3D modeling of normal skin and cutaneous squamous cell carcinoma. A comparative study in 2D cultures, spheroids, and 3D bioprinted systems.

The current cancer research and drug testing are primarily based on 2D cell cultures and animal models. However, these methods have limitations and yield distinct drug response patterns. This study addressed this gap by developing an innovativein vitrohuman three-dimensional (3D) normal skin model and a multicellular model of human cutaneous squamous cell carcinoma (cSCC) using 3D bioprinting technology. Comparative analyzes were performed between bioprinted 3D-cSCC model, consisting of HaCaT keratinocytes, primary normal human dermal fibroblasts and A431 cancer cells (tricellular), bioprinted 3D-A431 model composed of A431 cancer cells only (monocellular), A431 cancer cell spheroids, and conventional 2D models. The models were structurally characterized by light microscopy, immunofluorescence (LIVE/DEAD assay, confocal microscopy) and immunohistochemistry (hematoxylin/eosin, p63, vimentin, Ki67, epidermal growth factor receptor stainings). The spatial arrangement of the 3D models was analyzed using the ARIVIS scientific image analysis platform. All models were also functionally assessed by cetuximab (CTX) response testing with the MTS assay. 3D-cSCC models were maintained for up to 16 weeks. Morphological and histological examinations confirmed the presence of skin-like layers in the bioprinted 3D models of normal skin, and the intricate and diverse features of the bioprinted skin cancer model, replicating the criticalin vivocharacteristics. In both mono- and tricellular bioprinted tumor constructs, there was a gradual formation and continuous growth of spheroid-like clusters of cancer cells, significantly influencing the morphology of the models. Cancer cells in the 3D bioprinted constructs showed reduced sensitivity to CTX compared to spheroids and 2D cultures. This study underscores the potential of 3D multicellular models in elucidating drug responses and gaining a better understanding the intricate interplay of cellular components within the tumor microenvironment. Developing the multicellular 3D tumor model paves the way for new research critical to advancing fundamental cancer research and future clinical applications, particularly drug response testing.

Calcination and ion substitution improve physicochemical and biological properties of nanohydroxyapatite for bone tissue engineering applications.

Nanohydroxyapatite (nanoHAP) is widely used in bone regeneration, but there is a need to enhance its properties to provide stimuli for cell commitment and osteoconduction. This study examines the effect of calcination at 1200 °C on the physicochemical and biological properties of nanoHAP doped with magnesium (Mg2+), strontium (Sr2+), and zinc (Zn2+). A synergistic effect of dual modification on nanoHAP biological properties was investigated. The materials were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), BET analysis, Fourier-transform spectroscopy, and thermal analysis methods. Furthermore, ion release tests and in vitro biological characterization, including cytocompatibility, reactive oxygen species production, osteoconductive potential and cell proliferation, were performed. The XRD results indicate that the ion substitution of nanoHAP has no effect on the apatite structure, and after calcination, ß-tricalcium phosphate (ß-TCP) is formed as an additional phase. SEM analysis showed that calcination induces the agglomeration of particles and changes in surface morphology. A decrease in the specific surface area and in the ion release rate was observed. Combining calcination and nanoHAP ion modification is beneficial for cell proliferation and osteoblast response and provide additional stimuli for cell commitment in bone regeneration.

Effects of Sterilization and Hydrolytic Degradation on the Structure, Morphology and Compressive Strength of Polylactide-Hydroxyapatite Composites.

Composites based on polylactide (PLA) and hydroxyapatite (HA) were prepared using a thermally induced phase separation method. In the experimental design, the PLA with low weight-average molar mass (Mw) and high Mw were tested with the inclusion of HA synthesized as whiskers or hexagonal rods. In addition, the structure of HA whiskers was doped with Zn, whereas hexagonal rods were mixed with Sr salt. The composites were sterilized and then incubated in phosphate-buffered saline for 12 weeks at 37 °C, followed by characterization of pore size distribution, molecular properties, density and mechanical strength. Results showed a substantial reduction of PLA Mw for both polymers due to the preparation of composites, their sterilization and incubation. The distribution of pore size effectively increased after the degradation process, whereas the sterilization, furthermore, had an impact on pore size distribution depending on HA added. The inclusion of HA reduced to some extent the degradation of PLA quantitatively in the weight loss in vitro compared to the control without HA. All produced materials showed no cytotoxicity when validated against L929 mouse skin fibroblasts and hFOB 1.19 human osteoblasts. The lack of cytotoxicity was accompanied by the immunocompatibility with human monocytic cells that were able to detect pyrogenic contaminants.

