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Multifunctional nanoprobes open exciting possibilities for accurate diagnosis and therapy. In this research, we developed a (64)Cu-labeled GdVO4:4%Eu two-dimension (2D) tetragonal ultrathin nanosheets (NSs) that simultaneously possess radioactivity, fluorescence, and paramagnetic properties for multimodal imaging. The carboxyl-functionalized Eu(3+)-doped GdVO4 NSs were synthesized by a facile solvothermal reaction, followed by ligand exchange with polyacrylic acid (PAA). With ultrathin thickness of ∼5 nm and width of ∼150 nm, the carboxyl-functionalized NSs were further modified by DOTA chelator for (64)Cu labeling and Asp-Gly-Glu-Ala (DGEA) peptide for integrin α2β1 targeting. After initial evaluation of the cytotoxicity and targeting capability with PC-3 cells, the obtained multifunctional nanoprobes ((64)Cu-DOTA-GdVO4:4%Eu-DGEA) were further explored for targeted positron emission tomography (PET) and T1-weighted magnetic resonance imaging (MRI) of PC-3 tumor (prostate cancer, high integrin α2β1 expression) in vivo. Based on the strong fluorescence of the NSs, the particle distribution in mouse tissues was also determined by fluorescent microscopy. In summary, GdVO4:4%Eu NS is a potential multimodal multiscale nanoprobe that could not only be used for in vivo imaging, but also be tracked in cellular scale and ex vivo due to its fluorescent property., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Injectable delivery matrices hold promise in enhancing engraftment and the overall efficacy of cardiac cell therapies; however, the mechanisms responsible remain largely unknown. Here we studied the interaction of a collagen matrix with circulating angiogenic cells (CACs) in a mouse myocardial infarction model. CACs + matrix treatment enhanced CAC engraftment, and improved myocardial perfusion, viability and function compared to cells or matrix alone. Integrin-linked kinase (ILK) was up-regulated in matrix-cultured CACs. Integrin α2β1 blocking prevented ILK up-regulation, significantly reduced the adhesion, proliferation, and paracrine properties of matrix-cultured CACs, and negated the benefits of CACs + matrix therapy in vivo. Furthermore, integrin α5 was essential for the angiogenic potential of CACs on matrix. These findings indicate that the synergistic therapeutic effect of CACs + matrix therapy in MI requires the matrix to enhance CAC function via α2β1 and α5 integrin signaling mechanisms, rather than simply delivering the cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The function of type II collagen in cartilage is well documented and its importance for long bone development has been implicated. However, the involvement of type II collagen in bone marrow derived mesenchymal stem cell (BMSC) osteogenesis has not been well investigated. This study elucidated the pivotal role of type II collagen in BMSC osteogenesis and its potential application to bone healing. Type II collagen-coated surface was found to accelerate calcium deposition, and the interaction of osteogenic medium-induced BMSCs with type II collagen-coated surface was mainly mediated through integrin α2β1. Exogenous type II collagen directly activated FAK-JNK signaling and resulted in the phosphorylation of RUNX2. In a segmental defect model in rats, type II collagen-HA/TCP-implanted rats showed significant callus formation at the reunion site, and a higher SFI (sciatic function index) scoring as comparing to other groups were also observed at 7, 14, and 21 day post-surgery. Collectively, type II collagen serves as a better modulator during early osteogenic differentiation of BMSCs by facilitating RUNX2 activation through integrin α2β1-FAK-JNK signaling axis, and enhance bone defect repair through an endochondral ossification-like process. These results advance our understanding about the cartilaginous ECM-BMSC interaction, and provide perspective for bone defect repair strategies., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study, we evaluated the competence of a rationally designed collagen-like peptide (CLP-Cys) sequence - containing the minimal essential Glycine-Glutamic acid-Arginine (GER) triplet but lacking the hydroxyproline residue - for supporting human mesenchymal stem cell (hMSC) adhesion, spreading and proliferation. Cellular responses to the CLP-Cys sequence were analyzed by conjugating the peptide to two different substrates - a hard, planar glass surface and a soft hyaluronic acid (HA) particle-based hydrogel. Integrin-mediated cell spreading and adhesion were observed for hMSCs cultivated on the CLP-Cys functionalized surfaces, whereas on control surfaces lacking the peptide motif, cells either did not adhere or maintained a round morphology. On the glass surface, CLP-Cys-mediated spreading led to the formation of extended and well developed stress fibers composed of F-actin bundles and focal adhesion complexes while on the soft gel surface, less cytoskeletal reorganization organization was observed. The hMSCs proliferated significantly on the surfaces presenting CLP-Cys, compared to the control surfaces lacking CLP-Cys. Competitive binding assay employing soluble CLP-Cys revealed a dose-dependent inhibition of hMSC adhesion to the CLP-Cys-presenting surfaces. Blocking the α(2)β(1) receptor on hMSC also resulted in a reduction of cell