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Background: Multilayered osteochondral scaffolds are becoming increasingly utilized for the repair of knee joint surface lesions (KJSLs). However, the literature on predictive factors is rather limited. Purpose: To (1) evaluate the clinical outcomes and safety of a combined single-step approach using a biomimetic collagen-hydroxyapatite scaffold (CHAS) and filtered bone marrow aspirate (fBMA) for the treatment of KJSLs and (2) identify significant predictors of the treatment outcomes. Study Design: Case series; Level of evidence, 4. Methods: All patients who underwent surgery because of a KJSL (size ≥1.5 cm2; International Cartilage Regeneration & Joint Preservation Society grades 3-4) using the combination above were selected from a hospital registry database (100 patients; minimum 2-year follow-up). Patient characteristics, medical history, knee joint and lesion status, intraoperative details, and cellular parameters of the injected fBMA were collected. The arthroscopic evaluation of chondral and meniscal tissue quality in all knee compartments was performed using the Chondropenia Severity Score. Treatment outcomes were determined clinically using patient-reported outcome measures (Knee Injury and Osteoarthritis Outcome Score, EuroQol–5 Dimensions–3 Levels, EuroQol–Visual Analog Scale, and Tegner Activity Scale) and by assessing the occurrence of serious adverse events and graft failure. Multivariable regression analysis was performed to identify significant predictors of the treatment outcomes. Results: At a mean follow-up of 54.2 ± 19.4 months, 78 (87%) patients completed the questionnaires with significant improvements toward the baseline (P <.00625): KOOS Pain subscale from 62 ± 17 to 79 ± 18, KOOS Total score from 57 ± 16 to 70 ± 20, EuroQol–Visual Analog Scale from 61 ± 21 to 76 ± 16, EuroQol–5 Dimensions–3 Levels from 0.57 ± 0.20 to 0.80 ± 0.21, and Tegner Activity Scale from 2.8 ± 1.5 to 3.9 ± 1.9. The graft failure rate was 4%. A longer duration of preoperative symptoms, previous surgery, larger lesions, older age, and female sex were the main negative predictors for the treatment outcomes. The Chondropenia Severity Score and the number of fibroblast colony-forming units in fBMA positively influenced some of the clinical results and safety. Conclusion: A CHAS augmented with fBMA proved to be an adequate and safe approach for the treatment of KJSLs up to midterm follow-up. Based on the subanalysis of predictive factors, the surgical intervention should be performed in a timely and precise manner to prevent lesion enlargement, deterioration of the general knee cartilage status, and recurrent surgical procedures, especially in older and female patients. When a CHAS is used, the quantity of MSCs seems to play a role in augmentation. Registration: NCT06078072 (ClinicalTrials.gov identifier). [ABSTRACT FROM AUTHOR]

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets. [ABSTRACT FROM AUTHOR]

Purpose: To document clinical, radiologic, and cellular data of a prospective patient series treated by a tri-layer collagen-hydroxyapatite biomimetic osteochondral scaffold (CHAS) intra-operatively seeded with cultivated autologous chondrocytes (AC) or with filtered bone marrow stem/stromal cells (fBMSC) to address chronic osteochondral knee lesions. Methods: Thirty-six consecutive patients (15 to 59 years) with chronic osteochondral lesions (1.8–10 cm2) in the condylar or patellofemoral knee surfaces were enrolled. Lesions were covered with CHAS fixed with a fibrin glue. The superficial layer of CHAS was intra-operatively injected with active cells: in initial five patients, ACs were put directly onto dry CHAS (dry-AC); next, eight AC patients had CHAS moistened with cell culture media (media-AC), while the tourniquet was released allowing blood soaking of CHAS in the rest (14 blood-AC, 9 blood-fBMSC). Seventeen (50%) patients required different concomitant procedures. All patients were followed for serious adverse events (SAE) or graft failures; clinical, radiographic, and MRI evaluation was conducted. Cellular data on the injected cells were assessed. Results: At a follow-up of 39 months (16–81), 17 patients required an additional surgical intervention: seven graft-related SAE (early post-operative synovitis and/or arthrofibrosis) were registered (3 dry-AC, 3 media-AC, 1 blood-fBMSC). There were two graft failures (1 dry-AC, 1 blood-fBMSC) for secondary reasons. All clinical scores significantly improved from pre- to post-operative values: IKCD subjective 44 to 65; IKDC examination (9/17/5/5) to (20/10/5/1); KOOS (P61/S59/ADL67/Sp32/QoL31) to (P79/S75/ADL84/Sp55/QoL51); Tegner activity scale 3.3 to 4.4. There was evidence of radiographic osteoarthritis progression—Kellgren-Lawrence 1.0 to 1.5. MOCART scores at the final follow-up averaged 71 (10 to 95). Graft-type analysis demonstrated an increased rate of graft-related SAE in dry-AC and media-AC, but their final outcomes were equivalent. Cellular data of AC at the implantation were as follows: cells in suspension 9.2 × 106, viability 95%. In blood-fBMSC group, a cell suspension with 87% viability was injected, which contained 1156 CFU-Fs. Conclusion: CHAS with intra-operative seeding of active cells, either AC or fBMSC, led to an overall successful outcome for the treatment of chronic osteochondral lesions in the knee. Blood soaking of CHAS in situ before cell seeding significantly decreased early post-operative adverse events, such as synovitis and arthrofibrosis. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions. Experimental Procedures: MicroRNA profiling of 734 human and human-associated viral miRNAs was performed on blood plasma samples from 16 healthy individuals and 16 patients with GBM, using the nCounter miRNA Expression Assay Kits. Results: We identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients’ survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM. Conclusion: The GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa strains resistant towards all currently available antibiotics are increasingly encountered, raising the need for new anti-pseudomonal drugs. We therefore conducted a medium-throughput screen of a small-molecule collection resulting in the identification of the N -alkylated 3,6-dihalogenocarbazol 1-( sec -butylamino)-3-(3,6-dichloro-9 H -carbazol-9-yl)propan-2-ol (MIC = 18.5 μg mL −1 ). This compound, compound 1 , is bacteriostatic towards a broad spectrum of Gram-positive and Gram-negative pathogens, including P. aeruginosa . Importantly, 1 also eradicates mature biofilms of P. aeruginosa . 1 displays no cytotoxicity against various human cell types, pointing to its potential for further development as a novel antibacterial drug. [ABSTRACT FROM AUTHOR]