Immobilization of RGD Peptide onto the Surface of Apatite-Wollastonite Ceramic for Enhanced Osteoblast Adhesion and Bone Regeneration

The arginine-glycine-aspartic （RGD） acid peptide was grafted to the surface of apatitewollastonite （AW） ceramic in an effort to improve its cell adhesion, proliferation and osteoinduction. RGD peptide was covalently immobilized onto the surface of AW ceramic via the synthetic cross linker AA.PTS-E and 1-ethyl-3-（3-dimethylaminopropyl）carbodiimide （EDC）. The modified surfaces were characterized by attenuated total reflectance Fourier transform infrared spectroscopy （ATR-FTIR） and X-ray photoelectron spectroscopy （XPS）. The chemical analysis indicated that RGD peptide had been immobilized onto the AW surface successfully. The growth of osteoblast-like cells （MG63） showed that modifying the AW surface with RGD peptide enhanced the cell adhesion and proliferation. And the histological evaluation of RGD-AW showed that the bone regeneration and remodeling process were significantly enhanced compared to the original AW ceramics after 2, 4 and 8 weeks implantation in rabbit＇s femoral condyles.

Surface Modification of Biomimetic PLGA-(ASP-PEG) Matrix with RGD-Containing Peptide:a New Non-Viral Vector for Gene Transfer and Tissue Engineering

RGD-containing peptide （ K16-GRGDSPC） , characterized as non-viral gene vectors, was fabricated to modify the surface of PLGA-[ASP- PEG] matrix, which offered the foundation for gene transfer with porous matrix of gene activated later. Peptide was synthesized and matrix was executed into chips A, B and chip C. Chip C was regarded as control. Chips A and B were reacted with cross-linker. Then chip A was reacted with peptide. MS and HPLC were ased to detect the .14W and purity of peptide. Sulphur, existing on the surface of biomaterials, was detected by XPS. The purity of un-reacted peptide in residual solution was detected by a spectrophotometer. HPLC shows that the peptide purity was 94%- 95% , and MS shows that the MW was 2 741. 3307. XPS reveals that the binding energy of sulphur was 164 eV and the ratio of carbon to sulphur （C/S） was 99. 746 ：0. 1014 in reacted chip A. The binding energy of sulphur in reacted chip B was 164 eV and 162 eV, C/ S was 99.574：0.4255, aM there was no sulphur in chip C. Peptide was manufactured and linked to the surface of biomimetic and 3-D matrix, which offered the possibilities for gene transfer and tissue engineering with this new kind of non-viral gene vector.

RGD peptide and graphene oxide co-functionalized PLGA nanofiber scaffolds for vascular tissue engineering

In recent years,much research has been suggested and examined for the development of tissue engineering scaffolds to promote cellular behaviors.In our study,RGD peptide and graphene oxide(GO)co-functionalized poly(lactide-co-glycolide,PLGA)(RGD-GO-PLGA)nanofiber mats were fabricated via electrospinning,and their physicochemical and thermal properties were characterized to explore their potential as biofunctional scaffolds for vascular tissue engineering.Scanning electron microscopy images revealed that the RGD-GO-PLGA nanofiber mats were readily fabricated and composed of randomoriented electrospun nanofibers with average diameter of 558nm.The successful co-functionalization of RGD peptide and GO into the PLGA nanofibers was confirmed by Fourier-transform infrared spectroscopic analysis.Moreover,the surface hydrophilicity of the nanofiber mats was markedly increased by co-functionalizing with RGD peptide and GO.It was found that the mats were thermally stable under the cell culture condition.Furthermore,the initial attachment and proliferation of primarily cultured vascular smoothmuscle cells(VSMCs)on the RGD-GO-PLGA nanofibermats were evaluated.It was revealed that the RGD-GO-PLGA nanofibermats can effectively promote the growth of VSMCs.In conclusion,our findings suggest that the RGD-GO-PLGA nanofiber mats can be promising candidates for tissue engineering scaffolds effective for the regeneration of vascular smooth muscle.

