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.

Enhanced vascularization of PCL porous scaffolds through VEGF-Fc modification.

To accelerate the vascularization of engineered tissue, an endothelial-specific fusion protein (VEGF-Fc), which consists of a human vascular endothelial growth factor (VEGF) and an immunoglobulin G Fc region, was fabricated and used to construct a bioactive interface in a porous scaffold. In this study, VEGF-Fc was immobilized on polycarprolactone (PCL) porous scaffolds by steeping, which is mediated by the hydrophobic binding of the Fc domain. The VEGF-Fc proteins were distributed stably and uniformly throughout the PCL porous scaffolds without affecting their surface morphology and mechanical properties. The immobilized VEGF-Fc activated the phosphorylation of VEGF2 receptor continuously, and further promoted the expressions of PI3K and MAPK, which effectively enhanced the adhesion and proliferation of human vascular endothelial cells (HUVECs). Furthermore, the immobilized VEGF-Fc promoted the migration of HUVECs into the scaffolds, and also enhanced the cellularization and ECM deposition in the subcutaneous implanted scaffolds in rats, which synergistically supported the vascularization of the scaffold in vivo. In view of the advantages of easy use, stability and efficiency, the VEGF-Fc fusion protein appeared to be a promising candidate for surface modification of porous scaffolds for tissue engineering.

Electrospun PDLLA/PLGA composite membranes for potential application in guided tissue regeneration.

With the aim to explore a membrane system with appropriate degradation rate and excellent cell-occlusiveness for guided tissue regeneration (GTR), a series of poly(D, L-lactic acid) (PDLLA)/poly(D, L-lactic-co-glycolic acid) (PLGA) (100/0, 70/30, 50/50, 30/70, 0/100, w/w) composite membranes were fabricated via electrospinning. The fabricated membranes were evaluated by morphological characterization, water contact angle measurement and tensile test. In vitro degradation was characterized in terms of the weight loss and the morphological change. Moreover, in vitro cytologic research revealed that PDLLA/PLGA composite membranes could efficiently inhibit the infiltration of 293 T cells. Finally, subcutaneous implant test on SD rat in vivo showed that PDLLA/PLGA (70/30, 50/50) composite membranes could function well as a physical barrier to prevent cellular infiltration within 13 weeks. These results suggested that electrospun PDLLA/PLGA (50/50) composite membranes could serve as a promising barrier membrane for guided tissue regeneration due to suitable biodegradability, preferable mechanical properties and excellent cellular shielding effects.

Carboxymethyl chitosan-poly(amidoamine) dendrimer core-shell nanoparticles for intracellular lysozyme delivery.

Intracellular delivery of native, active proteins is challenging due to the fragility of most proteins. Herein, a novel polymer/protein polyion complex (PIC) nanoparticle with core-shell structure was prepared. Carboxymethyl chitosan-grafted-terminal carboxyl group-poly(amidoamine) (CM-chitosan-PAMAM) dendrimers were synthesized by amidation and saponification reactions. (1)H NMR was used to characterize CM-chitosan-PAMAM dendrimers. The TEM images and results of lysozyme loading efficiency indicated that CM-chitosan-PAMAM dendrimers could self-assemble into core-shell nanoparticles, and lysozyme was efficiently encapsulated inside the core of CM-chitosan-PAMAM dendrimer nanoparticles. Activity of lysozyme was completely inhibited by CM-chitosan-PAMAM Dendrimers at physiological pH, whereas it was released into the medium and exhibited a significant enzymatic activity in an acidic intracellular environment. Moreover, the CM-chitosan-PAMAM dendrimer nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 3.16 mg/ml. The results indicated that these CM-chitosan-PAMAM dendrimers have excellent properties as highly potent and non-toxic intracellular protein carriers, which would create opportunities for novel applications in protein delivery.

hE-cadherin-Fc fusion protein coated surface enhances the adhesion and proliferation of human mesenchymal stem cells.

A fusion protein consisting of human E-cadherin extracellular domain and the immunoglobulin G Fc region (hE-cadherin-Fc) was prepared and used as a cell-cell adhesion biomimicking matrix for the in vitro expansion of human mesenchymal stem cells (hMSCs) for use in regenerative medicine. The hE-cadherin-Fc was stably immobilized onto a polystyrene plate due to the hydrophobicity of the Fc domain, enhancing the surface wettability and topography of the plate. The hE-cadherin-Fc matrix markedly promoted the cell adhesion and proliferation of hMSCs compared with the tissue culture-treated plate (TC-PS) and the gelatin-coated plate. Furthermore, the expanded hMSCs on the hE-cadherin-Fc were positive for CD105, similar to those from the gelatin. Additionally, the expression of E-cadherin and ß-catenin in the hMSCs was improved on the hE-cadherin-Fc matrix, suggesting that the interactions of the hE-cadherin-Fc matrix with the hMSCs were substitutes for the cell-cell adhesion junctions during the initial culture stage in the absence of intercellular interactions. The hE-cadherin-Fc was shown to be a promising artificial ECM for the in vitro expansion of hMSCs.