Rebeka Rudolf

Proinflammatory and immunoregulatory mechanisms in periapical lesions

Proinflammatory and immunoregulatory cytokines are important for the pathogenesis of periapical lesions. However, little is known about how their functions are balanced and controlled at different phases of lesion development. The aim of this study was to examine the relationship between the production of Th1, Th2, Th17 and T regulatory cell (T reg) cytokines by human periapical lesion mononuclear cells (PL-MNC) in culture and their correlation with cellular composition and clinical presentation of the lesions. We show that symptomatic lesions are characterized by the infiltration of neutrophils, high production of IL-17, positive correlation between IL-17 and IFN-gamma, but not between IL-17 and IL-23 production. Most IL-17(+) cells coexpressed IFN-gamma. Asymptomatic lesions were phenotypically heterogeneous. The lesions with the predominance of T cells over B cells/plasma cells expressed higher levels of IFN-gamma which correlated with higher production of IL-12 and the frequency of macrophages. In contrast, in most B-type lesions higher levels of IL-5 and TGF-beta were observed, as well as positive correlation between the production of TGF-beta and IL-10. The addition of Th cytokines in PL-MNC cultures confirmed that Th1, Th2 and Th17 cytokines are mutually antagonistic, except that IL-17, unexpectedly, augmented the production of IFN-gamma. IL-10 and TGF-beta inhibited the production of both Th1 and Th17 cytokines. Dendritic cells (DCs) from periapical lesions, composed of immature (CD83(-)), and mature (CD83(+)) myeloid type DCs and plasmacytoid (BDCA2(+)) DCs produced higher levels of IL-12 and IL-23 but lower levels of IL-10 and TNF-alpha than monocyte (Mo) -derived DCs. IL-23 stimulated the production of IL-17 by PL-MNC, whereas the secretion of IFN-gamma was enhanced by both IL-12 and IL-23. Cumulatively, these results suggest that: (1) Th1 immune response is most probably important for all stages of periapical lesion development; (2) Th2 and immunoregulatory cytokines are more significant for advanced types of lesions with the predominance of B cells/plasma cells; (3) Th17 immune response seems to play a dominant role in exacerbating inflammation.

Relationship between microstructure, cytotoxicity and corrosion properties of a Cu-Al-Ni shape memory alloy.

Cu-Al-Ni shape memory alloys (SMAs) have been investigated as materials for medical devices, but their biomedical application is still limited. The aim of this work was to compare the microstructure, corrosion and cytotoxicity in vitro of a Cu-Al-Ni SMA. Rapidly solidified (RS) thin ribbons, manufactured via melt spinning, were used for the tests. The control alloy was a permanent mould casting of the same composition, but without shape memory effect. The results show that RS ribbons are significantly more resistant to corrosion compared with the control alloy, as judged by the lesser release of Cu and Ni into the conditioning medium. These results correlate with the finding that RS ribbons were not cytotoxic to L929 mouse fibroblasts and rat thymocytes. In addition, the RS ribbon conditioning medium inhibited cellular proliferation and IL-2 production by activated rat splenocytes to a much lesser extent. The inhibitory effects were almost completely abolished by conditioning the RS ribbons in culture medium for 4 weeks. Microstructural analysis showed that RS ribbons are martensitic, with boron particles as a minor phase. In contrast, the control Cu-Al-Ni alloy had a complex multiphase microstructure. Examination of the alloy surfaces after conditioning by energy dispersive X-ray and Auger electron spectroscopy showed the formation of Cu and Al oxide layers and confirmed that the metals in RS ribbons are less susceptible to oxidation and corrosion compared with the control alloy. In conclusion, these results suggest that rapid solidification significantly improves the corrosion stability and biocompatibility in vitro of Cu-Al-Ni SMA ribbons.

Characterization and immunosuppressive properties of mesenchymal stem cells from periapical lesions.

AIM Mesenchymal stem cells (MSCs) isolated from healthy dental tissues are being investigated as an alternative source of MSCs for the treatment of damaged tissues and inflammatory diseases. Here we investigated whether MSCs from periapical lesions (PL-MSCs) also possess multi-lineage differentiation capacity and immunomodulatory properties. MATERIAL & METHODS PL-MSCs, isolated by collagenase/DNAse digestion from surgically extracted PLs, were compared with MSCs from non-inflamed dental pulp (DP-MSCs) and dental follicle (DF-MSCs) for their phenotype and multi-potent differentiation potential. The anti-inflammatory and immunomodulatory effects of PL-MSCs were studied in co-culture with peripheral blood mononuclear cells (PB-MNCs) and PL-inflammatory cells (PL-ICs). RESULTS PL-MSCs were characterized by typical MSCs phenotype, lower clonogenicity and self-renewal rate, compared to DF-MSCs and DP-MSCs. These cells possess the potential to differentiate into adipocyte-, osteoblast- and chondrocyte-like cells in vitro, which differs from that of DP-MSCs and DF-MSCs. PL-MSCs inhibited phytohemaglutinine-induced proliferation of PB-MNCs and production of IL-2, IFNγ and IL-5 in the co-culture, probably via TGF-β-dependent mechanisms. These cells also suppressed the production of IL-1β, IL-6, and TNF-α by PL-ICs via soluble mediators, whereas the suppression of IL-8 production required a direct cell-to-cell contact. CONCLUSION The differentiation potential of PL-MSCs and their immunosuppressive/anti-inflammatory properties could be beneficial for the treatment of chronic periodontal diseases.