Inhibition of LPAR6 overcomes sorafenib resistance by switching glycolysis into oxidative phosphorylation in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most threatening tumours in the world today. Pharmacological treatments for HCC mainly rely on protein kinase inhibitors, such as sorafenib and regorafenib. Even so, these approaches exhibit side effects and acquired drug resistance, which is an obstacle to HCC treatment. We have previously shown that selective lysophosphatidic acid receptor 6 (LPAR6) chemical antagonists inhibit HCC growth. Here, we investigated whether LPAR6 mediates resistance to sorafenib by affecting energy metabolism in HCC. To uncover the role of LPAR6 in drug resistance and cancer energy metabolism, we used a gain-of-function and loss-of-function approach in 2D tissue and 3D spheroids. LPAR6 was ectopically expressed in HLE cells (HLE-LPAR6) and knocked down in HepG2 (HepG2 LPAR6-shRNA). Measurements of oxygen consumption and lactate and pyruvate production were performed to assess the energy metabolism response of HCC cells to sorafenib treatment. We found that LPAR6 mediates the resistance of HCC cells to sorafenib by promoting lactic acid fermentation at the expense of oxidative phosphorylation (OXPHOS) and that the selective LPAR6 antagonist 9-xanthenyl acetate (XAA) can effectively overcome this resistance. Our study shows for the first time that an LPAR6-mediated metabolic mechanism supports sorafenib resistance in HCC and proposes a pharmacological approach to overcome it.

Mesenchymal Stem Cell Seeding on 3D Scaffolds.

Evaluation of mesenchymal stem cell seeding efficiency in three-dimensional (3D) scaffolds is a critical step for constructing a potent and useful tissue engineering product for regenerative medicine. To determine the quantity of cells seeded on a scaffold, their condition and viability, and/or to confirm cell adhesion to the scaffold surface, a number of cellular assays are used. The assays are most often based on a direct or indirect colorimetric-, fluorimetric-, bioluminescent-, or isotope-based measurement of changes reflecting the activity of cellular processes. This chapter presents a selection of assays measuring the efficiency of cell seeding on scaffolds, that is, the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assay, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, the ATP (adenosine triphosphate), DAPI (4',6-diamidino-2-phenylindole) assay, the Alamar Blue (7-hydroxy-10-oxidophenoxazin-10-ium-3-one, resazurin) assay and the Pico Green dsDNA (N'-[3-(dimethylamino)propyl]-N,N-dimethyl-N'-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]propane-1,3-diamine) assay. These assays monitor the number of viable cells, sometimes in conjunction with specifying cell membrane integrity, determine enzymatic activity associated with cell metabolism, measure cell proliferation rate, and assess the total protein or DNA content in the cell-scaffold construct. The choice of the appropriate methods and the details for testing 3D cultures are of utmost importance to properly evaluate tissue engineering products. Still, developing standards for assessment of cell-scaffold constructs remains a challenge in tissue engineering.

Scaffold vascularization method using an adipose-derived stem cell (ASC)-seeded scaffold prefabricated with a flow-through pedicle.