Effect of RGD-modified Silk Material on the Adhesion and Proliferation of Bone Marrow-derived Mesenchymal Stem Cells

In order to investigate the effect ofArg-Gly-Asp （RGD） peptide-modified silk biomaterial on the adhesion and proliferation of bone marrow-derived mesenchymal stem cells （MSCs）, MSCs of third generation were seeded onto the surface of RGD-decorated silk （silk-RGD group）, silk alone （silk group） or tissue culture plate （TCP group）. After incubation for 4 or 12 h, MSCs were examined quantitatively by using precipitation method for cell attachment. The cell proliferation, which was defined as cell density, was compared among the three groups after culture for 1, 2, 3, and 4 days. Cell skeleton, which was labeled fluorescently, was observed under laser confocal microscope after 24 h of culture. The results showed that cell adhesion rate in silk-RGD group was higher than in silk group （P〈0.05）, but similar to that in TCP group after incubation for 4 or 12 h （P〉0.05）. There were no sig- nificant differences in the cell proliferation among the three groups at different time points （P〉0.05 for all）. Laser confocal microscopy revealed that in silk-RGD group, MSCs, strongly fluorescently stained, spread fully, with stress fibers clearly seen, while in silk group, actin filaments were sparsely aligned and less stress fibers were found. It was concluded that RGD peptide could improve the ad- hesion of MSCs to the silk scaffold, but had no impact on the proliferation of the cells.

Micro-positron emission tomography imaging of angiogenesis based on ^(18)F-RGD for assessing liver metastasis of colorectal cancer

Background:Positron emission tomography (PET) imaging is a non-invasive method to visualize and quantify the tumor microenvironment.This study aimed to explore the feasibility of ^(18)F-AIF-NOTA-E[PEG_(4-c)(RGDfk)]_(2) (denoted as ^(18)F-RGD) PET quantitative parameters to distinguish the angiogenesis in colorectal cancer (CRC) mice which has different metastatic potential.Methods:Twenty Lo Vo and twenty LS174T of CRC liver metastases animal models were established by implantation of human CRC cell lines via intrasplenic injection.Radiotracer-based micro-PET imaging of animal model was performed and the uptake of ^(18)F-RGD tracer in the tumor tissues was quantified as tumor-to-liver maximum or mean standardized uptake value (SUVmax or SUVmean) ratio.Pearson correlation was used to analyze the relationship between radioactive parameters and tumor markers.Results:The SUVmax and SUVmean ratios of Lo Vo model were significantly higher than those of LS174T in both liver metastasis and primary tumor lesions (P<0.05).A significant difference was observed in both vascular endothelial growth factor (VEGF) and Ki67 expressions between Lo Vo and LS174T primary tumors (P<0.05).The tumor-to-liver SUVmax or SUVmean ratio of ^(18)F-RGD showed a moderate correlation with VEGF expression (r=0.5700,P=0.001 and r=0.6657,P<0.001,respectively),but the SUVmean ration showed a weak correlation with Ki67 expression (r=0.3706,P<0.05).The areas under the receiver operating characteristic (ROC) curves of ^(18)F-RGD SUVmean ratio,SUVmax ratio for differentiating Lo Vo from LS174T tumor were 0.801 and 0.759,respectively.Conclusions:The tumor-to-liver SUVmean ratio of ^(18)F-RGD was a promising image parameter for the process of monitoring tumor angiogenesis in CRC xenograft mice model.

