Osteogenic and angiogenic potential of molybdenum-containing mesoporous bioactive glass nanoparticles: An ionic approach to bone tissue engineering.

Biomaterials intended for application in bone tissue engineering (BTE) ideally stimulate osteogenesis and angiogenesis simultaneously, as both mechanisms are of critical importance for successful bone regeneration. Mesoporous bioactive glass nanoparticles (MBGNs) can be tailored towards specific biological needs, for example by addition of ions like Molybdenum (Mo). While Mo has been shown to enhance osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (BMSCs) as well as their ability to form and mature a primitive osseous extracellular matrix (ECM), there are contradictory findings regarding its impact on angiogenesis. In this study, the effects of Mo-MBGNs (mol%: 70 SiO2, 25 CaO, 5 MoO3) on viability, proliferation, osteogenic differentiation, ECM formation and angiogenic response of BMSCs were compared to undoped MBGNs (in mol%: 70 SiO2, 30 CaO) and a control group of BMSCs. Furthermore, a human umbilical vein endothelial cells tube formation assay and a chorioallantoic membrane-assay using fertilized chicken eggs were used to analyze angiogenic properties. Mo-MBGNs were cytocompatible and promoted the proliferation of BMSCs. Furthermore, Mo-MBGNs showed promising osteogenic properties as they enhanced osteogenic differentiation, ECM formation and maturation as well as the gene expression and protein production of relevant osteogenic factors in BMSCs. However, despite the promising outcome on osteogenic properties, the addition of Mo to MBGNs resulted in anti-angiogenic effects. Due to the high relevance of vascularization in-vivo, the anti-angiogenic properties of Mo-MBGNs might hamper their osteogenic properties and therefore might restrict their performance in BTE applications. These limitations can be overcome by the addition of ions with distinct pro-angiogenic properties to the Mo-MBGNs-composition. Due to their promising osteogenic properties, Mo-MBGNs constitute a suitable basis for further research in the field of ionic (growth factor free) BTE.

Biological effects of a zinc-substituted borosilicate bioactive glass on human bone marrow derived stromal cells in vitro and in a critical-size femoral defect model in rats in vivo.

The borosilicate 0106-B1-bioactive glass (BG) composition (in wt%: 37.5 SiO2, 22.6 CaO, 5.9 Na2O, 4.0P2O5, 12.0 K2O, 5.5 MgO, 12.5 B2O3) has shown favorable processing characteristics and bone regeneration ability. This study investigated the addition of zinc (Zn) to 0106-B1-BG as an approach to improve this BG's biological properties. Different proportions of ZnO were substituted for CaO in 0106-B1-BG, resulting in three new BG-compositions: 1-Zn-BG, 2-Zn-BG, 3-Zn-BG (in wt%: 37.5 SiO2, 21.6/20.1/17.6 CaO, 4.0 P2O5, 5.9 Na2O, 12.0 K2O, 5.5 MgO, 12.5 B2O3 and 1.0/2.5/5.0 ZnO). Effects of the BG compositions on cytocompatibility, osteogenic differentiation, extracellular matrix deposition, and angiogenic response of human bone marrow-derived mesenchymal stromal cells (BMSCs) were evaluated in vitro. Angiogenic effects were assessed using a tube formation assay containing human umbilical vein endothelial cells. The in vivo osteogenic and angiogenic potentials of 3-Zn-BG were investigated in comparison to the Zn-free 0106-B1-BG in a rodent critical-size femoral defect model. The osteogenic differentiation of BMSCs improved in the presence of Zn. 3-Zn-BG showed enhanced angiogenic potential, as confirmed by the tube formation assay. While Zn-doped BGs showed clearly superior biological properties in vitro, 3-Zn-BG and 0106-B1-BG equally promoted the formation of new bone in vivo; however, 3-Zn-BG reduced osteoclastic cells and vascular structures in vivo. The acquired data suggests that the differences regarding the in vivo and in vitro results may be due to modulation of inflammatory responses by Zn, as described in the literature. The inflammatory effect should be investigated further to promote clinical applications of Zn-doped BGs.

Boric acid and Molybdenum trioxide synergistically stimulate osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells.