BACKGROUND: Vascularization is important for the clinical application of tissue engineered products. Both adipose-derived stem cells (ASCs) and surgical prefabrication can be used to induce angiogenesis in scaffolds. Our aim was to compare the angiogenic potential of ASC-seeded scaffolds combined with scaffold prefabrication with that of non-seeded, non-prefabricated scaffolds. METHODS: For prefabrication, functional blood vessels were introduced into the scaffold using a flow-through pedicle system. ASCs were isolated from rat fat deposits. Three-dimensional-printed cylindrical poly-ε-caprolactone scaffolds were fabricated by fused deposition modelling. Three groups, each containing six rats, were investigated by using non-seeded, ASC-seeded, and osteogenic induced ASC-seeded scaffolds. In each group, one rat was implanted with two scaffolds in the inguinal region. On the right side, a scaffold was implanted subcutaneously around the inferior epigastric vessels (classic prefabrication group). On the left side, the inferior epigastric vessels were placed inside the prefabricated scaffold in the flow-through pedicle system (flow-through prefabrication group). The vessel density and vascular architecture were examined histopathologically and by µCT imaging, respectively, at 2 months after implantation. RESULTS: The mean vessel densities were 10- and 5-fold higher in the ASC-seeded and osteogenic induced ASC-seeded scaffolds with flow-through prefabrication, respectively, than in the non-seeded classic prefabricated group (p < 0.001). µCT imaging revealed functional vessels within the scaffold. CONCLUSION: ASC-seeded scaffolds with prefabrication showed significantly improved scaffold vasculogenesis and could be useful for application to tissue engineering products in the clinical settings.

Characterization and Optimization of the Seeding Process of Adipose Stem Cells on the Polycaprolactone Scaffolds.

The purpose of the current study was to evaluate the usefulness of adipose-derived stem cells (ASCs) for bone injury therapy. Lipoaspirates were collected from the abdomen regions of 17 healthy female donors (mean age 49 ± 6 years) using Coleman technique or Body-jet liposuction. In the present study, the primary objective was the in vitro characteristics of human ASCs. The secondary objective was the optimization of the cell seeding process on 3D-printed scaffolds using polycaprolactone (PCL) or polycaprolactone covered with tricalcium phosphate (PCL + 5% TCP). Biological evaluation of human ASC showed high efficiency of isolation obtaining a satisfying amount of homogeneous cell populations. Results suggest that ASCs can be cultured in vitro for a long time without impairing their proliferative capacity. Growth kinetics shows that the highest number of cells can be achieved in passage 5 and after the 16th passage; there is a significant decrease of cell numbers and their proliferative potential. The percentage of colony forming units from the adipose stem cells is 8% ± 0.63% (p < 0.05). It was observed that the accumulation of calcium phosphate in the cells in vitro, marked with Alizarin Red S, was increased along with the next passage. Analysis of key parameters critically related to the cell seeding process shows that volume of cell suspension and propagation time greatly improve the efficiency of seeding both in PCL and PCL + 5% TCP scaffolds. The cell seeding efficiency did differ significantly between scaffold materials and cell seeding methods (p < 0.001). Increased seeding efficiency was observed when using the saturation of cell suspension into scaffolds with additional incubation. Alkaline phosphatase level production in PCL + 5% TCP scaffold was better than in PCL-only scaffold. The study results can be used for the optimization of the seeding process and quantification methods determining the successful implementation of the preclinical model study in the future tissue engineering strategies.

Comparison of adipose stem cells sources from various locations of rat body for their application for seeding on polymer scaffolds.

Adipose tissue yields adult adipose stem cells (ASCs) in large quantities via less-invasive methods. These cells are of interest owing to their modulating properties and paracrine activities, which can be harnessed in regenerative medicine. Many studies on the use of rat fat tissue in an autologous animal model have been conducted; however, the different locations to obtain stromal vascular fraction of rat fat depots have not been fully characterized. The purpose of the current study was to identify optimal source of ASC from various locations of rat body. Animal experiments in vitro revealed that fat depots from cervical fat are an optimal ASC source. A high ASC yield facilitates subsequent studies on autologous transplantation in rats. The secondary objective was to compare the efficiency of osteoinductive media composition and evaluate of osteogenic potential of ASCs for seeding on scaffolds for bone repair. Scaffolds were assessed in vitro, using rat adipose stem cells and three-dimensional (3D) scaffolds comprising polycaprolactone (PCL) or polycaprolactone covered with tricalcium phosphate (PCL + 5%TCP). Seeded ASCs adhere to the surface and migrate to the scaffolds. Upon staining and determining alkaline phosphatase levels, PCL + 5%TCP scaffolds performed better than PCL scaffolds. Furthermore, growth factors such as BMP2 and FGF2 significantly increased ASC mineralization and induced osteogenesis (p < 0.05). Our results may help select and develop pre-clinical animal model for confirming the use of ASC, alone or in association with appropriate biomaterials for bone repair.

[Angiogenesis - possibilities, problems and perspectives]. / Angiogeneza - mozliwosci, problemy, perspektywy.

Angiogenesis is the formation of new blood vessels from existing vessels. This process occurs via budding endothelial cells in postnatal period, which is essential to many physiological phenomena (e.g. wound healing, formation of the placenta) and pathological ones such as cancer, ischemic diseases, and chronic inflammation. Various mechanisms of the formation of new blood vessels have been discovered and a number of pro-angiogenic and anti-angiogenic factors have been found. Understanding the function of these factors contributes to the creation of new tools and applications in the treatment of pathological processes. Article describes the regulation of angiogenesis and is a review of the most significant angiogenic factors and their inhibitors. It shows the selected mechanisms which underlie the action of currently used anti-angiogenic drugs and is a review of research which use these factors in anti-angiogenic therapy.

[Mesenchymal stem cells]. / Mezenchymalne komórki macierzyste.

The multipotential progenitor cells called ,Mesenchymal Stem Cells" (MSC) are capable of differrentiation at least into bone, cartilage, and adipose tissues. The commonly recognized role of these cells is the formation of connective tissue which participates in formation of every organ. The progeny of MSC produces also the hematopoietic microenvironment, recently it have been documented that these cells are capable of the modulation of the immune system activities. MSC are isolated from the tissues of fetal origin (umbilical cord, cord blood, or placenta), or from several adult donor sites, in particular from bone marrow and adipose tissue which are most useful for practical purposes. The capability of multipotential differentiation, immunomodulation, and the regulation of the endogenous tissue repair are the reasons why mesenchymal stem cells are widely applied for regenerative medicine purposes.

Direct targeting of Arabidopsis cysteine synthase complexes with synthetic polypeptides to selectively deregulate cysteine synthesis.

Biosynthesis of cysteine is one of the fundamental processes in plants providing the reduced sulfur for cell metabolism. It is accomplished by the sequential action of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they constitute the hetero-oligomeric cysteine synthase (CS) complex through specific protein-protein interactions influencing the rate of cysteine production. The aim of our studies was to deregulate the CS complex formation in order to investigate its function in the control of sulfur homeostasis and optimize cysteine synthesis. Computational modeling was used to build a model of the Arabidopsis thaliana mitochondrial CS complex. Several polypeptides based on OAS-TL C amino-acid sequence found at SAT-OASTL interaction sites were designed as probable competitors for SAT3 binding. After verification of the binding in a yeast two-hybrid assay, the most strongly interacting polypeptide was introduced to different cellular compartments of Arabidopsis cell via genetic transformation. Moderate increase in total SAT and OAS-TL activities, but not thiols content, was observed dependent on the transgenic line and sulfur availability in the hydroponic medium. Though our studies demonstrate the proof of principle, they also suggest more complex interaction of both enzymes underlying the mechanism of their reciprocal regulation.

Molecular analysis of WWOX expression correlation with proliferation and apoptosis in glioblastoma multiforme.

Glioblastoma multiforme is the most common type of primary brain tumor in adults. WWOX is a tumor suppressor gene involved in carcinogenesis and cancer progression in many different neoplasms. Reduced WWOX expression is associated with more aggressive phenotype and poor patient outcome in several cancers. We investigated alternations of WWOX expression and its correlation with proliferation, apoptosis and signal trafficking in 67 glioblastoma multiforme specimens. Moreover, we examined the level of WWOX LOH and methylation status in WWOX promoter region. Our results suggest that loss of heterozygosity (relatively frequent in glioblastoma multiforme) along with promoter methylation may decrease the expression of this tumor suppressor gene. Our experiment revealed positive correlations between WWOX and Bcl2 and between WWOX and Ki67. We also confirmed that WWOX is positively correlated with ErbB4 signaling pathway in glioblastoma multiforme.