Regulatory T-cells in Periapical Lesions

CD4+CD25hiFoxp3+ regulatory T-cells (Tregs) are of crucial importance in regulating the immune response, including the control of any defense against infection. Their presence in periapical lesions has not been demonstrated, as yet. We hypothesized that Tregs infiltrate periapical lesions, where they inhibit T-cell proliferation. The aim of this study was to characterize Tregs in periapical lesions by confocal microscopy, flow cytometry, and functional assays. We showed that CD4+CD25hiFoxp3+ cells in periapical lesions expressed IL-10 and TGF-β. Their frequency was significantly higher than in peripheral blood and correlated with the levels of TGF-β and IL-10 in culture supernatants of periapical lesion mononuclear cells. Tregs inhibited the proliferation of responder T-cells in vitro, at least in part, by stimulating the production of IL-10. These findings suggest that CD4+CD25hiFoxp3+ cells in periapical lesions may play regulatory roles in controlling local immune/inflammatory processes.

Cytotoxicity of Gold Nanoparticles Prepared by Ultrasonic Spray Pyrolysis

The aim of this work was to study the cytotoxicity of different fractions of gold nanoparticles prepared by ultrasonic spray pyrolysis from gold scrap. The target cells were rat thymocytes, as a type of nonproliferating cells, and L929 mouse fibroblasts, as a type of continuous proliferating cells. Fractions 1 and 2, composed of pure gold nanoparticles, as determined by scanning electron microscopy with a combination of energy dispersive X-ray analysis, were nontoxic for thymocytes, but reduced moderately the proliferative activity of L929 cells. The inhibitory effect of fraction 2, containing particles smaller in size than fraction 1, was stronger. Fraction 3, composed of Au and up to 3% Cu was noncytotoxic for thymocytes, but was cytotoxic for L929 cells. Fraction 4, composed of Au and Ag nanoparticles, and fraction 5, composed of Au together with Cu, Ni, Zn, Fe, and In were cytotoxic for both thymocytes and L929 cells. These results suggest that USP enables the synthesis of pure gold nanoparticles with controlled size, even from gold scrap. However, microstructural analyses and biocompatibility testing are necessary for their proper selection from more cytotoxic gold nanoparticles, contaminated with other elements of gold alloys.

The influence of the microstructure of high noble gold-platinum dental alloys on their corrosion and biocompatibility in vitro

The aim of this work was to compare the microstructures of two high noble experimental Au-Pt alloys with similar composition with their corrosion and biocompatibility in vitro. We showed that Au-Pt II alloy, composed of 87.3 wt.% Au, 9.9 wt.% Pt, 1.7 wt.% Zn and 0.5 wt.% Ir + Rh + In, although possessing better mechanical properties than the Au-Pt I alloy (86.9 wt.% Au, 10.4 wt.% Pt, 1.5 wt.% Zn and 0.5 wt.% Ir + Rh + In), exerted higher adverse effects on the viability of L929 cells and the suppression of rat thymocyte functions, such as proliferation activity, the production of Interleukin-2 (IL-2), expression of IL-2 receptor and activation — induced apoptosis after stimulation of the cells with Concanavalin-A. These results correlated with the higher release of Zn ions in the culture medium. As Zn2+, at the concentrations which were detected in the alloy’s culture media, showed a lesser cytotoxic effect than the Au-Pt conditioning media, we concluded that Zn is probably not the only element responsible for alloy cytotoxicity. Microstructural characterization of the alloys, performed by means of scanning electron microscopy in addition to energy dispersive X-ray and X-ray diffraction analyses, showed that Au-Pt I is a two-phase alloy containing a dominant Au-rich α1 phase and a minor Pt-rich α2 phase. On the other hand, the Au-Pt II alloy additionally contained three minor phases: AuZn3, Pt3Zn and Au1.4Zn0.52. The highest content of Zn was identified in the Pt3Zn phase. After conditioning, the Pt3Zn and AuZn3 phases disappeared, suggesting that they are predominantly responsible for Zn loss, lower corrosion stability and subsequent lower biocompatibility of the Au-Pt II alloy.

