ABSTRACT
Objective@#To investigate the effect of pure titanium surface of large diameter TiO2 nanotubes modified by RGD peptide on the adhesion and proliferation of MG63 osteoblasts. to provide theoretical proof for developing titanium implants.@*Methods@#Commercially pure titanium discs were divided into four groups and treated with SLA to obtain a microrough surface (SLA group). Then, nanotubes were imposed on this microrough surface by anodization (SLA+80 group). The surface was then modified by dopamine (DOPA) (DOPA Group), after which bioactive RGD peptide layers were generated on the TiO2 nanotube surfaces via electrochemical and molecular self-assembly techniques (RGD group). The titanium surface morphology and elemental composition of each group were characterized by field emission scanning electron microscopy (FE-SEM) and X-ray photoelectron spectroscopy (XPS). MG63 cells were cultured in vitro to evaluate biological activities of titanium before and after treatment, including the evaluation of early-stage cell adhesion capacity by fluorescence microscopy, proliferation capacity by MTS assay, and mRNA expression of the cell osteoblast-related genes alkaline phosphatase (ALP) and osteocalcin (OCN) by qRT-PCR.@*Results@#FE-SEM and XPS showed that hierarchical micro/nanosurfaces decorated with TiO2 nanotubes were produced on titanium using sandblasting and large grit etching combined with anodization, dopamine was then self-polymerized to form a polydopamine film on the TiO2 nanotube surfaces, and RGD peptides were then conjugated to the polydopamine film, finally forming RGD peptide-modified bioactive layers. In vitro experiments showed that compared with the other three materials, the RGD-modified material was more conducive to cell adhesion and proliferation (P < 0.05). The expression levels of ALP and OCN mRNA in the RGD group were significantly higher than those in the SLA group and DOPA group (P < 0.05).@*Conclusion@#Hierarchical micro/nanosurfaces decorated with TiO2 nanotubes functionally modified with RGD peptides have good biocompatibility and could be used for developing titanium implants and further improving early osseointegration.
ABSTRACT
PURPOSE: The present paper reports a systematic study on the effect of bifunctional chelators (BFC) namely, NOTA, DOTA, and DTPA, on the radiochemical formulation, in vitro stability, and in vivo biological properties of ⁶⁸Ga-labeled RGD peptide derivatives.METHODS: The three RGD conjugates namely, NOTA-Bn-E-[c(RGDfk)]₂, DOTA-Bn-E-[c(RGDfk)]₂, and DTPA-Bn-E-[c(RGDfk)]₂ were radiolabeled with ⁶⁸Ga and the radiolabeling was optimized with respect to the ligand amount, radiolabeling time, and temperature. Further, the ⁶⁸Ga complexes were assessed for their in vitro and in vivo stabilities. The biodistribution studies of the three radiolabeled conjugates were carried out in C57BL/6 mice bearing melanoma tumor at 30 min and 1 h post-adimistration.RESULTS: NOTA-Bn-E-[c(RGDfk)]₂ could be radiolabeled with ⁶⁸Ga at room temperature while DOTA-Bn-E-[c(RGDfk)]₂ and DTPA-Bn-E-[c(RGDfk)]₂ were radiolabeled at high temperature. ⁶⁸Ga-NOTA-Bn-E-[c(RGDfk)]₂ was found to be the most kinetically rigid in in vitro stability assay. The uptake of the three radiolabeled peptide conjugates in melanoma tumor was comparable at 1 h post-administration (NOTA; DOTA; DTPA (% I.D./g):: 2.78 ± 0.38; 3.08 ± 1.1; 3.36 ± 0.49). However, the tumor/background ratio of ⁶⁸Ga-NOTA-Bn-E-[c(RGDfk)]₂ was the best amongst the three radiotracers. ⁶⁸Ga-complexes of NOTA-Bn-E-[c(RGDfk)]₂ and DOTABn-E-[c(RGDfk)]₂ showed excellent in vivo stability while ⁶⁸Ga-DTPA-Bn-E-[c(RGDfk)]₂ showed significant metabolic degradation.CONCLUSION: These studies show that ⁶⁸Ga-NOTA-Bn-E-[c(RGDfk)]₂ would be the most appropriate ⁶⁸Ga-labeled radiotracer and the most amenable for kit formulation.
Subject(s)
Animals , Mice , Chelating Agents , In Vitro Techniques , Melanoma , Pentetic Acid , PeptidesABSTRACT
Objective To investigate the effect of argenin-glycin-aspartic acid (RGD) peptides on islet viability and function. Methods Rats were randomly divided into 2 groups: 1640 group and RGD group. Ficoll was used to purify islet in a discontinuous-density-gradient way. Islet concentration is 27% ,25% ,23% ,20.5% and 11% respectively. Acridine orange/ethidium bromide(AO/EB) fluorescent staining method was adopted to observe the effect of RGD peptides on islet viability. Radioimmunoassay was adopted to detect insulin level and measure insulin secretion index (SI). Caspase-9 cells and phospho-Akt 473-positive cells were determined by flow cytometry to investigate the influence of RGD peptides on caspase-9 activity and Akt. Results 600-700 IEQ islet was extracted from each rat by Ficoll purification through modified gradient centrifugation. AO/EB stain showed islet survival rate was more than 95% immediately after separation. Islets cultured in the medium of RPMI-1640 showed SI of 1.64 ±0.28 after 1 week, while islets cultured in the medium of RPMI-1640 containing RGD peptides showed SI of 2.28 ± 0.16 (P ＜ 0.05 ). Flow cytometry showed the level of activated caspase-9 was ( 22.66 ± 3.56 ) % if islet was cultured in RPMI-1640 alone for 1 week while the level was( 10.54 ± 1.96) % if islet was cultured in RPMI-1640 containing RGD peptides. There was statistical difference ( P ＜ 0. 05 ). The effect of RGD on Akt phosphorylation was also detected. Akt phosphorylation proportion was (31.47 ±4.08)% 1 week after cultured in RPMI-1640 while the value was(61. 054 ±6.03)% if cultured in RPMI-1640 containing RGD peptides. The difference had statistical significance (P ＜ 0. 05). Conclusion RGD peptides could inhibit apoptosis through the phosphorylation of Akt/PKB.