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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 cm 2 ; 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)., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: M.V. and L.G. are employed by Educell, which is a commercial cell and tissue company that provides separated mesenchymal stem cells and other advanced cell therapies. M.D. is a clinical consultant for Finceramica. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

Background: Biomaterials augmented with Bone Marrow Aspirate Concentrate (BMAC) are becoming increasingly utilized in the cartilage treatment. However, the potential role of cellular parameters in the intraoperatively applied BMAC have yet to be elucidated., Purpose: (A) To evaluate clinical outcomes and safety of a combined single-step approach with scaffolds (fibrin glues, collagen gels, collagen-hydroxyapatite membrane) and filtered Bone Marrow Aspirate (fBMA) for the treatment of osteochondral lesions of the talus (OLTs). (B) To identify significant factors for postoperative improvements, considering cellular parameters as potential predictors., Methods: All the patients operated on due to OLTs by the combination above were selected from the hospital registry database (35 pts, years 16-55, and minimally 1 year follow-up). Treatment outcomes were followed clinically with Patient-reported outcome measures (PROMs), and by pursuing serious adverse events (SAE) and graft failures (GF). Cellular parameters of the injected fBMA were determined. Pre- and postoperative PROMs values were compared to evaluate postoperative improvements. Multivariable regression models were applied to identify potential factors (demographics, medical history, joint and lesion characteristics, scaffold type, surgical and cellular parameters) that predict the treatment outcomes., Results: At the mean follow-up of 32.2 (12.5) months, all Foot and Ankle Outcome Score (FAOS) and European Quality of Life in Five Dimensions Three-Level (EQ-5D-3 L) values improved significantly. 4 (11%) SAE (3 arthrofibrosis, one hardware removal), and 3 (9%) GF occurred. Female gender and concomitant procedures were the main negative predictors for postoperative outcomes. The number of fibroblast colony forming units (CFU-F) or their proportion among total nucleated cells (CFU-F/TNC) were positively correlated with the improvements of some PROMs., Conclusions: Scaffolds augmented with fBMA proved as an adequate and safe approach for OLTs treatment. Cellular parameters seem to influence the treatment outcomes, thus further attention should be given to the intraoperatively applied products., Level of Evidence: Level IV.

Background: Stem cell therapy is a promising new approach to wound healing. Stromal vascular fraction is a heterogeneous collection of cells, including adipose-derived stem cells, which are traditionally isolated using a manual collagenase-based technique. To our knowledge, this is the first human study that histologically assesses the potential of intraoperative intradermal injection of stromal vascular fraction on skin regeneration., Methods: In this controlled study, 20 patients undergoing deep inferior epigastric perforator flap breast reconstruction and bilateral flank liposuction were included. Stromal vascular fraction was injected intradermally into one side of the abdominal suture line, while the other side served as a control. Outcome measures included analysis of stromal vascular fraction by flow cytometry, histological analysis of scar tissue, and scar photography., Results: Cell yield for application and cell viability were 55.9 ± 28.5 × 10 6 and 75.1% ± 14.5%, respectively. Age and body mass index were positively correlated with the number of cells for application and adipose-derived stem cells. Mean vascular density, elastic fiber content, collagen maturity (scar index), epidermal thickness, and number of rete ridges all showed higher values on the treated side. Furthermore, the injected number of adipose-derived stem cells and pericytes positively correlated with vascular density., Conclusions: It is safe to speculate that intradermal stromal vascular fraction injection at the beginning of the healing process increases vascular density, collagen maturity and organization, elastic fiber content, epidermal thickness, epidermal-dermal anchoring of the scarring skin and is therefore responsible for improved skin regeneration. It is a viable and safe method that can be used as an adjunctive treatment in plastic surgery procedures where suboptimal wound healing is anticipated., Level of Evidence Iv: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 ., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature and International Society of Aesthetic Plastic Surgery.)

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 cm 2 ) 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 × 10 6 , 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.

Background: Osteochondral injury is a very common orthopaedic pathology, mainly affecting young, active population, with limited current treatment options. Herein we are presenting cellular and early clinical data of a patient series treated for chronic osteochondral lesions in the knee with a filter-based intra-operative bone marrow aspirate (BMA) separation device., Methods: Fifteen patients with chronic knee osteochondral lesions (60% females, 19-59 years) were included in this prospective case series. Filtered BMA (f-BMA), containing mesenchymal stem/stromal cells (MSCs), was combined with a biomimetic collagen-hydroxyapatite scaffold (CHAS) and implanted into the site of the lesion. Harvested BMA and post-separation f-BMA were analysed for blood cell counts, flow cytometry, and fibroblast colony forming units (CFU-Fs). Patients were followed for serious adverse events and graft failures. Clinical evaluation was assessed using the knee injury and osteoarthritis outcome score (KOOS). In 8 patients a magnetic resonance imaging (MRI)/arthroscopy were performed., Results: Cell suspension contained 0.027% CD271 + CD45 - 7-AAD - cells, 0.15% CD73 + CD90 + CD105 + cells and 0.0012% CFU-Fs of all nucleated cells with 86% viability. Filtration process resulted in 12.8 (4.0-40.8) fold enrichment in terms of CFU-F content in comparison to initial BMA. No serious adverse events related directly to the osteochondral treatment were reported. After an average follow-up of 20 months (14-25) all KOOS subscales (Symptoms/Pain/Daily activities/Sport and recreation/Quality of life) increased significantly from pre-operative 55/56/67/30/30 to post-operative 73/76/79/51/52 (p values < 0.05), respectively. MRI or arthroscopic evaluation revealed nearly normal to normal overall International Cartilage Repair Society assessment in 7/8 patients., Conclusion: The filter-based BMA separation procedure significantly increased the frequency of mesenchymal stem/stromal cells (MSCs), however their concentration was not increased. The clinical evaluation revealed high safety profile of the treatment and resulted in improved clinical status of the patients.

