Fallopian tube abnormalities in uterine serous carcinoma.

OBJECTIVE: Uterine serous carcinoma (USC) is presumed to arise from endometrial intra-epithelial carcinoma (EIC), whereas tubo-ovarian high-grade serous carcinomas have similar precursor lesions in the Fallopian tube, i.e. serous tubal intra-epithelial carcinoma (STIC). The presence of Fallopian tube abnormalities and their clonal relationship to the concurrent USC was investigated. METHODS: In this multicenter study, all patients treated for USC between 1992 and 2017 were retrospectively identified. Histopathological diagnosis of USC, EIC and STIC was revised by an expert pathologist. Additionally, all Fallopian tube sections were immunohistochemically stained (p53 and Ki-67). Fallopian tube abnormalities were classified as either p53 signature, serous tubal intra-epithelial lesion (STIL) or STIC. The USCs and Fallopian tube abnormalities were analyzed by targeted next-generation sequencing. RESULTS: In 168 included patients, Fallopian tube abnormalities were found in 27.4% (46/168): p53-signatures in 17.9% (30/168), STILs in 3.0% (5/168) and STICs in 6.5% (11/168). In subgroup analysis, STICs were found in 9.5% (11/115) of patients with at least one section of the fimbriated end embedded. Next-generation sequencing showed identical TP53-mutations in the STIC and corresponding USC. CONCLUSIONS: In conclusion, the presence of Fallopian tube abnormalities was shown in a high percentage of patients with USC, representing either true precursor lesions or metastasized disease.

Single-Stage Autologous Chondrocyte-Based Treatment for the Repair of Knee Cartilage Lesions: Two-Year Follow-up of a Prospective Single-Arm Multicenter Study.

BACKGROUND: There is an unmet need for a single-stage cartilage repair treatment that is cost-effective and chondrocyte-based. PURPOSE: To evaluate the safety and preliminary efficacy of autologous freshly isolated primary chondrocytes and bone marrow mononucleated cells (MNCs) seeded into a PolyActive scaffold in patients with symptomatic cartilage lesions of the knee. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: A total of 40 patients with symptomatic knee cartilage lesions were treated with freshly isolated autologous chondrocytes combined with bone marrow MNCs delivered in a biodegradable load-bearing scaffold. The treatment requires only 1 surgical intervention and is potentially a cost-effective alternative to autologous chondrocyte implantation. The primary chondrocytes and bone marrow MNCs were isolated, washed, counted, mixed, and seeded into a load-bearing scaffold in the operating room. Patients were followed up at 3, 6, 12, 18, and 24 months. Primary endpoints were treatment-related adverse events up to 3 months, adverse implant effects between 3 and 24 months, and the implant success rate at 3 months as measured by lesion filling. RESULTS: Successful lesion filling (≥67% on magnetic resonance imaging) was found in 40 patients at 3 months and in 32 of the 32 patients analyzed at 24 months. Significant improvement over baseline was found for visual analog scale for pain from 3 months onward; Knee injury and Osteoarthritis Outcome Score (KOOS)-Pain and KOOS-Activities of Daily Living from 6 months onward; for KOOS-Symptoms and Stiffness, KOOS-Quality of Life and International Knee Documentation Committee from 12 months onward; and for KOOS-Sport and Recreation from 18 months onward. Hyaline-like repair tissue was found in 22 of 31 patients available for biopsy. Arthralgia and joint effusion were the most common adverse events. Scaffold delamination and adhesions led to removal of the implant in 2 patients. CONCLUSION: The treatment of knee cartilage lesions with autologous primary chondrocytes and bone marrow MNCs, both isolated and seeded into a load-bearing PolyActive scaffold within a single surgical intervention, is safe and clinically effective. Good lesion fill and sustained clinically important and statistically significant improvement in all patient-reported outcome scores were found throughout the 24-month study. Hyaline-like cartilage was observed on biopsy specimen in at least 22 of the 40 patients. REGISTRATION: NCT01041885 (ClinicalTrials.gov identifier).

Novel bilayer bacterial nanocellulose scaffold supports neocartilage formation in vitro and in vivo.

