Function and Mechanism of RGD in Bone and Cartilage Tissue Engineering

Bone and cartilage injury is common, tissue engineered scaffolds are potential means to repair. Because most of the scaffold materials used in bone and cartilage tissue engineering are bio-inert, it is necessary to increase the cellular adhesion ability of during tissue engineering reconstruction. The Arginine - Glycine - Aspartic acid (Arg-Gly-Asp, RGD) peptide family is considered as a specific recognition site for the integrin receptors. Integrin receptors are key regulators of cell-cell and cell-extracellular microenvironment communication. Therefore, the RGD polypeptide families are considered as suitable candidates for treatment of a variety of diseases and for the regeneration of various tissues and organs. Many scaffold material for tissue engineering and has been approved by US Food and Drug Administration (FDA) for human using. The application of RGD peptides in bone and cartilage tissue engineering was reported seldom. Only a few reviews have summarized the applications of RGD peptide with alloy, bone cements, and PCL in bone tissue engineering. Herein, we summarize the application progress of RGD in bone and cartilage tissue engineering, discuss the effects of structure, sequence, concentration, mechanical stimulation, physicochemical stimulation, and time stimulation of RGD peptide on cells differentiation, and introduce the mechanism of RGD peptide through integrin in the field of bone and cartilage tissue engineering.

Possible Treatment of Myocardial Infarct Based on Tissue Engineering Using a Cellularized Solid Collagen Scaffold Functionalized with Arg-Glyc-Asp (RGD) Peptide

Currently, the clinical impact of cell therapy after a myocardial infarction (MI) is limited by low cell engraftment due to low cell retention, cell death in inflammatory and poor angiogenic infarcted areas, secondary migration. Cells interact with their microenvironment through integrin mechanoreceptors that control their survival/apoptosis/differentiation/migration and proliferation. The association of cells with a three-dimensional material may be a way to improve interactions with their integrins, and thus outcomes, especially if preparations are epicardially applied. In this review, we will focus on the rationale for using collagen as a polymer backbone for tissue engineering of a contractile tissue. Contractilities are reported for natural but not synthetic polymers and for naturals only for: collagen/gelatin/decellularized-tissue/fibrin/Matrigel™ and for different material states: hydrogels/gels/solids. To achieve a thick/long-term contractile tissue and for cell transfer, solid porous compliant scaffolds are superior to hydrogels or gels. Classical methods to produce solid scaffolds: electrospinning/freeze-drying/3D-printing/solvent-casting and methods to reinforce and/or maintain scaffold properties by reticulations are reported. We also highlight the possibility of improving integrin interaction between cells and their associated collagen by its functionalizing with the RGD-peptide. Using a contractile patch that can be applied epicardially may be a way of improving ventricular remodeling and limiting secondary cell migration.

615 Reactivating antitumor immunity in gliomas with osteopontin/integrin blocking peptide

BackgroundGlioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Despite improvements in imaging, surgical techniques, radiotherapy and chemotherapy, the prognosis of patients with GBM remains poor with a median overall survival of 15 months [1,2]. GBM is immunologically a ”cold” tumor with low infiltration of functional T and NK cells, which imposes poor responsiveness of GBM patients to immunotherapies. The immunosuppressive microenvironment in GBM is created by the malignant cells and tumor-associated macrophages (TAMs), such as resident brain microglia and recruited peripheral myeloid cells [3]. Osteopontin/Spp1 is one of glioma-derived factors that is responsible for the protumorigenic reprogramming of TAMs [4]. SPP1 expression is highly elevated in tumor tissues and sera from GBM patients, and inversely correlates with patient survival [5]. Cross-talk between malignant cells and TAMs relays on osteopontin binding to integrin receptors (mainly αvβ3 and αvβ5) via its RGD motif [6]. Thus, with the use of a RGD peptide (our in-house designed competitor of binding to integrins) we interfered with glioma-microglia interaction in vitro and evaluated the in vivo antitumor efficacy of integrin blockade as a monotherapy and in combination with an immune check-point inhibitor.MethodsThe efficacy of the RGD peptide to block microglia-dependent glioma invasion was determined in a Matrigel invasion assay. Antitumor activity of the peptide was assessed in a murine syngeneic orthotopic GL261 glioma model. RGD peptide was administrated intratumorally via osmotic pomps. For combination therapy, the animals received anti-PD-1 or isotype IgG antibody (4 inj. x 10 mg/kg i.p.). Tumor volume was measured using MRI. Heterogeneity of the immune cells compartment of glioma microenvironment was analysed by flow cytometry. The transcriptomes of CD11b+ cells immunosorted from tumor-bearing mouse brains were evaluated using RNAseq. Cytokine levels in the blood and the brain homogenates were measured using Luminex bead-based assays.ResultsThe microglia-stimulated invasion of GL261 glioma cells was reduced significantly in the presence of the RGD peptide in the in vitro co-culture system. The RGD peptide administrated to tumor-bearing mice induced proinflammatory reprogramming of TAMs. Combination of the RGD peptide with anti-PD-1 therapy increased the production of proinflammatory cytokines and the percentage of effector CD8+(CD44+CD62L-) cells in the tumors.ConclusionsThese results demonstrate that blockade of osteopontin/integrin signaling using the RGD peptide can mitigate the immunosuppressive microenvironment, reactivate the antitumor immunity and lay ground for improved response to immunotherapy in GBM.ReferencesJemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, Thun MJ: Cancer statistics, 2005. CA Cancer J Clin 2005, 55(1):10–30.Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K et al: Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC- NCIC trial. Lancet Oncol 2009, 10(5):459–466.Woroniecka KI, Rhodin KE, Chongsathidkiet P, Keith KA, Fecci PE: T-cell Dysfunction in Glioblastoma: Applying a New Framework. Clin Cancer Res 2018, 24(16):3792–3802Denhardt, D.T., M. Noda, A.W. O’Regan, D. Pavlin, and J.S. Berman. 2001. Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 107:1055–1061.Grassinger, J., D.N. Haylock, M.J. Storan, G.O. Haines, B. Williams, G.A. Whitty, et al. 2009. Thrombin-cleaved osteopontin regulates hemopoietic stem and progenitor cell functions through interactions with alpha9beta1 and alpha4beta1 integrins. Blood 114:49–59.Anborgh, P.H., J.C. Mutrie, A.B. Tuck, and A.F. Chambers. 2010. Role of the metastasis-promoting protein osteopontin in the tumour microenvironment. Journal of cellular and molecular medicine 14:2037–2044Ethics ApprovalAll research protocols conformed to the Guidelines for the Care and Use of Laboratory Animals (European and national regulations 2010/63/UE September 22, 2010 and Dz. Urz. UE L276/20.10.2010, respectively). Animals were decapitated by a qualified researcher. The First Warsaw Local Ethics Committee for Animal Experimentation approved the study (approval no. 812/2019).

