Alginate Improves the Chondrogenic Capacity of 3D PCL Scaffolds In Vitro: A Histological Approach.

Polycaprolactone (PCL) scaffolds have demonstrated an effectiveness in articular cartilage regeneration due to their biomechanical properties. On the other hand, alginate hydrogels generate a 3D environment with great chondrogenic potential. Our aim is to generate a mixed PCL/alginate scaffold that combines the chondrogenic properties of the two biomaterials. Porous PCL scaffolds were manufactured using a modified salt-leaching method and embedded in a culture medium or alginate in the presence or absence of chondrocytes. The chondrogenic capacity was studied in vitro. Type II collagen and aggrecan were measured by immunofluorescence, cell morphology by F-actin fluorescence staining and gene expression of COL1A1, COL2A1, ACAN, COL10A1, VEGF, RUNX1 and SOX6 by reverse transcription polymerase chain reaction (RT-PCR). The biocompatibility of the scaffolds was determined in vivo using athymic nude mice and assessed by histopathological and morphometric analysis. Alginate improved the chondrogenic potential of PCL in vitro by increasing the expression of type II collagen and aggrecan, as well as other markers related to chondrogenesis. All scaffolds showed good biocompatibility in the in vivo model. The presence of cells in the scaffolds induced an increase in vascularization of the PCL/alginate scaffolds. The results presented here reinforce the benefits of the combined use of PCL and alginate for the regeneration of articular cartilage.

Cadaveric Adipose-Derived Stem Cells for Regenerative Medicine and Research.

Advances in regenerative medicine have enabled the search for new solutions to current health problems in so far unexplored fields. Thus, we focused on cadaveric subcutaneous fat as a promising source of adipose-derived stem cells (ADSCs) that have potential to differentiate into different cell lines. With this aim, we isolated and characterized ADSCs from cadaveric samples with a postmortem interval ranging from 30 to 55 h and evaluated their ability to differentiate into chondrocytes or osteocytes. A commercial ADSC line was used as reference. Morphological and protein expression analyses were used to confirm the final stage of differentiation. Eight out of fourteen samples from patients were suitable to complete the whole protocol. Cadaveric ADSCs exhibited features of stem cells based upon several markers: CD29 (84.49 ± 14.07%), CD105 (94.38 ± 2.09%), and CD44 (99.77 ± 0.32%). The multiparametric assessment of differentiation confirmed the generation of stable lines of chondrocytes and osteocytes. In conclusion, we provide evidence supporting the feasibility of obtaining viable postmortem human subcutaneous fat ADSCs with potential application in tissue engineering and research fields.

Morphological Characterization of Human Lung Cancer Organoids Cultured in Type I Collagen Hydrogels: A Histological Approach.

The malignity of lung cancer is conditioned by the tumor microenvironment (TME), in which cancer-associated fibroblasts (CAFs) are relevant. In this work, we generated organoids by combining A549 cells with CAFs and normal fibroblasts (NF) isolated from adenocarcinoma tumors. We optimized the conditions for their manufacture in a short time. We evaluated the morphology of organoids using confocal microscopy analysis of F-actin, vimentin and pankeratin. We determined the ultrastructure of the cells in the organoids via transmission electron microscopy and the expression of CDH1, CDH2 and VIM via RT-PCR. The addition of stromal cells induces the self-organization of the organoids, which acquired a bowl morphology, as well as their growth and the generation of cell processes. They also influenced the expression of genes related to epithelial mesenchymal transition (EMT). CAFs potentiated these changes. All cells acquired a characteristic secretory phenotype, with cohesive cells appearing inside the organoids. In the periphery, many cells acquired a migratory phenotype, especially in organoids that incorporated CAFs. The deposit of abundant extracellular matrix could also be observed. The results presented here reinforce the role of CAFs in the progression of lung tumors and could lay the foundation for a useful in vitro pharmacological model.

New roles for AP-1/JUNB in cell cycle control and tumorigenic cell invasion via regulation of cyclin E1 and TGF-ß2.

BACKGROUND: JUNB transcription factor contributes to the formation of the ubiquitous transcriptional complex AP-1 involved in the control of many physiological and disease-associated functions. The roles of JUNB in the control of cell division and tumorigenic processes are acknowledged but still unclear. RESULTS: Here, we report the results of combined transcriptomic, genomic, and functional studies showing that JUNB promotes cell cycle progression via induction of cyclin E1 and repression of transforming growth factor (TGF)-ß2 genes. We also show that high levels of JUNB switch the response of TGF-ß2 stimulation from an antiproliferative to a pro-invasive one, induce endogenous TGF-ß2 production by promoting TGF-ß2 mRNA translation, and enhance tumor growth and metastasis in mice. Moreover, tumor genomic data indicate that JUNB amplification associates with poor prognosis in breast and ovarian cancer patients. CONCLUSIONS: Our results reveal novel functions for JUNB in cell proliferation and tumor aggressiveness through regulation of cyclin E1 and TGF-ß2 expression, which might be exploited for cancer prognosis and therapy.