Immobilization of RGD Peptidcs onto Decellularized Valve Scaffolds to Promote Cell Adhesion

Porcine aortic valves were decellularized with trypsinase/EDTA and Triton-100. With the help of a coupling reagent Sulfo-LC-SPDP, the biological valve scaffolds were immobilized with one of RGD （arginine-glycine-aspartic acid） containing peptides, called GRGDSPC peptide. Myofibroblasts harvested from rats were seeded onto them. Based on the spectra of X-ray photoelectron spectroscopy, we could find conjugation of GRGDSPC peptide and the scaffolds. Cell count by both microscopy and MTT assay showed that myofibroblasts were easier to adhere to the modified scaffolds. It is proved that it is feasible to immobilize RGD peptides onto decellularized valve scaffolds, and effective to promote cell adhesion, which is beneficial for constructing tissue engineering heart valves in vitro.

Integrin-targeting with peptide-bioconjugated semiconductor-magnetic nanocrystalline heterostructures

Binary asymmetric nanocrystals （BNCs）, composed of a photoactive TiO2 nanorod joined with a superparamagnetic γ-Fe203 spherical domain, were embedded in polyethylene glycol modified phospholipid micelle and successfully bioconjugated to a suitably designed peptide containing an RGD motif. BNCs represent a relevant multifunctional nanomaterial, owing to the coexistence of two distinct domains in one particle, characterized by high photoactivity and magnetic properties, that is particularly suited for use as a phototherapy and hyperthermia agent as well as a magnetic probe in biological imaging. We selected the RGD motif in order to target integrin expressed on activated endothelial cells and several types of cancer cells. The prepared RGD-peptide/BNC conjugates, comprehensively monitored by using complementary optical and structural techniques, demon- strated a high stability and uniform dispersibility in biological media. The cytotoxicity of the RGD-peptide/BNC conjugates was studied in vitro. The cellular uptake of RGD-peptide conjugates in the cells, assessed by means of two distinct approaches, namely confocal microscopy analysis and emission spectroscopy determination in cell lysates, displayed selectivity of the RGD-peptide-BNC conjugate for the cw]33 integrin. These RGD-peptide-BNC conjugates have a high potential for theranostic treatment of cancer.

Adhesive peptides conjugated PAMAM dendrimer as a coating polymeric material enhancing cell responses

This present work aims to functionalize poly（amidoamine） （PAMAM） dendrimers with various reported adhesive peptides, including Arg-Gly-Asp （RGD）, Tyr-lle-Gly-Ser-Arg （YIGSR）, and Ile-Lys-Val-Ala-Val （IKVAV） for enhancing cell responses. The RGD, YIGSR, or IKVAV functionalized PAMAM coated substrate could promote cell adhesion of bone marrow mesenchymal stem cells （BMSCs） within 1 h after incubation. The neurite differentiation and proliferation of pheochromocytoma （PC12） cells were also significantly enhanced after culturing on the peptide functionalized PAMAM dendrimers for two and foul days. This peptide functionalized PAMAM dendrimers are considered as the potential candidates for various tissue engineering applications.

Influence of mixed organosilane coatings with variable RGD surface densities on the adhesion and proliferation of human osteosarcoma Saos-2 cells to magnesium alloy AZ31

In the last decade,the use of magnesium and its alloys as biodegradable implant materials has become increasingly accepted.However,surface modification of these materials to control the degradation rate in the early stages of healing and improve their biocompatibility is crucial to the successful implementation of magnesium alloy implants in medicine.Cell adhesion and proliferation at the implant surface is a vital factor for successful integration of a biomaterial within the body.Cells accomplish this task by binding to ligands such as the arginine-glycine-aspartic acid peptide sequence(RGD)commonly found on adhesive proteins present in the extracellular matrix.In this paper,we report a biomimetic surface modification strategy involving deposition of a mixed organosilane layer on Mg AZ31 followed by covalent immobilization of RGD peptides through a heterobifunctional cross-linker molecule.Our results indicate that with optimized deposition conditions uniform organosilane coatings were successfully deposited on the Mg AZ31 substrate.Furthermore,we have demonstrated that the surface density of immobilized RGD can be varied by depositing organosilane layers from solutions containing two different organosilanes in specified ratios.Increases in cell adhesion and cell proliferation were observed on the surface modified substrates.