adhesion on both types of CLP-Cys surfaces, confirming the affinity of CLP-Cys to α(2)β(1) receptors. These results established the competence of the hydroxyproline-free CLP-Cys for eliciting integrin-mediated cellular responses including adhesion, spreading and proliferation. Thus, CLP-Cys-modified HA hydrogels are attractive candidates as bioactive scaffolds for tissue engineering applications., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Osteoblasts grown on microstructured Ti surfaces enhance osteointegration by producing local factors that regulate bone formation as well as bone remodeling, including the RANK ligand decoy receptor osteoprotegerin (OPG). The objective of this study was to explore the mechanism by which surface microstructure and surface energy mediate their stimulatory effects on OPG expression. Titanium disks were manufactured to present different surface morphologies: a smooth pretreatment surface (PT, Ra<0.2microm), microstructured sandblasted/acid etched surface (SLA, Ra=3-4microm), and a microstructured Ti plasma-sprayed surface (TPS, Ra=4microm). Human osteoblast-like MG63 cells were cultured on these substrates and the regulation of OPG production by TGF-beta1, PKC, and alpha2beta1 integrin signaling determined. Osteoblasts produced increased amounts of OPG as well as active and latent TGF-beta1 and had increased PKC activity when grown on SLA and TPS. Exogenous TGF-beta1 increased OPG production in a dose-dependent manner on all surfaces, and this was prevented by adding blocking antibody to the TGF-beta type II receptor or by reducing TGF-beta1 binding to the receptor by adding exogenous soluble type II receptor. The PKC inhibitor chelerythrine inhibited the production of OPG in a dose-dependent manner, but only in cultures on SLA and TPS. shRNA knockdown of alpha2 or a double knockdown of alpha2beta1 also reduced OPG, as well as production of TGF-beta1. These results indicate that substrate-dependent OPG production is regulated by TGF-beta1, PKC, and alpha2beta1 and suggest a mechanism by which alpha2beta1 signaling increases PKC, resulting in TGF-beta1 production and TGF-beta1 then acts on its receptor to increase transcription of OPG.

Implant osseointegration is a prerequisite for clinical success in orthopaedic and dental applications, many of which are restricted by loosening. Biomaterial surface modification approaches, including calcium-phosphate ceramic coatings and macro/microporosity, have had limited success in promoting integration. To improve osseointegration, titanium surfaces were coated with the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) collagen-mimetic peptide, selectively promoting alpha2beta1 integrin binding, a crucial event for osteoblastic differentiation. Titanium surfaces presenting GFOGER triggered osteoblastic differentiation and mineral deposition in bone marrow stromal cells, leading to enhanced osteoblastic function compared to unmodified titanium. Furthermore, this integrin-targeted coating significantly improved in vivo peri-implant bone regeneration and osseointegration, as characterized by bone-implant contact and mechanical fixation, compared to untreated titanium in a rat cortical bone-implant model. GFOGER-modified implants also significantly enhanced osseointegration compared to surfaces modified with full-length type I collagen, highlighting the importance of presenting specific biofunctional domains within the native ligand. In addition, this biomimetic implant coating is generated using a simple, single-step procedure that readily translates to a clinical environment with minimal processing and cytotoxicity concerns. Therefore, this study establishes a biologically active and clinically relevant implant-coating strategy that enhances bone repair and orthopaedic implant integration.

This work describes the cytotoxicity, and the cell adhesion behavior of K562 cell line from human erythroleukemia transfected with the DNA for the alpha(2)beta(1) integrin over type-I collagen matrices with variable degree of carboxyl group and wettability. The results showed that type-I collagen materials with variable degree of carboxyl group prepared by selective hydrolysis of carboxyamide side chains of Asn and Gln residues present in the protein, independently from the extent of side chain hydrolysis, was characterized by preserved triple helix structure for materials with a carboxyl group content up to 87 +/- 17. Imbibition and wettability increased linearly with increasing carboxyl group content from 46 +/- 12 to 87 +/- 17, and no signs of cytotoxicity were detected. Nevertheless, in comparison to native collagen, K562 cell adhesion to PACMs was significantly improved by factors ranging from 1.60 to 1.47x, with the reduction in cell adhesion observed with increasing carboxyl content attributed to a balance between the inhibition of increasing negative charge and the stimulation by increased wettability. On the other hand, the overall improvement of K562 cell adhesion to polyanionic collagen was attributed to the introduction of new distinct motifs described as the minimal active recognition sequence for alpha(2)beta(1) integrins binding with type-I collagen produced as a result of Asn-Gly Glu-Ala alpha2(I)294-297, and Gly Gln-Arg-Gly Val-Val carboxyamide side chains hydrolysis., (Copyright 2002 Elsevier Science Ltd.)