Synthesis and Evaluation of a Dimeric RGD Peptide as a Preliminary Study for Radiotheranostics with Radiohalogens

We recently developed 125I- and 211At-labeled monomer RGD peptides using a novel radiolabeling method. Both labeled peptides showed high accumulation in the tumor and exhibited similar biodistribution, demonstrating their usefulness for radiotheranostics. This study applied the labeling method to a dimer RGD peptide with the aim of gaining higher accumulation in tumor tissues based on improved affinity with αvβ3 integrin. We synthesized an iodine-introduced dimer RGD peptide, E[c(RGDfK)] (6), and an 125/131I-labeled dimer RGD peptide, E[c(RGDfK)]{[125/131I]c[RGDf(4-I)K]} ([125/131I]6), and evaluated them as a preliminary step to the synthesis of an 211At-labeled dimer RGD peptide. The affinity of 6 for αvβ3 integrin was higher than that of a monomer RGD peptide. In the biodistribution experiment at 4 h postinjection, the accumulation of [125I]6 (4.12 ± 0.42% ID/g) in the tumor was significantly increased compared with that of 125I-labeled monomer RGD peptide (2.93 ± 0.08% ID/g). Moreover, the accumulation of [125I]6 in the tumor was greatly inhibited by co-injection of an excess RGD peptide. However, a single injection of [131I]6 (11.1 MBq) did not inhibit tumor growth in tumor-bearing mice. We expect that the labeling method for targeted alpha therapy with 211At using a dimer RGD peptide could prove useful in future clinical applications.

Solvent Effect on the Structure and Properties of RGD Peptide (1FUV) at Body Temperature (310 K) Using Ab Initio Molecular Dynamics

The structure and properties of the arginine-glycine-aspartate (RGD) sequence of the 1FUV peptide at 0 K and body temperature (310 K) are systematically investigated in a dry and aqueous environment using more accurate ab initio molecular dynamics and density functional theory calculations. The fundamental properties, such as electronic structure, interatomic bonding, partial charge distribution, and dielectric response function at 0 and 310 K are analyzed, comparing them in dry and solvated models. These accurate microscopic parameters determined from highly reliable quantum mechanical calculations are useful to define the range and strength of complex molecular interactions occurring between the RGD peptide and the integrin receptor. The in-depth bonding picture analyzed using a novel quantum mechanical metric, the total bond order (TBO), quantifies the role played by hydrogen bonds in the internal cohesion of the simulated structures. The TBO at 310 K decreases in the dry model but increases in the solvated model. These differences are small but extremely important in the context of conditions prevalent in the human body and relevant for health issues. Our results provide a new level of understanding of the structure and properties of the 1FUV peptide and help in advancing the study of RGD containing other peptides.

The Synthesis and Evaluation of RGD−Conjugated Chitosan Gel as Daily Supplement for Body Weight Control

(1) Background: Obesity is one of the most widespread chronic diseases and increases the risk of several other chronic diseases, especially type 2 diabetes. (2) Methods: Endobarrier is a new medical device what is worn in the small intestines for the treatment of type 2 diabetes and obesity. However, given the invasive and other adverse effects of the Endobarrier, we propose the use of RGD peptide conjugated with chitosan (RC) as an alternative. (3) Results: The FTIR and NMR spectrum showed RGD peptide was successfully conjugated on chitosan and RGD−CT is retained in the small intestine even after digestion. In vitro of wst-1 and live and dead staining studies show that the RGD−CT gel is highly biocompatible and non-toxic. Rats treated with the RGD−CT gel for a short term showed significant decrease change more than 30% in body weight, while the blood and hematic biometrics were within normal values. (4) Conclusions: The RGD−CT gel is safe, suitable for the short-term, reducing visceral fat rate health food to control weight. In the future, it is expected to develop a safe, long-term effective, flexibility of use and low-side-effect anti-obesity therapy in the era of precision medicine by further modification.