Osteogenic differentiation of bone marrow mesenchymal stem cells on chitosan/gelatin scaffolds: gene expression profile and mechanical analysis.

In the present study we explore the extracellular matrix (ECM) produced by human bone marrow mesenchymal stem/stromal cells (BM-MSCs) induced to undergo osteogenic differentiation within porous chitosan/gelatin (CS:Gel) scaffolds by investigating their multiple gene expression profile and mechanical behavior. Initially, the efficiency of the BM-MSCs osteogenic differentiation within the constructs was confirmed by the significant rise in the expression of the osteogenesis associated genes DLX5, RUNX2, ALP and OSC. In line with these findings, OSC and Col1A1 protein expression was also detected in BM-MSCs on the CS:Gel scaffolds at day 14 of osteogenic differentiation. We then profiled, for the first time, the expression of 84 cell adhesion and ECM molecules using PCR arrays. The arrays, which were conducted at day 14 of osteogenic differentiation, demonstrated that 49 genes including collagens, integrins, laminins, ECM proteases, catenins, thrombospondins, ECM protease inhibitors and cell-cell adhesion molecules were differentially expressed in BM-MSCs seeded on scaffolds compared to tissue culture polystyrene control. Moreover, we performed dynamic mechanical analysis of the cell-loaded scaffolds on days 0, 7 and 14 to investigate the correlation between the biological results and the mechanical behavior of the constructs. Our data demonstrate a significant increase in the stiffness of the constructs with storage modulus values of 2 MPa on day 7, compared to 0.5 MPa on day 0, following a drop of the stiffness at 0.8 MPa on day 14, that may be attributed to the significant increase of specific ECM protease gene expression such as MMP1, MMP9, MMP11 and MMP16 at this time period.

Chitosan/gelatin scaffolds support bone regeneration.

Chitosan/Gelatin (CS:Gel) scaffolds were fabricated by chemical crosslinking with glutaraldehyde or genipin by freeze drying. Both crosslinked CS:Gel scaffold types with a mass ratio of 40:60% form a gel-like structure with interconnected pores. Dynamic rheological measurements provided similar values for the storage modulus and the loss modulus of the CS:Gel scaffolds when crosslinked with the same concentration of glutaraldehyde vs. genipin. Compared to genipin, the glutaraldehyde-crosslinked scaffolds supported strong adhesion and infiltration of pre-osteoblasts within the pores as well as survival and proliferation of both MC3T3-E1 pre-osteoblastic cells after 7 days in culture, and human bone marrow mesenchymal stem cells (BM-MSCs) after 14 days in culture. The levels of collagen secreted into the extracellular matrix by the pre-osteoblasts cultured for 4 and 7 days on the CS:Gel scaffolds, significantly increased when compared to the tissue culture polystyrene (TCPS) control surface. Human BM-MSCs attached and infiltrated within the pores of the CS:Gel scaffolds allowing for a significant increase of the osteogenic gene expression of RUNX2, ALP, and OSC. Histological data following implantation of a CS:Gel scaffold into a mouse femur demonstrated that the scaffolds support the formation of extracellular matrix, while fibroblasts surrounding the porous scaffold produce collagen with minimal inflammatory reaction. These results show the potential of CS:Gel scaffolds to support new tissue formation and thus provide a promising strategy for bone tissue engineering.

Minor populations of paroxysmal nocturnal hemoglobinuria-type cells in patients with chronic idiopathic neutropenia.

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by increased apoptosis of the bone marrow (BM) granulocytic progenitor cells under the influence of pro-inflammatory mediators and oligoclonal/monoclonal T-lymphocytes. Because patients with immune-mediated BM failure display frequently paroxysmal nocturnal hemoglobinuria (PNH)-type cells in the peripheral blood (PB), we investigated the possible existence of PNH-type cells in 91 patients with CIN using flow cytometry. The patients displayed increased proportions of PNH-type glycophorin A+ /CD59dim and glycophorin A+ /CD59- red blood cells (RBCs), FLAER- /CD24- granulocytes, and FLAER- /CD14- monocytes, compared to controls (n = 55). A positive correlation was found between the proportions of PNH-type RBCs, granulocytes, and monocytes and an inverse correlation between the number of PB neutrophils and the proportions of PNH-type cell populations. The number of patients, displaying percentages of PNH-type cells above the highest percentage observed in the control group, was significantly increased among patients with skewed compared to those with normal T-cell receptor repertoire suggesting that T-cell-mediated immune processes underlie the emergence of PNH-type cells in CIN. Our findings suggest that patients with CIN display PNH-type cells in the PB at a high frequency corroborating the hypothesis that CIN belongs to the immune-mediated BM failure syndromes.