Impact of Anti-Mycotic Drugs on the Osteogenic Response of Bone Marrow Mesenchymal Stem Cells In Vitro.

The treatment of fungal bone infections and infected non-unions is a huge challenge in modern trauma and orthopedics, which normally contain the local and systemic administration of anti-fungal drugs. Although frequently used, little is known about the impact of systemic and locally administered fungicides on the osteogenic regenerative capabilities of infected bone tissue, especially upon the osteogenesis of human bone marrow mesenchymal stem cells (BM-hMSCs). This study evaluates the effects of the three most common fungicides for the systemic treatment of bone infections, Voriconazole (VOR), liposomal Amphotericin B (LAMB), and Fluconazole (FLU), as well as the effects of VOR and LAMB-loaded Polymethylmethacrylate (PMMA) cement chips in different concentrations upon the osteogenic response of BM-hMSCs in vitro. Within this study, we compared the ability of BM-hMSC to differentiate into osteoblast-like cells and synthesize hydroxyapatite as assessed by radioactive 99mTechnetium-Hydroxydiphosphonate (99mTc-HDP) labeling, cell proliferation, and analyses of supernatants upon various osteogenic parameters. Our results revealed that VOR added to the cell culture medium affects the osteogenic potential of BM-hMSC negatively, while there were no detectable effects of LAMB and FLU. Moreover, we showed dose-dependent negative effects of high- and extended-dose fungicide-loaded PMMA cement due to cytotoxicity, with a higher cytotoxic potential of VOR than LAMB, while low-dose fungicide-loaded PMMA had no significant effect on the osteogenic potential of BM-hMSC in vitro.

Impact of Antibiotic-Loaded PMMA Spacers on the Osteogenic Potential of hMSCs.

Antibiotic-loaded PMMA bone cement is frequently used in modern trauma and orthopedic surgery. Although many of the antibiotics routinely applied are described to have cytotoxic effects in the literature, clinical experience shows no adverse effects for bone healing. To determine the effects of antibiotic-loaded PMMA spacers on osteogenesis in vitro, we cultivated human bone marrow mesenchymal stem cells (BM-hMSCs) in the presence of PMMA spacers containing Gentamicin, Vancomycin, Gentamicin + Clindamycin as well as Gentamicin + Vancomycin in addition to a blank control (agarose) and PMMA containing no antibiotics. The cell number was assessed with DAPI staining, and the osteogenic potential was evaluated by directly measuring the amount of hydroxyapatite synthesized using radioactive 99mTc-HDP labelling as well as measuring the concentration of calcium and phosphate in the cell culture medium supernatant. The results showed that Gentamicin and Vancomycin as well as their combination show a certain amount of cytotoxicity but no negative effect on osteogenic potential. The combination of Gentamicin and Clindamycin, on the other hand, led to a drastic reduction in both the cell count and the osteogenic potential.

99mTc-HDP Labeling-A Non-Destructive Method for Real-Time Surveillance of the Osteogenic Differentiation Potential of hMSC during Ongoing Cell Cultures.

99-Metastabil Technetium (99mTc) is a radiopharmaceutical widely used in skeletal scintigraphy. Recent publications show it can also be used to determine the osteogenic potential of human mesenchymal stem cells (hMSCs) by binding to hydroxyapatite formed during bone tissue engineering. This field lacks non-destructive methods to track live osteogenic differentiation of hMSCs. However, no data about the uptake kinetics of 99mTc and its effect on osteogenesis of hMSCs have been published yet. We therefore evaluated the saturation time of 99mTc by incubating hMSC cultures for different periods, and the saturation concentration by using different amounts of 99mTc activity for incubation. The influence of 99mTc on osteogenic potential of hMSCs was then evaluated by labeling a continuous hMSC culture three times over the course of 3 weeks, and comparing the findings to cultures labeled once. Our findings show that 99mTc saturation time is less than 0.25 h, and saturation concentration is between 750 and 1000 MBq. Repeated exposure to γ-radiation emitted by 99mTc had no negative effects on hMSC cultures. These new insights can be used to make this highly promising method broadly available to support researchers in the field of bone tissue engineering using this method to track and evaluate, in real-time, the osteogenic differentiation of hMSC, without any negative influence on the cell viability, or their osteogenic differentiation potential.

Effects of Different Basal Cell Culture Media upon the Osteogenic Response of hMSCs Evaluated by 99mTc-HDP Labeling.

