Dextran-coated Fluorapatite Nanorods Doped With Lanthanides In Labelling And Directing Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objective:BMSCs have been demonstrated as an attractive cell source for tissue-engineering applications because of their ability to be easily isolated and expanded from adult bone marrow aspirates and their versatility for pluripotent differentiation into mesenchymal tissues. To date, however, an ideal agent for monitoring and tracking of transplanted BMSCs is still lacking. The organic fluorescent dyes CM-Dil and BrdU are usually used to track cells in vivo, while it is difficult to achieve long-term efficacy. Terbium (Tb)-or europium (Eu)-doped fluorapatite nanorods (Tb/Eu-FHA) were prepared using the hydrothermal method. The surfaces of the Tb/Eu-FHA nanorods were further conjugated with hydrophilic cationic polymers, such as dextran, to enhance hydrophilicity, biocompatibility and cell penetration. Then, the dextran-coated nanorods were cocultured with bone marrow mesenchymal stem cells (BMSCs). A luminescence signal in the cells was detected after12hours with a laser scanning confocal microscope (LSCM). Our current work attempts to provide an excellent fluorescent cell labelling agent for BMSCs in bone tissue engineering.Methods:1. Eu3+or Tb3+-doped FHA nanorods were synthesized via the hydrothermal method. Ca(NO3)2·4H2O, Eu(NO3)3or Tb(NO3)3, Na3PO4·12H2O, octadecylamine, oleic acid, ethanol, NaF and Na3PO4were added sequentially and mixed. The obtained Tb/Eu-FHA nanorods doped were collected by centrifugation.2. The structure, morphology, and luminescence of the products were determined using X-ray diffraction (XRD), field emission scanning electron microscopy (ESEM), transmission electron microscopy (TEM), and photoluminescence spectra (PL). Surfaces of the Tb-FHA nanorods were further conjugated with hydrophilic cationic polymers such as dextran to enhance the hydrophilic, biocompatibility and cell penetrations.3. Bone marrow aspirates were harvested from anterior superior iliac spine of the beagle dog. The specific cell surface antigen markers of cBMSCs were examined by flow cytometry (FCM). CD29, CD44, CD34and CD45were examined. Biocompatibility evaluation of dextran-coated Tb/Eu-THA nanorods on BMSCs were performed using MTS assay.4. The cBMSCs and dextran-coated Tb/FHA at a concentration of100μg/mL were cocultured, and fluorescent images of cBMSCs were collected via laser scanning confocal microscope (LSCM). The mRNA levels of ALP, COLlal, and ON were analysed via real-time PCR assay on days14and21of differentiation. The ALP activity was detected by ELISA on day14and day21of differentiation.5. The labelled BMSCs were seeded on sterilized PCL scaffold in a concentration of1x107cells/ml, and then implanted subcutaneously into nude mice. Samples were harvested after1and3months of incubation. Specimens were used to make rapid frozen sections that were then stained with haematoxylin and eosin (HE). Images were collected via inverted fluorescence microscope.Result:1. Typical FHA nanorods doped with Tb3+are straight nanorods with a diameter of about20nm and lengths ranging from110to170nm. The nanorods possessed uniform morphology and good crystallinity, and hard to soluble in water.2. Tb-FHA nanorods become luminescent with a maximum emission intensity at543nm (5D4-7F5), and the emission intensity of Eu-FHA nanorods is617nm (5D0-7F2). The result of XRD shows all diffraction peaks can be indexed as typical hexagonal FHA.3. The green/red fluorescence of cBMSCs can be clearly seen in confocal images with an excitation wavelength of405nm and488nm. No obvious cytotoxicity of Tb-FHA group was observed of the MTS assay. The fluorapatite nanorods act synergistically with the biochemical reagents in osteogenic medium to promote osteogenic differentiation.4. The mRNA levels of ALP, COLlal, and ON mRNA detected on day14and day21are all up-regulated. Consistent with real-time PCR result, ELSIA assay also shows an increase of ALP protein level.5. Samples were harvested after1and3months of incubation. Fluorescent images of the rapidly frozen sections were gathered via inverted fluorescence microscope and tipical green fluorescence are shown.Conclusions:1. The fluoride substituted hydroxyapatite nanorods doped with lanthanides synthesized using hydrothermal method possess excellent luminescent properties, uniform morphology, good crystallinity, and the uniformity of sizes. The luminescence shows high photostability, high resistance to photobleaching, and long luminescent lifetime.2. BMSCs can be efficient isolat from the bone marrow of beagle dogs suing whole bone marrow culture method. The cBMSCs show the potential of osteogenic, adipogenic and chondrogenic differentiation. Immunephenotype matches bone marrow mesenchymal stem cell characteristics. 3. Dextran-coated Tb/Eu-FHA nanorods proved to be successful in labelling cBMSCs. The nanorods possess excellent biocompatibility, concentration of100μg/mL is recommended, high concentration is useless.4. Dextran-coated Tb/Eu-FHA nanorods promote osteogenic differentiation of cBMSCs. There is a synergistic effect between the fluorapatite nanorods and biochemical reagents in osteogenic medium on osteogenic differentiation.5. Dextran-coated Tb/Eu-FHA nanorods can be used as an in vivo tracer, the fluorescence can persist for a long time in vivo.6. Lower costs, improved performance, simplicity of manufacture, facilitation of use and storage make the nanorods an make the nanorods may become excellent fluorescent cell labelling agent for BMSCs in bone tissue engineering.