In Vitro Biocompatibility of MC3T3-E1 Osteoblast-like Cells on Arg-Gly-Asp Acid Peptides Immobilized Graphite-like Carbon Coating on Carbon/Carbon Composites

Carbon/carbon （C/C） composites were deposited with graphite-like carbon （GLC） coating, and then, Arg-Gly- Asp acid （RGD） peptides were successfully immobilized onto the functionalized GLC coating. GLC coating was utilized to prevent carbon particles releasing and create a uniform surface condition for C/C composites. RGD peptides were utilized to improve biocompatibility of GLC coating. Surface chemical characterizations of functionalized GLC coating were detected by contact angle measurement, X-ray photoelectron spectroscopy and Raman spectra. Optical morphology of GLC coatings was observed by confocal laser scanning microscopy. In vitro biological performance was determined using samples seeded with MC3T3-E1 osteoblast-like cells and cultured for 1 week. Surface characterizations and morphological analysis indicated that C/C composites were covered by a dense and uniform GLC coating. Contact angle of GLC coating was reduced to 27.2° when it was functionalized by H202 oxidation at 40 ℃ for 1 h. In vitro cytological test showed that the RGD peptides immobilized GLC coating had a significant improvement in biocompatibility. It was suggested that RGD peptides provided GLC coating with a bioactive surface to improve cell adhesion and proliferation on C/C composites.

Biomolecular functionalization for enhanced cell–material interactions of poly(methyl methacrylate)surfaces 5th China-Europe Symposium on Biomaterials in Regenerative Medicine(CESB 2015)Hangzhou,China April 7–10,2015

The integration of implants or medical devices into the body tissues requires of good cell–material interactions.However,most polymeric materials used for these applications lack on biological cues,which enhanced mid-and long-term implant failure due to weak integration with the surrounding tissue.Commonly used strategies for tissue–material integration focus on functionalization of the material surface by means of natural proteins or short peptides.However,the use of these biomolecules involves major drawbacks such as immunogenic problems and oversimplification of the constructs.Here,designed elastin-like recombinamers(ELRs)are used to enhance poly(methyl methacrylate)surface properties and compared against the use of short peptides.In this study,cell response has been analysed for different functionalization conditions in the presence and absence of a competing protein,which interferes on surface–cell interaction by unspecific adsorption on the interface.The study has shown that ELRs can induce higher rates of cell attachment and stronger cell anchorages than short peptides,being a better choice for surface functionalization.

Integrin α_(v)β_(3)-targeted polydopamine-coated gold nanostars for photothermal ablation therapy of hepatocellular carcinoma

Photothermal therapy(PTT)has emerged as a promising cancer therapeutic method.In this study,Arg-Gly-Asp(RGD)peptide-conjugated polydopamine-coated gold nanostars(Au@PDA-RGD NPs)were prepared for targeting PTT of hepatocellular carcinoma(HCC).A polydopamine(PDA)shell was coated on the surface of gold nanostars by the oxidative self-polymerization of dopamine(termed as Au@PDA NPs).Au@PDA NPs were further functionalized with polyethylene glycol and RGD peptide to improve biocompatibility as well as selectivity toward the HCC cells.Au@PDARGD NPs showed an intense absorption at 822 nm,which makes them suitable for near-infraredexcited PTT.Our results indicated that the Au@PDA-RGD NPs were effective for the PTT therapy of the α_(v)β_(3) integrin receptor-overexpressed HepG2 cells in vitro.Further antitumor mechanism studies showed that the Au@PDA-RGD NPs-based PTT induced human liver cancer cells death via the mitochondrial-lysosomal and autophagy pathways.In vivo experiments showed that Au@PDARGD NPs had excellent tumor treatment efficiency and negligible side effects.Thus,our study showed that Au@PDA-RGD NPs could offer an excellent nanoplatform for PTT of HCC.