Surface Stiffness Has No Impact on MCF-7 Sensitivity to Doxorubicin.

Resistance to the chemotherapeutic agents in the clinical management of cancer remains a significant challenge, and the mechanical environment of cancer cells is one of the major determinants of this. Stiffening of the environment is usually associated with increased chemoresistance of cancer cells, although this process depends on the type of cancer. Breast cancer is the most frequently diagnosed cancer, and more than half a million people die from it each year worldwide. In this study, we used the most frequent (70% of diagnosed cases) breast cancer phenotype, representing the MCF-7 cell line, to investigate the influence of surface stiffness on its sensitivity to one of the most commonly used anticancer drugs-doxorubicin. We showed that the mechanical environment affected MCF-7 proliferation, adhesion, and the expression and activation of mitogen-activated protein kinases (MAPKs). Furthermore, the role of MAPKs in response to doxorubicin was dependent on surface stiffness; nevertheless, surface stiffness did not affect MCF-7 resistance to doxorubicin.

Dental pulp stem cell-derived extracellular matrix: autologous tool boosting bone regeneration.

BACKGROUND AIMS: To facilitate artificial bone construct integration into a patient's body, scaffolds are enriched with different biologically active molecules. Among various scaffold decoration techniques, coating surfaces with cell-derived extracellular matrix (ECM) is a rapidly growing field of research. In this study, for the first time, this technology was applied using primary dental pulp stem cells (DPSCs) and tested for use in artificial bone tissue construction. METHODS: Rat DPSCs were grown on three-dimensional-printed porous polylactic acid scaffolds for 7 days. After the predetermined time, samples were decellularized, and the remaining ECM detailed proteomic analysis was performed. Further, DPSC-secreated ECM impact to mesenchymal stromal cells (MSC) behaviour as well as its role in osteoregeneration induction were analysed. RESULTS: It was identified that DPSC-specific ECM protein network ornamenting surface-enhanced MSC attachment, migration and proliferation and even promoted spontaneous stem cell osteogenesis. This protein network also demonstrated angiogenic properties and did not stimulate MSCs to secrete molecules associated with scaffold rejection. With regard to bone defects, DPSC-derived ECM recruited endogenous stem cells, initiating the bone self-healing process. Thus, the DPSC-secreted ECM network was able to significantly enhance artificial bone construct integration and induce successful tissue regeneration. CONCLUSIONS: DPSC-derived ECM can be a perfect tool for decoration of various biomaterials in the context of bone tissue engineering.

The Role of Cardiac T-Cadherin in the Indicating Heart Failure Severity of Patients with Non-Ischemic Dilated Cardiomyopathy.

Background and objectives: T-cadherin (T-cad) is one of the adiponectin receptors abundantly expressed in the heart and blood vessels. Experimental studies show that T-cad sequesters adiponectin in cardiovascular tissues and is critical for adiponectin-mediated cardio-protection. However, there are no data connecting cardiac T-cad levels with human chronic heart failure (HF). The aim of this study was to assess whether myocardial T-cad concentration is associated with chronic HF severity and whether the T-cad levels in human heart tissue might predict outcomes in patients with non-ischemic dilated cardiomyopathy (NI-DCM). Materials and Methods: 29 patients with chronic NI-DCM and advanced HF were enrolled. Patients underwent regular laboratory investigations, echocardiography, coronary angiography, and right heart catheterization. TNF-α and IL6 in serum were detected by enzyme-linked immunosorbent assay (ELISA). Additionally, endomyocardial biopsies were obtained, and the levels of T-cad were assessed by ELISA and CD3, CD45Ro, CD68, and CD4- immunohistochemically. Mean pulmonary capillary wedge pressure (PCWP) was used as a marker of HF severity, subdividing patients into two groups: mean PCWP > 19 mmHg vs. mean PCWP < 19 mmHg. Patients were followed-up for 5 years. The study outcome was composite: left ventricular assist device implantation, heart transplantation, or death from cardiovascular causes. Results: T-cad shows an inverse correlation with the mean PCWP (rho = -0.397, p = 0.037). There is a tendency towards a lower T-cad concentration in patients with more severe HF, as indicated by the mean PCWP > 19 mmHg compared to those with mean PCWP ≤ 19 mmHg (p = 0.058). Cardiac T-cad levels correlate negatively with myocardial CD3 cell count (rho = -0.423, p = 0.028). Conclusions: Univariate Cox regression analysis did not prove T-cad to be an outcome predictor (HR = 1, p = 0.349). However, decreased T-cad levels in human myocardium can be an additional indicator of HF severity. T-cad in human myocardium has an anti-inflammatory role. More studies are needed to extend the role of T-cad in the outcome prediction of patients with NI-DCM.

Bioavailability of Different Vitamin D Oral Supplements in Laboratory Animal Model.