INTRODUCTION: Metals and their metal ions have been shown to exhibit certain biological functions that make them attractive for use in biomaterials, for example in bone tissue engineering (BTE) applications. Recent data shows that Molybdenum (Mo) is a potent inducer of osteogenic differentiation in human bone marrow-derived mesenchymal stromal cells (BMSCs). On the other hand, while boron (B) has been shown to enhance vascularization in BTE applications, its impact on osteogenic differentiation is volatile: while improved osteogenic differentiation has been described, other data show that B might slow down osteogenic differentiation or reduce the calcification of the extracellular matrix (ECM) when applied in higher doses. Still, the combination of pro-osteogenic Mo and pro-angiogenic B is certainly attractive in the context of biomaterials intended for the use in BTE. METHODS: Therefore, the combined effect of molybdenum trioxide and boric acid at different ratios was investigated in this study to evaluate the effects on the viability, proliferation, osteogenic differentiation, ECM production and maturation of BMSCs. RESULTS: Mo ions proved to be stronger osteoinductive compared to B, in fact, while some osteogenic differentiation markers were downregulated in the presence of B, the presence of Mo provided compensation. The combined application of B and Mo indicated a combination of individual effects, partially even enhancing the expected combined performance of the single stimulations. CONCLUSIONS: The combination of B and Mo might be beneficial for BTE applications since the limited osteogenic properties of B can be compensated by Mo. Furthermore, since B is known to be pro-angiogenic, the combination of both substances may synergistically lead to improved vascularization and bone regeneration. Future studies should assess the angiogenic performance of this combination in greater detail.

Molybdenum trioxide enhances viability, osteogenic differentiation and extracellular matrix formation of human bone marrow-derived mesenchymal stromal cells.

BACKGROUND: Metals and their ions allow specific modifications of the biological properties of bioactive materials that are intended for application in bone tissue engineering. While there is some evidence about the impact of particles derived from orthopedic Cobalt-Chromium-Molybdenum (Co-Cr-Mo) alloys on cells, there is only limited data regarding the influence of the essential trace element Mo and its ions on the viability, osteogenic differentiation as well as on the formation and maturation of the primitive extracellular matrix (ECM) of primary human bone marrow-derived stromal cells (BMSCs) available so far. METHODS: In this study, the influence of a wide range of molybdenum (VI) trioxide (MoO3), concentrations on BMSC viability was evaluated via measurement of fluorescein diacetate metabolization. Thereafter, the impact of three non-cytotoxic concentrations of MoO3 on the cellular osteogenic differentiation as well as on ECM formation and maturation of BMSCs was assessed. RESULTS: MoO3 had no negative influence on BMSC viability in most tested concentrations, as viability was in fact even enhanced. Only the highest concentration (10 mM) of MoO3 showed cytotoxic effects. Cellular osteogenic differentiation, measured via the marker enzyme alkaline phosphatase was enhanced by the presence of MoO3 in a concentration-dependent manner. Furthermore, MoO3 showed a positive influence on the expression of relevant marker genes for osteogenic differentiation (osteopontin, osteocalcin and type I collagen alpha 1) and on the formation and maturation of the primitive ECM, as measured by collagen deposition and ECM calcification. CONCLUSION: MoO3 is considered as an attractive candidate for supplementation in biomaterials and qualifies for further research.

Ionic dissolution products of Cerium-doped bioactive glass nanoparticles promote cellular osteogenic differentiation and extracellular matrix formation of human bone marrow derived mesenchymal stromal cells.

Cerium (Ce) is a promising candidate ion for application in bone tissue engineering (BTE) since it reduces the presence of reactive oxygen species. Ce-doped mesoporous bioactive glass nanoparticles (MBGNs) serving as vectors for the local application of Ce already demonstrated stimulating effects on the expression of pro-osteogenic genes in Saos-2 cells. So far, there is no evidence available about the effects of Ce-doped MBGNs on the viability, osteogenic differentiation and the formation of the osseous extracellular matrix (ECM) of primary human bone marrow-derived mesenchymal stromal cells (BMSCs). Therefore, in this study, the biocompatibility of the ionic dissolution products (IDPs) of MBGNs containing increasing concentrations of CeO2(0.05 MCe-MBGNs, composition in mol%: 86.6SiO2-12.1CaO-1.3CeO2; and 0.2 MCe-MBGNs, composition in mol%: 86.0SiO2-11.8CaO-2.2CeO2) and unmodified MBGNs (composition in mol%: 86SiO2-14CaO) was evaluated using human BMSCs. Eventually, the impact of the MBGNs' IDPs on the cellular osteogenic differentiation and their ability to build and mature a primitive osseous ECM was assessed. The Ce-doped MBGNs had a positive influence on the viability and stimulated the cellular osteogenic differentiation of human BMSCs evaluated by analyzing the activity of alkaline phosphate as a marker enzyme for osteoblasts in the present setting. Furthermore, the formation and calcification of a primitive osseous ECM was significantly stimulated in the presence of Ce-doped MBGNs in a positive concentration-dependent manner as demonstrated by an elevated presence of collagen and increased ECM calcification. The results of thisin-vitrostudy show that Ce-doped MBGNs are attractive candidates for further application in BTE.