Mesenchymal stem cells (MSCs) are heterogeneous population of cells with great potential for regenerative medicine. MSCs are relatively easy to expand in a cell culture, however determination of their concentration in harvested tissue is more complex and is not implemented as routine procedure. To identify MSCs collected from bone marrow we have used two combinations of cell markers (CD45 - /CD73 + /CD90 + /CD105 + and CD45 - /CD271 + ) and fibroblast colony-forming unit (CFU-F) assay. Further, in donors of various ages, mesenchymal stem cell concentration was compared with the result of CFU-F assay and with hematopoietic stem cell concentration, determined by a standardized flow cytometric assay. A positive correlation of MSC populations to the CFU-F numbers is observed, the population of the CD45 - /CD271 + cells correlates better with CFU-F numbers than the population of the CD45 - /CD73 + /CD90 + /CD105 + cells. The relationship between the hematopoietic CD45 dim /CD34 + cell concentration and mesenchymal CFU-Fs or CD45 - /CD271 + cells shows a positive linear regression. An age-related quantitative reduction of hematopoietic CD45 dim /CD34 + , mesenchymal CD45 - /CD73 + /CD90 + /CD105 + and CD45 - /CD271 + stem cells, and CFU-F numbers were noted. Additionally, statistically significant higher CFU-F numbers were observed when bone marrow samples were harvested from three different sites from the anterior iliac crest instead of harvesting the same sample amount only from one site.

Smooth muscle cells (SMCs) are essential for tissue engineering strategies to fabricate organs such as blood vessels, the oesophagus and bladder, and to create disease models of these systems. In order for such therapies and models to be feasible, SMCs must be sourced effectively to enable production of large numbers of functional cells. In vitro, SMCs divide slowly and demonstrate short proliferative lifespans compared with other types of cells, including stem cells and fibroblasts, limiting the number of cells that can be derived from expansion in culture of a primary isolation. As such, it would be beneficial to better understand the factors underlying induction and maintenance of SMC phenotypes, in order to produce new sources of SMCs for tissue engineering and disease modelling. Here we report the ability of human dermal fibroblasts to display patterns of gene expression resembling contractile SMCs when cultured under conditions that are known to promote a contractile phenotype in SMCs, including culture on collagen IV, low-serum culture, TGF-β1 treatment and hypoxia. These factors drive expression of the myogenic transcription factor myocardin, as well as expression of several of its gene targets that are known contributors to contractile phenotype in SMCs, including smooth muscle alpha actin, calponin, and myosin heavy chain. Our results suggest that culture conditions associated with culture of SMCs may be sufficient to induce myogenic gene expression patterns and potential myogenic function in non-muscle cells., (Copyright © 2017 John Wiley & Sons, Ltd.)

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.

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-9H-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.

Background: Glioblastoma multiforme (GBM) is a brain tumour with a very high patient mortality rate, with a median survival of 47 weeks. This might be improved by the identification of novel diagnostic, prognostic and predictive therapy-response biomarkers, preferentially through the monitoring of the patient blood. The aim of this study was to define the impact of GBM in terms of alterations of the plasma protein levels in these patients., Materials and Methods: We used a commercially available antibody array that includes 656 antibodies to analyse blood plasma samples from 17 healthy volunteers in comparison with 17 blood plasma samples from patients with GBM., Results: We identified 11 plasma proteins that are statistically most strongly associated with the presence of GBM. These proteins belong to three functional signalling pathways: T-cell signalling and immune responses; cell adhesion and migration; and cell-cycle control and apoptosis. Thus, we can consider this identified set of proteins as potential diagnostic biomarker candidates for GBM. In addition, a set of 16 plasma proteins were significantly associated with the overall survival of these patients with GBM. Guanine nucleotide binding protein alpha (GNAO1) was associated with both GBM presence and survival of patients with GBM., Conclusions: Antibody array analysis represents a useful tool for the screening of plasma samples for potential cancer biomarker candidates in small-scale exploratory experiments; however, clinical validation of these candidates requires their further evaluation in a larger study on an independent cohort of patients.

We previously identified a decapeptide from the model plant Arabidopsis thaliana, OSIP108, which is induced upon fungal pathogen infection. In this study, we demonstrated that OSIP108 interferes with biofilm formation of the fungal pathogen Candida albicans without affecting the viability or growth of C. albicans cells. OSIP108 displayed no cytotoxicity against various human cell lines. Furthermore, OSIP108 enhanced the activity of the antifungal agents amphotericin B and caspofungin in vitro and in vivo in a Caenorhabditis elegans-C. albicans biofilm infection model. These data point to the potential use of OSIP108 in combination therapy with conventional antifungal agents. In a first attempt to unravel its mode of action, we screened a library of 137 homozygous C. albicans mutants, affected in genes encoding cell wall proteins or transcription factors important for biofilm formation, for altered OSIP108 sensitivity. We identified 9 OSIP108-tolerant C. albicans mutants that were defective in either components important for cell wall integrity or the yeast-to-hypha transition. In line with these findings, we demonstrated that OSIP108 activates the C. albicans cell wall integrity pathway and that its antibiofilm activity can be blocked by compounds inhibiting the yeast-to-hypha transition. Furthermore, we found that OSIP108 is predominantly localized at the C. albicans cell surface. These data point to interference of OSIP108 with cell wall-related processes of C. albicans, resulting in impaired biofilm formation.

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.