Background and Objectives: The major cause of vitamin D deficiency is inadequate exposure to sunlight. It is difficult to supplement it with food because sufficient concentrations of vitamin D naturally occur only in a handful of food products. Thereby, deficiency of this vitamin is commonly corrected with oral supplements. Different supplement delivery systems for improved vitamin D stability and bioavailability are proposed. In this study, we compared efficiency of three vitamin D delivery systems: microencapsulated, micellized, and oil-based. Materials and Methods: As a model in this medical testing, laboratory rats were used for the evaluation of bioavailability of different vitamin D vehicles. Animals were divided into three groups: the first one was given microencapsulated vitamin D3, the second-oil-based vitamin D3, and the third-micellized vitamin D3. Test substances were given per os to each animal for 7 days, and vitamin D concentration in a form of 25-hydroxyvitamin D (25(OH)D) in the blood was checked both during the vitamin delivery period and later, up to the 24th day. Results: Comparison of all three tested products showed that the microencapsulated and oil-based vitamin D3 vehicles were the most bioavailable in comparison to micellized vitamin D3. Even more, the effect of the microencapsulated form of vitamin D3 remained constant for the longest period (up to 14 days). Conclusions: The results of this study suggest that the oral vitamin D supplement vehicle has an impact on its bioavailability, thus it is important to take into account how much of the suppled vitamin D will be absorbed. To maximize the full exploit of supplement, the best delivery strategy should be employed. In our study, the microencapsulated form of vitamin D was the most bioavailable.

Development of multilayered artificial urethra graft for urethroplasty.

To enhance the treatment of patients' urethral defects, such as strictures and hypospadias, we investigated the potential of using artificial urethral tissue. Our study aimed to generate this tissue and assess its effectiveness in a rabbit model. Two types of bioprinted grafts, based on methacrylated gelatin-silk fibroin (GelMA-SF) hydrogels, were produced: acellular, as well as loaded with autologous rabbit stem cells. Rabbit adipose stem cells (RASC) were differentiated toward smooth muscle in the GelMA-SF hydrogel, while rabbit buccal mucosa stem cells (RBMC), differentiated toward the epithelium, were seeded on its surface, forming two layers of the cell-laden tissue. The constructs were then reinforced with polycaprolactone-polylactic acid meshes to create implantable multilayered artificial urethral grafts. In vivo experiments showed that the cell-laden tissue integrated into the urethra with less fibrosis and inflammation compared to its acellular counterpart. Staining to trace the implanted cells confirmed integration into the host organism 3 months postsurgery.

Inflammation-suppressing cornea-in-a-syringe with anti-viral GF19 peptide promotes regeneration in HSV-1 infected rabbit corneas.

Pathophysiologic inflammation, e.g., from HSV-1 viral infection, can cause tissue destruction resulting in ulceration, perforation, and ultimately blindness. We developed an injectable Cornea-in-a-Syringe (CIS) sealant-filler to treat damaged corneas. CIS comprises linear carboxylated polymers of inflammation-suppressing 2-methacryloyloxyethyl phosphorylcholine, regeneration-promoting collagen-like peptide, and adhesive collagen-citrate glue. We also incorporated GF19, a modified anti-viral host defense peptide that blocked HSV-1 activity in vitro when released from silica nanoparticles (SiNP-GF19). CIS alone suppressed inflammation when tested in a surgically perforated and HSV-1-infected rabbit corneal model, allowing tissue and nerve regeneration. However, at six months post-operation, only regenerated neocorneas previously treated with CIS with SiNP-GF19 had structural and functional features approaching those of normal healthy corneas and were HSV-1 virus-free. We showed that composite injectable biomaterials can be designed to allow regeneration by modulating inflammation and blocking viral activity in an infected tissue. Future iterations could be optimized for clinical application.

Synthesis and characterization of UV curable biocompatible hydrophilic copolymers containing siloxane units.

Tissues are highly three-dimensional structure complexes composed of different cell types and their interactions. One of the main challenges in tissue engineering is the inability to produce large, highly perfused scaffolds in which cells can grow at a high cell density and viability. Poly(dimethyl siloxane) (PDMS) is used as a flexible, biocompatible cell culture substrate with tunable mechanical properties. However, its fragility and hydrophobicity still pose a challenge. Here, we present a new strategy for the three-step one-pot synthesis of novel biocompatible hydrophilic copolymers containing siloxane units. In the first step, free radical copolymerization of acrylic acid (AA), butyl methacrylate (BMA), and 2-hydroxyethyl methacrylate (HEMA) was carried out in dioxane (DO) solution in the presence of 2,2'-azodiisobutyronitrile (AIBN). In the second step, the copolymers were modified with diepoxypropoxypropyl-terminated polydimethylsiloxane (DE-PDMS), and in the third step, the copolymers were additionally modified with glycidyl methacrylate (GMA). The modified copolymers were characterized by FTIR, NMR spectroscopy and elemental analysis. Films of modified copolymers were prepared by UV curing. SEM studies revealed microphase separated morphology with distribution of PDMS domains. The mechanical properties of the films depended on the amount of incorporated silicone modifier. The films were more hydrophilic than PDMS films. All novel copolymers showed high biocompatibility.

Genetic Ablation of Ankrd1 Mitigates Cardiac Damage during Experimental Autoimmune Myocarditis in Mice.

