Terbium-Doped Hydroxyapatite Nanoparticles For Labeling And Tracing Human Adipose-Derived Stem Cells

Objective:Stem cell therapy offers huge potential in treating degenerative diseases or injury which traditional pharmacology and medical treatments has yet to be able to help to any great extent. In order to achieve the most treatment effect, we should understand the course of survival, multiplication, differentiation and homing of stem cells. Cell labeling and tracking are the major methords for observing the changes of the stem cells. In current, the common labeling agent includes organic dyes, superparamagnetic iron oxide(SPIO), quantum dots(QDs). But there are a lot of problems, such as photobleaching, cytotoxicity, instability. Lanthanide-doped fluorescent nanoparticles as novel biomarker applied to the biomedical field. Compared to the traditional fluorescent markers, the lanthanide nanoparticles improve the detection sensitivity. So we attempts to synthesize Tb3+doped hydroxyapatite nanoparticles (Tb-nHA) and label human adipose-derived stem cells(hADSCs). We aim to find a new nanoparticle for long-term stem cell tracking through evaluation of the influence on the cell and labeling efficiency.Methods:1. Tb3+doped hydroxyapatite nanoparticles(Tb-nHA) were synthesized via the hydrothermal synthesis with Ca(NO3) 2 ·4H2O, Tb (NO3) 3 ·6H2O, Na3PO4 · 12H2O, oleic acid, ethanol and octadecylamine. The nanoparticles were collected by centrifugation.2. The morphology, structure and luminescence of the nanoparticles were determined using transmission electron microscope (TEM), X-ray diffractometer (XRD), fourier transform infrared spectrometer (FTIR) and photoluminescence spectra(PL).3. The adipose tissue samples were obtained from the patients undergoing liposuction and digested with collagenase type I. The human adipose-derived stem cells(hADSCs) were examined by cytochemical staining, flowcytometry and real-time PCR.4. The hADSCs and Tb-nHA at different concentration were cocultured, and the influence of Tb-nHA on hADSCs was evaluated with MTT, cytochemical staining, flowcytometry and realtime PCR.5. The fluorescent images of different passages of hADSCs were collected with confocal microscope(LSCM),and the fluorescent indensities were compared. The localization of intracellular nanoparticles was observed using colocalization with LAMP-1 and TEM.6. The P2 Tb-nHA labeling hADSCs were seeded on CHA scaffold in a concentration of 2.0×107/ml. after osteogenesis for 2W in vitro, the cells and the scaffold were inlanted subcutaneously into node mice. After 3M and 6M of operation, the samples were harvested and examined with HE stained and immunochemical stain, while the images were collected via fluorescence microscope.Result:1. Tb-nHA were short nanorods with a diameter of 13.05±1.34nm and average length about 173.56±23.84nm.2. Tb-nHA possessed good crysallinity and the structure conformed to the typical HA. When excited at 265nm, Tb-nHA exhibited distinct fluorescence peak at 544nm.3. The hASCs were isolated by digestion with collagenase type I. The cells presented similar immunophenotypes, osteogenic and adipogenic differentiations. Meanwhile Tb-nHA had neither cytotoxicity to the hADSCs nor effect to multipotent differentiations.4. The result of colocalization showed the two locations of the green fluorescent of Tb-nHA and the red fluorescent of LAMP-1 coincided. TEM showed the nanopartiles were uptaken into the lysosomes.5. After 3M and 6M implantation histology of Tb-nHA labeled hASCs showed a lot of cells in the scaffold, and the green fluorescent was obtained by fluorescent microscope. The labeled cells were confirmed as hASCs by immunochemical stain.Conclusions:1. The HA nanoparticle doped with Terbium were synthesized via the hydrothermal synthesis possessed uniform morphology, good crystallinity and excellent luminescent.2. The characteristics of hADSCs accorded with MSCs. Tb-nHA did not change the native characteristics of hADSCs, and were allowed to label and trace hADSCs.3. Tb-nHA was uptaken into hADSCs by endocytosis and located in the lysosomes.4. Because of the excellent luminescent, Tb-nHA was a ideal long-term tracer for stem cells in vivo.