Tissue engineering provides a promising alternative therapy to the complex surgical reconstruction of auricular cartilage by using ear-shaped autologous costal cartilage. Bacterial nanocellulose (BNC) is proposed as a promising scaffold material for auricular cartilage reconstruction, as it exhibits excellent biocompatibility and secures tissue integration. Thus, this study evaluates a novel bilayer BNC scaffold for auricular cartilage tissue engineering. Bilayer BNC scaffolds, composed of a dense nanocellulose layer joined with a macroporous composite layer of nanocellulose and alginate, were seeded with human nasoseptal chondrocytes (NC) and cultured in vitro for up to 6 weeks. To scale up for clinical translation, bilayer BNC scaffolds were seeded with a low number of freshly isolated (uncultured) human NCs combined with freshly isolated human mononuclear cells (MNC) from bone marrow in alginate and subcutaneously implanted in nude mice for 8 weeks. 3D morphometric analysis showed that bilayer BNC scaffolds have a porosity of 75% and mean pore size of 50 ± 25 µm. Furthermore, endotoxin analysis and in vitro cytotoxicity testing revealed that the produced bilayer BNC scaffolds were non-pyrogenic (0.15 ± 0.09 EU/ml) and non-cytotoxic (cell viability: 97.8 ± 4.7%). This study demonstrates that bilayer BNC scaffolds offer a good mechanical stability and maintain a structural integrity while providing a porous architecture that supports cell ingrowth. Moreover, bilayer BNC scaffolds provide a suitable environment for culture-expanded NCs as well as a combination of freshly isolated NCs and MNCs to form cartilage in vitro and in vivo as demonstrated by immunohistochemistry, biochemical and biomechanical analyses.

An improved cartilage digestion method for research and clinical applications.

Enzymatic isolation of chondrocytes from a cartilage biopsy is the first step to establish in vitro models of chondrogenesis or to generate cell-based grafts for cartilage repair. Such process is based on manually operated procedures and typically results in yields lower than 20% of the total available cells. In this study, we hypothesized that, as compared to conventionally used protocols, the enzymatic digestion of human articular cartilage in the presence of ascorbic acid 2-phosphate (AscA2P) or of sodium chloride (NaCl), in combination with the use of a perfusion bioreactor system, leads to a higher and more reproducible yield of cell populations with high proliferation and chondrogenic capacity. The addition of AscA2P within the enzymatic digestion medium did not significantly increase the cell yield, but resulted in a significant decrease of the intradonor variability in cell yield (-17.8% ± 10.7%, p = 0.0247) and in a significant increase of the proliferation rate of the isolated chondrocytes (+19.0% ± 1.4%, p < 0.05) with respect to the control group. The addition of NaCl during cartilage digestion did not modulate cell yield. When the cartilage digestion was further performed under direct perfusion flow, beneficial synergistic effects were achieved, with an overall increase of 34.7% ± 6.8% (p < 0.001) in the cell yield and an average decrease of 57.8% ± 11.2% (p < 0.01) in the coefficient of variation with respect to the control group. Importantly, by implementing this strategy it was possible to retrieve clonal subpopulations more efficiently capable of undergoing chondrogenesis, both in vitro and in vivo. Our findings bear relevance for the preparation of human chondrocytes for laboratory investigations, and in the perspective of efficient and streamlined manufacturing of cell/tissue grafts for articular cartilage repair.

Concise review: unraveling stem cell cocultures in regenerative medicine: which cell interactions steer cartilage regeneration and how?

Cartilage damage and osteoarthritis (OA) impose an important burden on society, leaving both young, active patients and older patients disabled and affecting quality of life. In particular, cartilage injury not only imparts acute loss of function but also predisposes to OA. The increase in knowledge of the consequences of these diseases and the exponential growth in research of regenerative medicine have given rise to different treatment types. Of these, cell-based treatments are increasingly applied because they have the potential to regenerate cartilage, treat symptoms, and ultimately prevent or delay OA. Although these approaches give promising results, they require a costly in vitro cell culture procedure. The answer may lie in single-stage procedures that, by using cell combinations, render in vitro expansion redundant. In the last two decades, cocultures of cartilage cells and a variety of (mesenchymal) stem cells have shown promising results as different studies report cartilage regeneration in vitro and in vivo. However, there is considerable debate regarding the mechanisms and cellular interactions that lead to chondrogenesis in these models. This review, which included 52 papers, provides a systematic overview of the data presented in the literature and tries to elucidate the mechanisms that lead to chondrogenesis in stem cell cocultures with cartilage cells. It could serve as a basis for research groups and clinicians aiming at designing and implementing combined cellular technologies for single-stage cartilage repair and treatment or prevention of OA.

In vivo screening of extracellular matrix components produced under multiple experimental conditions implanted in one animal.