The osteogenic differentiation of mesenchymal stem cells is now a standard procedure in modern bone tissue engineering. As this is a promising field for future clinical applications, many cell culture media exist to promote osteogenic differentiation. Prior to differentiation, cells must be expanded to obtain sufficient numbers for experiments. Little evidence is available regarding the optimal media combination for expansion and differentiation to maximize the osteogenic response. Therefore, human BM-MSCs (n = 6) were expanded in parallel in DMEM (Dulbecco's Modified Eagle Medium) LG (Low Glucose) and α-MEM (Minimum Essential Media alpha-modification), followed by simultaneous monolayer differentiation toward the osteogenic lineage in: 1. DMEM LG (Low Glucose), 2. DMEM HG (High Glucose), 3. α-MEM, 4. "Bernese medium", and 5. "Verfaillie medium", with a corresponding negative control (total 20 groups). As a marker for osteogenic differentiation, hydroxyapatite was accessed using radioactive 99mTc-HDP labeling and quantitative alizarin red staining. The results indicate that all media except "Bernese medium" are suitable for osteogenic differentiation, while there was evidence that DMEM LG is partly superior when used for expansion and differentiation of BM-hMSCs. Using "Verfaillie medium" after DMEM LG expansion led to the highest grade of osteogenic differentiation. Nevertheless, the difference was not significant. Therefore, we recommend using DMEM LG for robust osteogenic differentiation, as it is highly suitable for that purpose, economical compared to other media, and requires little preparation time.

Impact of High-Dose Anti-Infective Agents on the Osteogenic Response of Mesenchymal Stem Cells.

Treatment of infected nonunions and severe bone infections is a huge challenge in modern orthopedics. Their treatment routinely includes the use of anti-infective agents. Although frequently used, little is known about their impact on the osteogenesis of mesenchymal stem cells. In a high- and low-dose set-up, this study evaluates the effects of the antibiotics Gentamicin and Vancomycin as well as the antifungal agent Voriconazole on the ability of mesenchymal stem cells to differentiate into osteoblast-like cells and synthesize hydroxyapatite in a monolayer cell culture. The osteogenic activity was assessed by measuring calcium and phosphate concentrations as well as alkaline phosphatase activity and osteocalcin concentration in the cell culture medium supernatant. The amount of hydroxyapatite was measured directly by radioactive 99mTechnetium-HDP labeling. Regarding the osteogenic markers, it could be concluded that the osteogenesis was successful within the groups treated with osteogenic cell culture media. The results revealed that all anti-infective agents have a cytotoxic effect on mesenchymal stem cells, especially in higher concentrations, whereas the measured absolute amount of hydroxyapatite was independent of the anti-infective agent used. Normed to the number of cells it can therefore be concluded that the above-mentioned anti-infective agents actually have a positive effect on osteogenesis while high-dose Gentamycin, in particular, is apparently capable of boosting the deposition of minerals.

Intra-operative use of one-step nucleic acid amplification (OSNA) for detection of the tumor load of sentinel lymph nodes in breast cancer patients.

BACKGROUND: The purpose of this single-center study was to determine the practicability of the intra-operative use of one-step nucleic acid amplification (OSNA) as the only method for detection of SLN. The OSNA system has been well described and is supposed to be as accurate as conventional histology. METHODS: Three hundred and thirty SLNs from 143 breast cancer patients were analyzed in an intra-operative setting. The CK19-copy number was determined by OSNA and divided into 3 results ("-" no metastasis; "+" micrometastasis; "++" marcometastasis). If OSNA gave a positive result, an axillary lymph node dissection was carried out during the same session. The central 1-mm slice of each node was obtained for permanent histology. Additionally, the results were correlated to clinicopathological factors, and the time for the intra-operative use was evaluated. RESULTS: Thirty-nine of the 143 patients were OSNA positive, 22 with macrometastatic and 17 with micrometastatic spread. The mean time for the OSNA run with one SLN was 34.4 min. We could show a correlation between the tumor size and OSNA positivity as well as between the numbers of OSNA positive SLNs with the tumor load of associated non-SLNs. Furthermore, we found that a cutoff CK19 copy number of 7,900/µL indicates a positive non-SLN result with the highest sensitivity (91 %) and specificity (61 %). CONCLUSION: We found OSNA to be very helpful for the intra-operative determination of the tumor load of a SLN as a basis for decision-making concerning further surgical axillary intervention. OSNA allows precise differentiation of micro- from macrometastasis, and the CK19 copy number predicts the probability of tumor load in other axillary lymph nodes and might help to find adequate adjuvant treatment options. This objective method is well suitable for everyday use and may reduce the pathologic workload and the risk of secondary operative interventions with all associated costs and stress for the patients.