In Vitro Effect of Δ9-Tetrahydrocannabinol and Cannabidiol on Cancer-Associated Fibroblasts Isolated from Lung Cancer.

There is evidence that demonstrates the effect of cannabinoid agonists inhibiting relevant aspects in lung cancer, such as proliferation or epithelial-to-mesenchymal transition (EMT). Most of these studies are based on evidence observed in in vitro models developed on cancer cell lines. These studies do not consider the complexity of the tumor microenvironment (TME). One of the main components of the TME is cancer-associated fibroblasts (CAFs), cells that are relevant in the control of proliferation and metastasis in lung cancer. In this work, we evaluated the direct effects of two cannabinoid agonists, tetrahydrocannabinol (THC) and cannabidiol (CBD), used alone or in combination, on CAFs and non-tumor normal fibroblasts (NFs) isolated from adenocarcinoma or from healthy lung tissue from the same patients. We observed that these compounds decrease cell density in vitro and inhibit the increase in the relative expression of type 1 collagen (COL1A1) and fibroblast-specific protein 1 (FSP1) induced by transforming growth factor beta (TGFß). On the other hand, we studied whether THC and CBD could modulate the interactions between CAFs or NFs and cancer cells. We conditioned the culture medium with stromal cells treated or not with THC and/or CBD and cultured A549 cells with them. We found that culture media conditioned with CAFs or NFs increased cell density, induced morphological changes consistent with EMT, inhibited cadherin-1 (CDH1) gene expression, and induced an increase in the relative expression of cadherin-2 (CDH2) and vimentin (VIM) genes in A549 cells. These changes were inhibited or decreased by THC and CBD administered alone or in combination. In another series of experiments, we conditioned culture media with A549 cells treated or not with THC and/or CBD, in the presence or absence of TGFß. We observed that culture media conditioned with A549 in the presence of TGFß induced an increase in the expression of COL1A1 and VIM, both in CAFs and in non-tumor NFs. Both THC and CBD ameliorated these effects. In summary, the results presented here reinforce the usefulness of cannabinoid agonists for the treatment of some relevant aspects of lung cancer pathology, and demonstrate in a novel way their possible effects on CAFs as a result of their relationship with cancer cells. Likewise, the results reinforce the usefulness of the combined use of THC and CBD, which has important advantages in relation to the possibility of using lower doses, thus minimizing the psychoactive effects of THC.

BMP-2 Enhances Osteogenic Differentiation of Human Adipose-Derived and Dental Pulp Stem Cells in 2D and 3D In Vitro Models.

Bone tissue provides support and protection to different organs and tissues. Aging and different diseases can cause a decrease in the rate of bone regeneration or incomplete healing; thus, tissue-engineered substitutes can be an acceptable alternative to traditional therapies. In the present work, we have developed an in vitro osteogenic differentiation model based on mesenchymal stem cells (MSCs), to first analyse the influence of the culture media and the origin of the cells on the efficiency of this process and secondly to extrapolate it to a 3D environment to evaluate its possible application in bone regeneration therapies. Two osteogenic culture media were used (one commercial from Stemcell Technologies and a second supplemented with dexamethasone, ascorbic acid, glycerol-2-phosphate, and BMP-2), with human cells of a mesenchymal phenotype from two different origins: adipose tissue (hADSCs) and dental pulp (hDPSCs). The expression of osteogenic markers in 2D cultures was evaluated in several culture periods by means of the immunofluorescence technique and real-time gene expression analysis, taking as reference MG-63 cells of osteogenic origin. The same strategy was extrapolated to a 3D environment of polylactic acid (PLA), with a 3% alginate hydrogel. The expression of osteogenic markers was detected in both hADSCs and hDPSCs, cultured in either 2D or 3D environments. However, the osteogenic differentiation of MSCs was obtained based on the culture medium and the cell origin used, since higher osteogenic marker levels were found when hADSCs were cultured with medium supplemented with BMP-2. Furthermore, the 3D culture used was suitable for cell survival and osteogenic induction.

Chondrogenic Potential of Human Dental Pulp Stem Cells Cultured as Microtissues.

