Dual-functional gelatin-capped silver nanoparticles for antibacterial and antiangiogenic treatment of bacterial keratitis.

Staphylococcus aureus (S. aureus) is a leading cause of keratitis worldwide and a significant threat to healthy vision. Pathological manifestations of bacterial keratitis (BK) caused by S. aureus involve stromal opacity, edema and neovascularization of an inflamed cornea, requiring immediate medical attention. Thus, S. aureus-induced keratitis is a devastating ocular infection that can lead to blindness if effective and timely treatment is not initiated. In this study, we demonstrate gelatin-capped silver nanoparticles (G-Ag NPs) as anti-infective therapeutics for the treatment of S. aureus-induced keratitis. G-Ag NPs were prepared by simple mixing of silver nitrate, maltose and gelatin. The gelatin molecules are capped in situ on the Ag NPs (∼14 nm). Compared to uncapped Ag NPs, the G-Ag NPs possess superior stability and antibacterial activity against S. aureus. We further demonstrate that G-Ag NPs possess effective inhibition of the proliferation, migration and tube formation of human umbilical vein endothelial cells, as well as strong disturbance of the angiogenesis in chick chorioallantoic membrane and rabbit corneal neovascularization. Furthermore, intrastromal administration of highly biocompatible G-Ag NPs alleviates S. aureus-induced bacterial keratitis in rabbit eyes and bacterial infection-induced corneal neovascularization. Our results demonstrate G-Ag NPs as a promising dual functional (antimicrobial and antiangiogenic) nanotherapeutic for preclinical treatment of eye-related microbial infections.

Matrix metalloprotease-3 expression in the medial plica and pannus-like tissue in knees from patients with medial compartment osteoarthritis.

AIMS: The severity of cartilage degeneration is positively correlated with the severity of the pathologic change of medial plica. However, knowledge of the pathogenic mechanisms and the impact of plica on cartilage destruction is limited. The aim of the present study was therefore to investigate matrix metalloprotease-3 (MMP-3) expression in the plica isolated from patients with medial compartment osteoarthritis of the knee. METHODS AND RESULTS: Immunohistochemistry showed that MMP-3 was highly expressed in pannus-like tissue and the plica. Western blotting of culture supernatants showed that interleukin-1ß (IL-1ß) treatment induced MMP-3 release by cells isolated from pannus tissue or the plica. Furthermore, reverse transcriptase polymerase chain reaction and real-time polymerase chain reaction analysis showed that MMP-3 mRNA levels were increased after IL-1ß treatment of the cultured cells. MMP-3 and IL-1ß mRNAs were expressed in the plica and pannus-like tissue, with MMP-3 mRNA being expressed at significantly higher levels in the plica than in normal synovial membrane and highly expressed in the plica at different stages in osteoarthritis (OA) patients. CONCLUSION: Pannus-like tissue and the plica express IL-1ß and MMP-3. Moreover, MMP-3 mRNA and protein expression in the plica may contribute to the pathogenesis of OA.

Apoptosis in chondrogenesis of human mesenchymal stem cells: effect of serum and medium supplements.

Apoptosis is an inevitable process during development and is evident in the formation of articular cartilage and endochondral ossification of growth plate. Mesenchymal stem cells (MSCs) can serve as alternative sources for cell therapy in focal chondral lesions or diffuse osteoarthritis. But there are few, if any, studies investigating apoptosis during chondrogenesis by MSCs. The aim of this study was to find the better condition to prevent apoptosis during chondrogenesis by MSCs. Apoptosis were evaluated in MSCs induced in different chondrogenic media by the use of Annexin V, TUNEL staining, lysosomal labeling with lysotracker and immunostaining of apoptotic markers. We found apparent apoptosis was demonstrated by Annexin V, TUNEL staining and lysosomal labeling during chondrogenesis. Meanwhile, the degree of apoptosis was related to the reagents of the defined chondrogenic medium. Adding serum in medium increased apoptosis, however, TGF-beta1 inhibited apoptosis. The apoptosis was associated with the activation of caspase-3, the increase in the Bax/Bcl-2 ratio, the loss of lysosomal integrity, and the increase of PARP-cleavage. Pro-inflammatory cytokines, IL-1alpha, IL-1beta and TNFalpha did not induce any increase in apoptosis. Interestingly, the inhibition of apoptosis by serum free medium supplemented with ITS was also associated with an increase in the expression of type II collagen, and a decrease in the expression of type X collagen, Runx2, and other osteogenic genes, while TGF-beta1 increased the expression of Sox9, type II and type X collagen and decreased the expression of osteogenic genes. These data suggest apoptosis occurs during chondrogenesis by MSCs by cell death intrinsic pathway activation and this process may be modulated by culture conditions.

Three-dimensional collagen fiber remodeling by mesenchymal stem cells requires the integrin-matrix interaction.

Tissue engineering aiming to repair or regenerate damaged tissues necessitates fabricating three-dimensional biomaterial scaffolds with controlled porosity for delivering cells. To facilitate cell distribution, a strategy using stem cell-based fabrication of biomaterials was tested in type II collagen fibers. Human mesenchymal stem cells when delivered in type II collagen assembled and reorganized these matrices and differentiated into spherical chondrocytes with the synthesis of cartilage proteins. The cell-mediated assembly and reorganization of collagen fibers was not limitless and only restricted to an appropriate ratio of cell number and collagen amount. The blocking of alpha2 or beta1-integrin function with specific antibodies significantly impeded the collagen-assembly effects. In vitro chondrogenesis or in vivo cartilage formation of human mesenchymal stem cells was also dependent on the interactions between cells and surrounding matrices. This method for three-dimensional fabricating collagen fibers may generally be applied to other biomaterials, when combined with surface modification or ligand addition for cell adhesion.

Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells.

The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor beta1 (TGF-beta1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-beta1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.