Animal experiments help to progress and ensure safety of an increasing number of novel therapies, drug development and chemicals. Unfortunately, these also lead to major ethical concerns, costs and limited experimental capacity. We foresee a coercion of all these issues by implantation of well systems directly into vertebrate animals. Here, we used rapid prototyping to create wells with biomaterials to create a three-dimensional (3D) well-system that can be used in vitro and in vivo. First, the well sizes and numbers were adjusted for 3D cell culture and in vitro screening of molecules. Then, the functionality of the wells was evaluated in vivo under 36 conditions for tissue regeneration involving human mesenchymal stem cells (hMSCs) and bovine primary chondrocytes (bPCs) screened in one animal. Each biocompatible well was controlled to contain µl-size volumes of tissue, which led to tissue penetration from the host and tissue formation under implanted conditions. We quantified both physically and biologically the amounts of extracellular matrix (ECM) components found in each well. Using this new concept the co-culture of hMSCs and bPCs was identified as a positive hit for cartilage tissue repair, which was a comparable result using conventional methods. The in vivo screening of candidate conditions opens an entirely new range of experimental possibilities, which significantly abates experimental animal use and increases the pace of discovery of medical treatments.

Co-culture in cartilage tissue engineering.

For biotechnological research in vitro in general and tissue engineering specifically, it is essential to mimic the natural conditions of the cellular environment as much as possible. In choosing a model system for in vitro experiments, the investigator always has to balance between being able to observe, measure or manipulate cell behaviour and copying the in situ environment of that cell. Most tissues in the body consist of more than one cell type. The organization of the cells in the tissue is essential for the tissue's normal development, homeostasis and repair reaction. In a co-culture system, two or more cell types brought together in the same culture environment very likely interact and communicate. Co-culture has proved to be a powerful in vitro tool in unravelling the importance of cellular interactions during normal physiology, homeostasis, repair and regeneration. The first co-culture studies focused mainly on the influence of cellular interactions on oocytes maturation to a pre-implantation blastocyst. Therefore, a brief overview of these studies is given here. Later on in the history of co-culture studies, it was applied to study cell-cell communication, after which, almost immediately as the field of tissue engineering was recognized, it was introduced in tissue engineering to study cellular interactions and their influence on tissue formation. This review discusses the introduction and applications of co-culture systems in cell biology research, with the emphasis on tissue engineering and its possible application for studying cartilage regeneration.

Effect of stratified culture compared to confluent culture in monolayer on proliferation and differentiation of human articular chondrocytes.

With conventional tissue culture of cells, it is generally assumed that when the available 2D substrate is fully occupied, growth ceases or is greatly reduced.However, in nature wound repair mostly involves proliferation of cells that are attracted to the defect site in a 3D environment.Hence, proliferation continues in 3D until the defect site is filled with cells contributing to repair tissue. With this in mind,we examined the growth behavior of human articular chondrocytes during stratified culture as opposed to routine culture to confluency. Additionally, we studied the influence of growth factors on proliferation during stratified culture and differentiation thereafter. Chondrocytes were cultured in monolayer on tissue culture plastic to confluency or stratified for an additional 7 days. Culture medium was based on DMEM with 10% serum and either supplemented with high concentrations of nonessential amino acids (NEAA) and ascorbic acid (AsAP), or instead with basic fibroblastic growth factor (bFGF), platelet-derived growth factor (PDBF-BB), and/or transforming growth factor beta1 (TGF-beta). After expansion, cells were harvested, counted, and their differentiation capacity was examined in pellet culture assay. It was shown that chondrocytes, cultured stratified proliferate exponentially for up to an additional 4 days and that cell yield increased 5-fold. Furthermore, during stratified culture the number of cells increased further in the presence of bFGF, PDBF-BB, and TGFbeta1 or high concentrations of NEAA and AsAP. Depending on donor variation and factors supplemented the cell yield ranged from 0.06 up to 1.1 million cells/cm2 at the second passage. During stratified culture in the presence of either bFGF and PDGF or high concentrations of NEAA and AsAP, exponential growth continued for up to 7 days. Finally, cells maintained their differentiation capacity when cultured stratified with or without growth factors (bFGF, TGF-beta, and PDGF), but not when cultured with high levels of AsAP and NEAA. In contrast to other 3D culture techniques like microcarrier or suspension culture, nutrient consumption remained the same as with conventional expansion. Because this allows culturing of clinically relevant amounts of chondrocytes without increasing the amount of serum, chondrocytes can be fully expanded in the presence autologous serum, avoiding the risk of viral and/or prion disease transmission associated with the use of animal-derived serum or serum replacers with animal-derived constituents.