Several tissue engineering stem cell-based procedures improve hyaline cartilage repair. In this work, the chondrogenic potential of dental pulp stem cell (DPSC) organoids or microtissues was studied. After several weeks of culture in proliferation or chondrogenic differentiation media, synthesis of aggrecan and type II and I collagen was immunodetected, and SOX9, ACAN, COL2A1, and COL1A1 gene expression was analysed by real-time RT-PCR. Whereas microtissues cultured in proliferation medium showed the synthesis of aggrecan and type II and I collagen at the 6th week of culture, samples cultured in chondrogenic differentiation medium showed an earlier and important increase in the synthesis of these macromolecules after 4 weeks. Gene expression analysis showed a significant increase of COL2A1 after 3 days of culture in chondrogenic differentiation medium, while COL1A1 was highly expressed after 14 days. Cell-cell proximity promotes the chondrogenic differentiation of DPSCs and important synthesis of hyaline chondral macromolecules.

High-Speed Video Microscopy for Primary Ciliary Dyskinesia Diagnosis: A Study of Ciliary Motility Variations with Time and Temperature.

Primary ciliary dyskinesia (PCD) is a rare disease resulting from a defect in ciliary function that generates, among other issues, chronic upper and lower respiratory tract infections. European guidelines recommend studying ciliary function (pattern (CBP) and frequency (CBF)), together with characteristic clinical symptoms, as one of the definitive tests. However, there is no "gold standard". The present study aims to use high-speed video microscopy to describe how CBF and CBP alter over time and at different temperatures to reduce the error rate in the diagnosis of PCD. Samples of nasal epithelium from 27 healthy volunteers were studied to assess CBF and CBP at 0, 3, 24, 48, and 72 h, at room temperature and 4 °C. It was observed that CBF increased while CBP became dyskinetic, both at room temperature and at 4 °C, as time passed, especially after 3 h. In order to preserve all ciliary function parameters and to perform a reliable analysis to improve the diagnostic process of PCD, analysis should be performed within the first 3 h of sample collection, preferably in reference centers.

Alginate-Agarose Hydrogels Improve the In Vitro Differentiation of Human Dental Pulp Stem Cells in Chondrocytes. A Histological Study.

Matrix-assisted autologous chondrocyte implantation (MACI) has shown promising results for cartilage repair, combining cultured chondrocytes and hydrogels, including alginate. The ability of chondrocytes for MACI is limited by different factors including donor site morbidity, dedifferentiation, limited lifespan or poor proliferation in vitro. Mesenchymal stem cells could represent an alternative for cartilage regeneration. In this study, we propose a MACI scaffold consisting of a mixed alginate-agarose hydrogel in combination with human dental pulp stem cells (hDPSCs), suitable for cartilage regeneration. Scaffolds were characterized according to their rheological properties, and their histomorphometric and molecular biology results. Agarose significantly improved the biomechanical behavior of the alginate scaffolds. Large scaffolds were manufactured, and a homogeneous distribution of cells was observed within them. Although primary chondrocytes showed a greater capacity for chondrogenic differentiation, hDPSCs cultured in the scaffolds formed large aggregates of cells, acquired a rounded morphology and expressed high amounts of type II collagen and aggrecan. Cells cultured in the scaffolds expressed not only chondral matrix-related genes, but also remodeling proteins and chondrocyte differentiation factors. The degree of differentiation of cells was proportional to the number and size of the cell aggregates that were formed in the hydrogels.

Optimization of a decellularization protocol of porcine tracheas. Long-term effects of cryopreservation. A histological study.

OBJECTIVE: The aim of this study was to optimize a decellularization protocol in the trachea of Sus scrofa domestica (pig) as well as to study the effects of long-term cryopreservation on the extracellular matrix of decellularized tracheas. METHODS: Porcine tracheas were decellularized using Triton X-100, SDC, and SDS alone or in combination. The effect of these detergents on the extracellular matrix characteristics of decellularized porcine tracheas was evaluated at the histological, biomechanical, and biocompatibility level. Morphometric approaches were used to estimate the effect of detergents on the collagen and elastic fibers content as well as on the removal of chondrocytes from decellularized organs. Moreover, the long-term structural, ultrastructural, and biomechanical effect of cryopreservation of decellularized tracheas were also estimated. RESULTS: Two percent SDS was the most effective detergent tested concerning cell removal and preservation of the histological and biomechanical properties of the tracheal wall. However, long-term cryopreservation had no an appreciable effect on the structure, ultrastructure, and biomechanics of decellularized tracheal rings. CONCLUSION: The results presented here reinforce the use of SDS as a valuable decellularizing agent for porcine tracheas. Furthermore, a cryogenic preservation protocol is described, which has minimal impact on the histological and biomechanical properties of decellularized porcine tracheas.