A nano-scaled and multi-layered recombinant fibronectin/cadherin chimera composite selectively concentrates osteogenesis-related cells and factors to aid bone repair.

Easily accessible and effective bone grafts are in urgent need in clinic. The selective cell retention (SCR) strategy, by which osteogenesis-related cells and factors are enriched from bone marrow into bio-scaffolds, holds great promise. However, the retention efficacy is limited by the relatively low densities of osteogenesis-related cells and factors in marrow; in addition, a lack of satisfactory surface modifiers for scaffolds further exacerbates the dilemma. To address this issue, a multi-layered construct consisting of a recombinant fibronectin/cadherin chimera was established via a layer-by-layer self-assembly technique (LBL-rFN/CDH) and used to modify demineralised bone matrix (DBM) scaffolds. The modification was proven stable and effective. By the mechanisms of physical interception and more importantly, chemical recognition (fibronectin/integrins), the LBL-rFN/CDH modification significantly improved the retention efficacy and selectivity for osteogenesis-related cells, e.g., monocytes, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), and bioactive factors, e.g., bFGF, BMP-2 and SDF-1α. Moreover, the resulting composite (designated as DBM-LBL-rFN/CDH) not only exhibited a strong MSC-recruiting capacity after SCR, but also provided favourable microenvironments for the proliferation and osteogenic differentiation of MSCs. Eventually, bone repair was evidently improved. Collectively, DBM-LBL-rFN/CDH presented a suitable biomaterial for SCR and a promising solution for tremendous need for bone grafts. STATEMENT OF SIGNIFICANCE: There is an urgent need for effective bone grafts. With the potential of integrating osteogenicity, osteoinductivity and osteoconductivity, selective cell retention (SCR) technology brings hope for developing ideal grafts. However, it is constrained by low efficacy and selectivity. Thus, we modified demineralized bone matrix with nano-scaled and multi-layered recombinant fibronectin/cadherin chimera (DBM-rFN/CDH-LBL), and evaluate its effects on SCR and bone repair. DBM-rFN/CDH-LBL significantly improved the efficacy and selectivity of SCR via physical interception and chemical recognition. The post-enriched DBM-rFN/CDH-LBL provided favourable microenvironments to facilitate the migration, proliferation and osteogenic differentiation of MSCs, thus accelerating bone repair. Conclusively, DBM-rFN/CDH-LBL presents a novel biomaterial with advantages including high cost-effectiveness, more convenience for storage and transport and can be rapidly constructed intraoperatively.

Mechanistic Projection of First-in-Human Dose for Bispecific Immunomodulatory P-Cadherin LP-DART: An Integrated PK/PD Modeling Approach.

A bispecific immunomodulatory biotherapeutic molecule (P-cadherin LP-DART) based on the Dual Affinity Re-Targeting (DART) scaffold has been developed as a potential antitumor treatment showing efficacy in preclinical testing. A minimal anticipated biological effect level (MABEL) approach was applied to project the first-in-human (FIH) dose, because of its immune agonistic properties following target engagement. The pharmacological activity of P-cadherin LP-DART is driven by binding to both P-cadherin on the tumor cells and CD3 on T cells. Therefore, the concentration of the tri-molecular synapse formed between drug, T cell, and tumor cell, rather than drug concentration, is responsible for efficacy. A mechanistic pharmacokinetic/pharmacodynamic (PK/PD)-driven approach was explored to understand the exposure-response relationship based on the synapse concentration to project the MABEL dose. Orthogonal approaches including PK-driven and receptor occupancy calculations were also investigated. This study showcases the application of PK/PD modeling in immune-oncology, and could potentially be implemented for other bispecific biotherapeutics.

Artificial biomimicking matrix modifications of nanofibrous scaffolds by hE-cadherin-Fc fusion protein to promote human mesenchymal stem cells adhesion and proliferation.

Extracellular matrix (ECM) plays a fundamental role in regulating cell attachment, proliferation, migration and differentiation. Both synthetic and biologically derived materials have been explored as an ECM in regenerative medicine and tissue engineering. To biomimick the extracellular matrix, we combined the advantages of the biological properties of nanofibrous scaffolds and the fusion protein to apply for the culture of human mesenchymal stem cells in vitro. In this study, we fabricated well random-oriented/aligned nanofibrous scaffolds with PCL, modified with hE-cadherin-Fc fusion protein and studied the synergistic effect of the scaffolds. The random-oriented/aligned architecture was observed in the nanofibrous scaffolds by SEM. XPS and WCA measurements evidenced that hE-cadherin-Fc was successfully modified on the PCL nanofibrous scaffolds and hydrophilicity of the scaffolds was well improved after fusion protein coating. The hE-cadherin-Fc modified markedly promoted the adhesion and proliferation of hMSCs and guided hMSCs to a spindlier morphology compared with unmodified nanofibrous scaffolds. Furthermore, hMSCs on the hE-cadherin-Fc-coated nanofibrous scaffolds also had differentiation potential. These results suggested that the combination of PCL nanofibrous scaffolds and hE-cadherin-Fc fusion protein may be a promising artificial ECM for the behavior of hMSCs in vitro.

Complex protein nanopatterns over large areas via colloidal lithography.

The patterning of biomolecules at the nanoscale provides a powerful method to investigate cellular adhesion processes. A novel method for patterning is presented that is based on colloidal monolayer templating combined with multiple and angled deposition steps. Patterns of gold and SiO2 layers are used to generate complex protein nanopatterns over large areas. Simple circular patches or more complex ring structures are produced in addition to hierarchical patterns of smaller patches. The gold regions are modified through alkanethiol chemistry, which enables the preparation of extracellular matrix proteins (vitronectin) or cellular ligands (the extracellular domain of E-cadherin) in the nanopatterns, whereas the selective poly(l-lysine)-poly(ethylene glycol) functionalization of the SiO2 matrix renders it protein repellent. Cell studies, as a proof of principle, demonstrate the potential for using sets of systematically varied samples with simpler or more complex patterns for studies of cellular adhesive behavior and reveal that the local distribution of proteins within a simple patch critically influences cell adhesion.

VEGF controls endothelial-cell permeability by promoting the beta-arrestin-dependent endocytosis of VE-cadherin.

How vascular endothelial growth factor (VEGF) induces vascular permeability, its first described function, remains poorly understood. Here, we provide evidence of a novel signalling pathway by which VEGF stimulation promotes the rapid endocytosis of a key endothelial cell adhesion molecule, VE-cadherin, thereby disrupting the endothelial barrier function. This process is initiated by the activation of the small GTPase Rac by VEGFR-2 through the Src-dependent phosphorylation of Vav2, a guanine nucleotide-exchange factor. Rac activation, in turn, promotes the p21-activated kinase (PAK)-mediated phosphorylation of a highly conserved motif within the intracellular tail of VE-cadherin. Surprisingly, this results in the recruitment of beta-arrestin2 to serine-phosphorylated VE-cadherin, thereby promoting its internalization into clathrin-coated vesicles and the consequent disassembly of intercellular junctions. Ultimately, this novel biochemical route by which VEGF promotes endothelial permeability through the beta-arrestin2-dependent endocytosis of VE-cadherin may help identify new therapeutic targets for the treatment of many human diseases that are characterized by vascular leakage.