Immunomodulatory Activity of IL-27 in Human Periapical Lesions

IL-27, a cytokine with pro-inflammatory and anti-inflammatory properties, is a new member of the IL-6/IL-12 family, whose function in periapical lesions is unknown. We hypothesized that the production of IL-27 and its effect depend upon the type of immune/inflammatory response and clinical presentation of periapical lesions. We tested this hypothesis by studying the expression and function of IL-27 in human periapical lesions, both in situ and in culture. Immunohistochemistry demonstrated the strongest expression of IL-27 by endothelial cells and mononuclear phagocytes. Its production by periapical lesion mononuclear cells (PL-MNC), especially in symptomatic lesions, was significantly higher compared with that in peripheral blood MNC and correlated with the frequency of CD14+ and CD3+ cells. Exogenous IL-27 stimulated Th1 and down-regulated Th17 cytokine production by PL-MNC from symptomatic lesions, but down-regulated Th1 and Th2 responses in asymptomatic lesions. These findings suggest that IL-27 is an immunomodulatory cytokine in periapical lesions, with complex biological effects.

Why do some titanium-alloy total hip arthroplasty modular necks fail?

BACKGROUND Increased modularity in total hip arthroplasty (THA) through extra junction between the neck and the femoral stem is gaining popularity among orthopaedic community. However, the advantage of the additional junction is shadowed by an increased risk of mechanical failure. The aim of this study was to describe the exact mechanism of fracture of the modular femoral neck in an uncemented stem. METHODS Clinical, metallurgical, and mechanical analysis including finite-element modelling and elemental-sensitive tissue analysis with the micro-PIXE method was performed on two patients treated with fully modular primary THA made from Titanium alloy of the same oval taper-cone design. In patient A revision was performed 7.8 years after the unilateral primary procedure because of modular femoral neck fracture, while patient B was left-side revised 15 years after the bilateral primary procedure because of aseptic loosening of the femoral stem. RESULTS Body weight was 30% higher and the arm of implanted modular femoral neck was 51% longer in patient A compared to patient B. Therefore, the stress ratio on the modular femoral neck of patient A was calculated to be 2.45 times higher than in patient B, preventing cold welding and producing taper damage and degradation at the neck-stem junction. Large clusters of metallic debris containing Titanium and Vanadium from the alloy were present in the periprosthetic soft tissues of patient A. CONCLUSIONS Patients with higher body mass index treated with fully modular Ti-alloy THA may be at increased risk to experience catastrophic failure of the device. Orthopaedic surgeons should avoid using long necks whenever possible, as these are especially prone to develop a vicious circle starting with the fretting process and crevice corrosion at the taper-cone connection, leading to crack initiation and crack propagation, accelerated by the increased vulnerability of the Ti-alloy in biologic media, ultimately ending as fracture at the typical site. Serum Ti concentration may represent a rough estimation of taper degradation and patients with elevated levels should be warned and followed accordingly.

Titanium alloy femoral neck fracture—clinical and metallurgical analysis in 6 cases

We present a case series of 6 hips in 6 male patients (average age 50 (37–56) years) who underwent revision of total hip arthroplasty (THA) as a result of a modular femoral neck fracture of an otherwise stable THA (Table 1). All 6 patients presented with acute-onset pain at a mean of 5.6 (2.3–12) years after the index procedure, following an uneventful and asymptomatic initial recovery period. The pain was located in the groin, the patients were unable to tolerate weight bearing, the involved leg was shortened, the hip was in external rotation, and emergency room radiographs revealed a fractured modular femoral neck slightly inside the female taper of the stem (Figure 1).

The Response of Macrophages to a Cu-Al-Ni Shape Memory Alloy

Cu—Al—Ni shape memory alloys (SMAs) have been investigated as materials for medical devices, but little is known about their biocompatibility. The aim of this work was to study the response of rat peritoneal macrophages (PMØ) to a Cu—Al—Ni SMA in vitro, by measuring the functional activity of mitochondria, necrosis, apoptosis, and production of proinflammatory cytokines. Rapidly solidified (RS) thin ribbons were used for the tests. The control alloy was a permanent mold casting of the same composition, but without the shape memory effect. Our results showed that the control alloy was severely cytotoxic, whereas RS ribbons induced neither necrosis nor apoptosis of PMØ. These findings correlated with the data that RS ribbons are significantly more resistant to corrosion compared to the control alloy, as judged by the lesser release of Cu and Ni in the conditioning medium. However, the ribbons generated intracellular reactive oxygen species and upregulated the production of IL-6 by PMØ. These effects were almost completely abolished by conditioning the RS ribbons for 5 weeks. In conclusion, RS significantly improves the corrosion stability and biocompatibility of Cu—Al—Ni SMA. The biocompatibility of this functional material could be additionally enhanced by conditioning the ribbons in cell culture medium.