-like cells, and analyzing their area and number of nuclei. Co-cultures were performed in the presence or absence of osteoclastogenic growth factors, M-CSF and RANKL. It was discovered that enzymatic isolation is not feasible in adult rats, but explant culture and explant culture with enzymatic pre-treatment were both successful. Explant culture with enzymatic pre-treatment yielded cells with a higher proliferation than explant culture in a growth medium. The differentiation medium reduced differences in proliferation during the culture. Some differences in metabolic activity and ALP activity were also found between the osteoblast-like cells isolated by explant culture or by explant culture with enzymatic pre-treatment, but only on some days of cultivation. According to microscopy, the presence of exogenous growth factors supporting osteoclastogenesis in co-cultures was necessary for the formation of osteoclast-like cells. In this case, the formation of a higher number of osteoclast-like cells with a larger area was observed in the co-culture with osteoblast-like cells isolated by explant culture compared to the explant culture with enzymatic pre-treatment. Apart from this observation, no differences in osteoclast markers were noted between the co-cultures with osteoblast-like cells isolated by explant culture and the explant culture with enzymatic pre-treatment. The TRAP and CA II activity was higher in the co-cultures with exogenous growth than that in the co-cultures without exogenous growth factors on day 7, but the opposite was true on day 14. To conclude, explant culture and explant culture with enzymatic pre-treatment are both suitable methods to yield osteoblast-like cells from adult rats capable of promoting osteoclastogenesis in a direct co-culture with peripheral blood mononuclear cells. Explant culture with enzymatic pre-treatment yielded cells with a higher proliferation. The explant culture yielded osteoblast-like cells which induced the formation of a higher number of osteoclast-like cells with a larger area compared to the explant culture with enzymatic pre-treatment when cultured with exogenous M-CSF and RANKL.

• Klíčová slova
• adult rat, co-culture, mature bone, osteoblast-like cell, osteoclast-like cell,
• MeSH
• buněčná diferenciace MeSH
• faktor stimulující kolonie makrofágů * metabolismus   MeSH
• kokultivační techniky MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• leukocyty mononukleární metabolismus   MeSH
• ligand RANK metabolismus   MeSH
• osteoblasty metabolismus   MeSH
• osteogeneze * MeSH
• osteoklasty metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• faktor stimulující kolonie makrofágů * MeSH
• ligand RANK MeSH

Human mesenchymal stem cells derived from Wharton's jelly (WJ-MSCs) were used for the treatment of the ischemic-compression model of spinal cord injury in rats. To assess the effectivity of the treatment, different dosages (0.5 or 1.5 million cells) and repeated applications were compared. Cells or saline were applied intrathecally by lumbar puncture for one week only, or in three consecutive weeks after injury. Rats were assessed for locomotor skills (BBB, rotarod, flat beam) for 9 weeks. Spinal cord tissue was morphometrically analyzed for axonal sprouting, sparing of gray and white matter and astrogliosis. Endogenous gene expression (Gfap, Casp3, Irf5, Cd86, Mrc1, Cd163) was studied with quantitative Real-time polymerase chain reaction (qRT PCR). Significant recovery of functional outcome was observed in all of the treated groups except for the single application of the lowest number of cells. Histochemical analyses revealed a gradually increasing effect of grafted cells, resulting in a significant increase in the number of GAP43+ fibers, a higher amount of spared gray matter and reduced astrogliosis. mRNA expression of macrophage markers and apoptosis was downregulated after the repeated application of 1.5 million cells. We conclude that the effect of hWJ-MSCs on spinal cord regeneration is dose-dependent and potentiated by repeated application.

• Klíčová slova
• Wharton’s jelly, astrogliosis, axonal growth, human mesenchymal stem cells, inflammatory response, neuroregeneration, spinal cord injury,
• MeSH
• apoptóza MeSH
• astrocyty MeSH
• axony metabolismus   MeSH
• bílá hmota metabolismus   patologie   MeSH
• biologické markery MeSH
• buněčná diferenciace MeSH
• exprese genu MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• lokomoce MeSH
• mezenchymální kmenové buňky cytologie   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• poranění míchy diagnóza   etiologie   metabolismus   terapie   MeSH
• šedá hmota metabolismus   patologie   MeSH
• transplantace mezenchymálních kmenových buněk * MeSH
• viabilita buněk MeSH
• Whartonův rosol cytologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH