Models including preoperative plasma levels of angiogenic factors, leptin and IL-8 as potential biomarkers of endometrial cancer.

Background: The diversity of endometrial cancer (EC) dictates the need for precise early diagnosis and pre-operative stratification to select treatment options appropriately. Non-invasive biomarkers invaluably assist clinicians in managing patients in daily clinical practice. Currently, there are no validated diagnostic or prognostic biomarkers for EC that could accurately predict the presence and extent of the disease. Methods: Our study analyzed 202 patients, of whom 91 were diagnosed with EC and 111 were control patients with the benign gynecological disease. Using Luminex xMAP™ multiplexing technology, we measured the pre-operative plasma concentrations of six previously selected angiogenic factors - leptin, IL-8, sTie-2, follistatin, neuropilin-1, and G-CSF. Besides basic statistical methods, we used a machine-learning algorithm to create a robust diagnostic model based on the plasma concentration of tested angiogenic factors. Results: The plasma levels of leptin were significantly higher in EC patients than in control patients. Leptin was higher in type 1 EC patients versus control patients, and IL-8 was higher in type 2 EC versus control patients, particularly in poorly differentiated endometrioid EC grade 3. IL-8 plasma levels were significantly higher in EC patients with lymphovascular or myometrial invasion. Among univariate models, the model based on leptin reached the best results on both training and test datasets. A combination of age, IL-8, leptin and G-CSF was determined as the most important feature for the multivariate model, with ROC AUC 0.94 on training and 0.81 on the test dataset. The model utilizing a combination of all six AFs, BMI and age reached a ROC AUC of 0.89 on both the training and test dataset, strongly indicating the capability for predicting the risk of EC even on unseen data. Conclusion: According to our results, measuring plasma concentrations of angiogenic factors could, provided they are confirmed in a multicentre validation study, represent an important supplementary diagnostic tool for early detection and prognostic characterization of EC, which could guide the decision-making regarding the extent of treatment.

Antibody Arrays Identified Cycle-Dependent Plasma Biomarker Candidates of Peritoneal Endometriosis.

Endometriosis is an estrogen-dependent inflammatory disease affecting women in their reproductive age. Due to non-specific symptoms, women with endometriosis are often misdiagnosed or are accurately diagnosed only after several years. Diagnosis of peritoneal endometriosis is especially challenging and relies only on laparoscopic surgery. To date, different molecules have been proposed as potential non-invasive biomarkers of endometriosis; however, none have been confirmed as clinically useful. Therefore, this study aimed to discover novel plasma biomarker candidates for peritoneal endometriosis using an antibody array platform. This study included patients with endometriosis-like symptoms characterized by the absence (controls) or presence of peritoneal endometriosis (cases) after laparoscopic surgery and histological evaluation. Patients were further divided into secretory and proliferative groups, according to the phase of their menstrual cycle. Their plasma samples were collected and analyzed on an antibody array platform targeting more than 1350 proteins with over 1820 antibodies. In the proliferative group, the analysis revealed three differential proteins between cases and controls: ITB3, ITA2B2, and ACVL-1. In the secretory group, none of the examined proteins reached the log-fold change (logFC) and significance thresholds simultaneously. The potential of the identified differential proteins as plasma biomarker candidates for peritoneal endometriosis should be evaluated on a larger cohort, and their role in endometriosis should be investigated in further studies.

Circulating estradiol and its biologically active metabolites in endometriosis and in relation to pain symptoms.

Objectives: Endometriosis (EM) is an estrogen-dominant inflammatory disease linked to infertility that affects women of reproductive age. EM lesions respond to hormonal signals that regulate uterine tissue growth and trigger inflammation and pain. The objective of this study was to evaluate whether estradiol (E2) and its biologically active metabolites are differentially associated with EM given their estrogenic and non-estrogenic actions including proliferative and inflammatory properties. Design: We performed a retrospective study of 209 EM cases and 115 women without EM. Methods: Pain-related outcomes were assessed using surveys with validated scales. Preoperative serum levels of estradiol (E2) and estrone (E1), their 2-, 4- and 16- hydroxylated (OH) and methylated (MeO) derivatives (n=16) were measured by mass spectrometry. We evaluated the associations between estrogen levels and EM anatomic sites, surgical stage, risk of EM, and symptoms reported by women. Spearman correlations established the relationships between circulating steroids. Results: Of the sixteen estrogens profiled, eleven were detected above quantification limits in most individuals. Steroids were positively correlated, except 2-hydroxy 3MeO-E1 (2OH-3MeO-E1). Higher 2OH-3MeO-E1 was linked to an increased risk of EM (Odd ratio (OR)=1.91 (95%CI 1.09-3.34); P=0.025). Ovarian EM cases displayed enhanced 2-hydroxylation with higher 2MeO-E1 and 2OH-E1 levels (P< 0.009). Abdominal, pelvic and back pain symptoms were also linked to higher 2OH-3MeO-E1 levels (OR=1.86; 95%CI 1.06-3.27; P=0.032). Conclusions: The 2-hydroxylation pathway emerges as an unfavorable feature of EM, and is associated with ovarian EM and pain related outcomes.

In vitro effects of ascorbic acid on viability and metabolism of patients' osteosarcoma stem cells.

Stagnation in novelties of osteosarcoma (OS) treatment indicates the need for new therapeutic methods. OS cancer stem cells (OS-CSC) are taught to have the ability to self-renew and develop mechanisms of anticancer drug resistance, and this is why it is difficult to eradicate them. Their metabolism has been recognized as a potential target of therapeutic action. Ascorbic acid (AA) is considered to act pro-oxidative against OS-CSC in vitro by oxidative effect and by inhibition of glycolysis. This study examined an in vitro impact of AA on OS-CSC metabolism isolated from patients' biopsies, with the aim of better understanding of OS-CSC metabolism and the action of AA on OS-CSC. OS-CSC were isolated using a sphere culture system and identified as stem cells using Hoechst 33342 exclusion assay. Determination of the dominant type of metabolism of OS-CSC, parental OS cells, human mesenchymal stem cells (hMSC) and U2OS OS lineage before and after AA treatment was done by Seahorse XF (Agilent). Cytotoxicity of high-dose AA was confirmed by the MTT test and was proven for all the examined cell types as well as HEK293. Seahorse technology showed that OS-CSC can potentially use both glycolysis and oxidative phosphorylation (OXPHOS), and can turn to glycolysis and slow metabolic potential in unfavorable conditions such as incubation in AA.

Morphological and Molecular Evaluation of the Tissue Repair following Nasal Septum Biopsy in a Sheep Model.

OBJECTIVE: Nasal septal pathologies requiring surgical intervention are common in the population. Additionally, nasal chondrocytes are becoming an important cell source in cartilage tissue engineering strategies for the repair of articular cartilage lesions. These procedures damage the nasal septal cartilage whose healing potential is limited due to its avascular, aneural, and alymphatic nature. Despite the high incidence of various surgical interventions that affect septum cartilage, limited nasal cartilage repair characterizations have been performed to date. METHODS: To evaluate the healing of the nasal septum cartilage perforation, a septal biopsy was performed in 14 sheep. Two and 6 months later, the tissue formed on the place of perforation was explanted and compared with the native tissue. Tissue morphology, protein and gene expression of explanted tissue was determined using histological, immunohistochemical and real-time quantitative polymerase chain reaction analysis. RESULTS: Tissue formed on the defect site, 2 and 6 months after the biopsy was characterized as mostly connective tissue with the presence of fibroblastic cells. This newly formed tissue contained no glycosaminoglycans and collagen type II but was positively stained for collagen type I. Cartilage-specific genes COL2, AGG, and COMP were significantly decreased in 2- and 6-month samples compared with the native nasal cartilage. Levels of COL1, COL4, and CRABP1 genes specific for perichondrium and connective tissue were higher in both test group samples in comparison with native cartilage. CONCLUSIONS: Newly formed tissue was not cartilage but rather fibrous tissue suggesting the role of perichondrium and mucosa in tissue repair after nasal septum injury.

Nasal Chondrocyte-Based Engineered Grafts for the Repair of Articular Cartilage "Kissing" Lesions: A Pilot Large-Animal Study.

BACKGROUND: Bipolar or "kissing" cartilage lesions formed on 2 opposite articular surfaces of the knee joint are commonly listed as exclusion criteria for advanced cartilage therapies. PURPOSE: To test, in a pilot large-animal study, whether autologous nasal chondrocyte (NC)-based tissue engineering, recently introduced for the treatment of focal cartilage injuries, could provide a solution for challenging kissing lesions. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral kissing lesions were freshly introduced into the knee joints of 26 sheep and covered with NC-based grafts with a low or high hyaline-like extracellular matrix; a control group was treated with a cell-free scaffold collagen membrane (SCA). The cartilage repair site was assessed at 6 weeks and 6 months after implantation by histology, immunohistochemistry, and magnetic resonance imaging evaluation. RESULTS: NC-based grafts, independently of their composition, induced partial hyaline cartilage repair with stable integrity in surrounding healthy tissue at 6 months after treatment. The SCA repaired cartilage to a similar degree to that of NC-based grafts. CONCLUSION: Kissing lesion repair, as evidenced in this sheep study, demonstrated the feasibility of the treatment of complex cartilage injuries with advanced biological methods. However, the potential advantages of an NC-based approach over a cell-free approach warrant further investigations in a more relevant preclinical model. CLINICAL RELEVANCE: NC-based grafts currently undergoing phase II clinical trials have a high potential to replace existing cartilage therapies that show significant limitations in the quality and reproducibility of the repair method. We have brought this innovative concept to the next level by addressing a new clinical indication.

Characterization of Chitosan-Based Scaffolds Seeded with Sheep Nasal Chondrocytes for Cartilage Tissue Engineering.

The treatment of cartilage defect remains a challenging issue in clinical practice. Chitosan-based materials have been recognized as a suitable microenvironment for chondrocyte adhesion, proliferation and differentiation forming articular cartilage. The use of nasal chondrocytes to culture articular cartilage on an appropriate scaffold emerged as a promising novel strategy for cartilage regeneration. Beside excellent properties, chitosan lacks in biological activity, such as RGD-sequences. In this work, we have prepared pure and protein-modified chitosan scaffolds of different deacetylation degree and molecular weight as platforms for the culture of sheep nasal chondrocytes. Fibronectin (FN) was chosen as an adhesive protein for the improvement of chitosan bioactivity. Prepared scaffolds were characterised in terms of microstructure, physical and biodegradation properties, while FN interactions with different chitosans were investigated through adsorption-desorption studies. The results indicated faster enzymatic degradation of chitosan scaffolds with lower deacetylation degree, while better FN interactions with material were achieved on chitosan with higher number of amine groups. Histological and immunohistochemical analysis of in vitro engineered cartilage grafts showed presence of hyaline cartilage produced by nasal chondrocytes.

Bioreactor-manufactured cartilage grafts repair acute and chronic osteochondral defects in large animal studies.

OBJECTIVES: Bioreactor-based production systems have the potential to overcome limitations associated with conventional tissue engineering manufacturing methods, facilitating regulatory compliant and cost-effective production of engineered grafts for widespread clinical use. In this work, we established a bioreactor-based manufacturing system for the production of cartilage grafts. MATERIALS & METHODS: All bioprocesses, from cartilage biopsy digestion through the generation of engineered grafts, were performed in our bioreactor-based manufacturing system. All bioreactor technologies and cartilage tissue engineering bioprocesses were transferred to an independent GMP facility, where engineered grafts were manufactured for two large animal studies. RESULTS: The results of these studies demonstrate the safety and feasibility of the bioreactor-based manufacturing approach. Moreover, grafts produced in the manufacturing system were first shown to accelerate the repair of acute osteochondral defects, compared to cell-free scaffold implants. We then demonstrated that grafts produced in the system also facilitated faster repair in a more clinically relevant chronic defect model. Our data also suggested that bioreactor-manufactured grafts may result in a more robust repair in the longer term. CONCLUSION: By demonstrating the safety and efficacy of bioreactor-generated grafts in two large animal models, this work represents a pivotal step towards implementing the bioreactor-based manufacturing system for the production of human cartilage grafts for clinical applications. Read the Editorial for this article on doi:10.1111/cpr.12625.

Bone Tissue Engineering in a Perfusion Bioreactor Using Dexamethasone-Loaded Peptide Hydrogel.

The main goal of this study was the formation of bone tissue using dexamethasone (DEX)-loaded [COCH3]-RADARADARADARADA-[CONH2] (RADA 16-I) scaffold that has the ability to release optimal DEX concentration under perfusion force. Bone-marrow samples were collected from three patients during a hip arthroplasty. Human mesenchymal stem cells (hMSCs) were isolated and propagated in vitro in order to be seeded on scaffolds made of DEX-loaded RADA 16-I hydrogel in a perfusion bioreactor. DEX concentrations were as follows: 4 × 10-3, 4 × 10-4 and 4 × 10-5 M. After 21 days in a perfusion bioreactor, tissue was analyzed by scanning electron microscopy (SEM) and histology. Markers of osteogenic differentiation were quantified by real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. Minerals were quantified and detected by the von Kossa method. In addition, DEX release from the scaffold in a perfusion bioreactor was assessed. The osteoblast differentiation was confirmed by the expression analysis of osteoblast-related genes (alkaline phosphatase (ALP), collagen I (COL1A1) and osteocalcin (OC). The hematoxylin/eosin staining confirmed the presence of cells and connective tissue, while SEM revealed morphological characteristics of cells, extracellular matrix and minerals-three main components of mature bone tissue. Immunocytochemical detection of collagen I is in concordance with given results, supporting the conclusion that scaffold with DEX concentration of 4 × 10-4 M has the optimal engineered tissue morphology. The best-engineered bone tissue is produced on scaffold loaded with 4 × 10-4 M DEX with a perfusion rate of 0.1 mL/min for 21 days. Differentiation of hMSCs on DEX-loaded RADA 16-I scaffold under perfusion force has a high potential for application in regenerative orthopedics.

Presence of phthalate esters in intravenous solution evaluated using gas chromatography-mass spectrometry method.

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer widely used in the production of poly-(vinyl) chloride (PVC) materials. It is a reproductive and developmental toxicant in animals and a suspected endocrine modulator in humans. DEHP is not covalently bound within the PVC molecule, which is why migration into a suitable medium can be expected. Since application of infusion solutions is one of the most common medical treatments, the objective of this study was to determine the migration of phthalates from softened PVC storage bags into infusion solution in different time periods within one year from date of production using a gas chromatography-mass spectrometry method. The measured values of DEHP ranged between 0.22 and 14.00 µg l(-1) , but the unexpected presence of other phthalate esters was also detected. It was concluded that values obtained in infusion solutions match the reference data and represent a minor risk for the patient. The presence of other phthalate esters leads to the conclusion that the pharmacopeic requirement for polymer cleanness was not fully met. Since phthalate esters are among the most extensively used industrial chemicals and are widely distributed in the environment, special precautions and further monitoring should be conducted to minimize any possible health risks.