Macrophage specificity of three anti-CD68 monoclonal antibodies (KP1, EBM11, and PGM1) widely used for immunohistochemistry and flow cytometry.

OBJECTIVES: To investigate the specificity of three anti-CD68 monoclonal antibodies (mAbs) for macrophages (Mphi) in immunohistochemistry (IHC) and flow cytometry (FACS). METHODS: IHC was performed on cryostat sections of rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes using the anti-CD68 mAbs KP1, EBM11, and PGM1, and the fibroblast (FB) markers CD90 and prolyl 4-hydroxylase. Expression of CD68 was also analysed by FACS on the monocytic cell lines THP-1 and U937, as well as on synovial fibroblasts (SFB), skin FB, and gingival FB (both surface and intracellular staining). RESULTS: In IHC, there was an overlap between CD68 (mAbs KP1 and EBM11) and the FB markers CD90/prolyl 4-hydroxylase in the lining layer, diffuse infiltrates, and stroma of RA and OA synovial membranes. In FACS analysis of THP-1 and U937 cells, the percentage of cells positive for the anti-CD68 mAbs KP1 and EBM11 progressively increased from surface staining of unfixed cells, to surface staining of pre-fixed cells, to intracellular staining of the cells. Upon intracellular FACS of different FB, nearly all cells were positive for KP1 and EBM11, but only a small percentage for PGM1. In surface staining FACS, a small percentage of FB were positive for all three anti-CD68 mAbs. CONCLUSION: An overlap between CD68 (mAbs KP1 or EBM11) and the FB markers CD90 or prolyl 4-hydroxylase may prevent unequivocal identification of Mphi in synovial tissue by IHC or in monocytic cells and FB upon intracellular FACS. This may be due to sharing of common markers by completely different cell lineages.

Mosaic chromosomal aberrations in synovial fibroblasts of patients with rheumatoid arthritis, osteoarthritis, and other inflammatory joint diseases.

Chromosomal aberrations were comparatively assessed in nuclei extracted from synovial tissue, primary-culture (P-0) synovial cells, and early-passage synovial fibroblasts (SFB; 98% enrichment; P-1, P-4 [passage 1, passage 4]) from patients with rheumatoid arthritis (RA; n = 21), osteoarthritis (OA; n = 24), and other rheumatic diseases. Peripheral blood lymphocytes (PBL) and skin fibroblasts (FB) (P-1, P-4) from the same patients, as well as SFB from normal joints and patients with joint trauma (JT) (n = 4), were used as controls. Analyses proceeded by standard GTG-banding and interphase centromere fluorescence in situ hybridization. Structural chromosomal aberrations were observed in SFB (P-1 or P-4) from 4 of 21 RA patients (19%), with involvement of chromosome 1 [e.g. del(1)(q12)] in 3 of 4 cases. In 10 of the 21 RA cases (48%), polysomy 7 was observed in P-1 SFB. In addition, aneusomies of chromosomes 4, 6, 8, 9, 12, 18, and Y were present. The percentage of polysomies was increased in P-4. Similar chromosomal aberrations were detected in SFB of OA and spondylarthropathy patients. No aberrations were detected in i) PBL or skin FB from the same patients (except for one OA patient with a karyotype 45,X[10]/46,XX[17] in PBL and variable polysomies in long-term culture skin FB); or ii) synovial tissue and/or P-1 SFB of normal joints or of patients with joint trauma. In conclusion, qualitatively comparable chromosomal aberrations were observed in synovial tissue and early-passage SFB of patients with RA, OA, and other inflammatory joint diseases. Thus, although of possible functional relevance for the pathologic role of SFB in RA, these alterations probably reflect a common response to chronic inflammatory stress in rheumatic diseases.

Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture--primary culture cells markedly differ from fourth-passage cells.

To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.