Myocarditis (MC) is an inflammatory disease of the myocardium that can cause sudden death in the acute phase, and dilated cardiomyopathy (DCM) with chronic heart failure as its major long-term outcome. However, the molecular mechanisms beyond the acute MC phase remain poorly understood. The ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic stress/stretch-inducible protein, which can modulate cardiac stress response during various forms of pathological stimuli; however, its involvement in post-MC cardiac remodeling leading to DCM is not known. To address this, we induced experimental autoimmune myocarditis (EAM) in ANKRD1-deficient mice, and evaluated post-MC consequences at the DCM stage mice hearts. We demonstrated that ANKRD1 does not significantly modulate heart failure; nevertheless, the genetic ablation of Ankrd1 blunted the cardiac damage/remodeling and preserved heart function during post-MC DCM.

Anti-inflammatory effect of different curcumin preparations on adjuvant-induced arthritis in rats.

BACKGROUND: Curcumin, a natural polyphenolic substance, has been known for more than two millennia as having strong anti-inflammatory activity towards multiple ailments, including arthritis. The main drawback of curcumin is its poor solubility in water, which leads to low intestinal absorption and minimal bioavailability. In this study, we aimed to compare the anti-arthritic in vivo effect of different curcumin preparations - basic curcumin extract, micellar curcumin, curcumin mixture with piperine, and microencapsulated curcumin. METHODS: Arthritis was induced in Wistar rats by complete Freund's adjuvant, and the severity of arthritis was evaluated daily using the arthritis score system. Curcumin preparations were given to animals per os daily for 20 consecutive days, starting at 6th day after arthritis induction. To determine the inflammatory background, pro-inflammatory cytokines were determined using the ELISA test. In addition, hematologic test, weight change, and limb swelling were tracked. RESULTS: Our results indicate that curcumin had a rather weak effect on arthritis progression in the Wistar rat model, microencapsulated curcumin effectively prevented the progression of arthritis - the disease stabilized after 10 days of supplementation. It also reduced the levels of immune cells (neutrophils and leukocytes), as well as pro-inflammatory cytokines - TNFα, IL-1, and IL-6, which levels were close to arthritis-free control. Other formulations of curcumin had lower or no effect on arthritis progression. CONCLUSION: Our study shows that the same concentrations of curcumin had a distinctly expressed positive anti-inflammatory effect depending on the form of its delivery. Specifically, we found that microencapsulated curcumin had the most promising effect for treatment.

Surface stiffness depended gingival mesenchymal stem cell sensitivity to oxidative stress.

Mesenchymal stem cells (MSCs) are widely used in the fields of cell therapy and tissue engineering, due to their wide spectrum of differentiation potential, immunomodulation function and ongoing oxidative stress (OS) reduction. Nevertheless, OS impact is often overlooked in these research fields. It is not only responsible for the induction and development of many ailments, e.g., diabetes, lung fibrosis, and cancer, moreover, OS causes stem cell death and senescence during cell therapy and tissue engineering practices. As MSCs are used to treat various tissues, they interact with different tissue-specific mechanical environments, thus it is important to understand how the mechanical environment impacts MSC sensitivity to OS. In this work, for the first time, as known to the authors, it was shown that gingival MSCs (GMSCs) sensitivity to OS depends on the stiffness of the surface, on which the cells are grown. Furthermore, the activity and expression of mitogen activated protein kinases ERK, JNK, and p38 were surface stiffness dependent. GMSCs isolated from intermediate/stiff gingiva tissue (~20 kPa) have shown the best proliferative and survival properties, then grown on the stiffest tissues mimicking polyacrylamide hydrogels (40 kPa). Therefore, MSC source might determine their sensitivity to OS in different stiffness environments and should be accounted when developing a treatment strategy.

The Effect of a Unique Region of Parvovirus B19 Capsid Protein VP1 on Endothelial Cells.

Parvovirus B19 (B19V) is a widespread human pathogen possessing a high tropism for erythroid precursor cells. However, the persistence or active replication of B19V in endothelial cells (EC) has been detected in diverse human pathologies. The VP1 unique region (VP1u) of the viral capsid has been reported to act as a major determinant of viral tropism for erythroid precursor cells. Nevertheless, the interaction of VP1u with EC has not been studied. We demonstrate that recombinant VP1u is efficiently internalized by rats' pulmonary trunk blood vessel-derived EC in vitro compared to the human umbilical vein EC line. The exposure to VP1u was not acutely cytotoxic to either human- or rat-derived ECs, but led to the upregulation of cellular stress signaling-related pathways. Our data suggest that high levels of circulating B19V during acute infection can cause endothelial damage, even without active replication or direct internalization into the cells.

Inflammation-Related Biomarkers Are Associated with Heart Failure Severity and Poor Clinical Outcomes in Patients with Non-Ischemic Dilated Cardiomyopathy.

Inflammation-related biomarkers are associated with clinical outcomes in mixed-etiology chronic heart failure populations. Inflammation-related markers tend to be higher in ischemic than in non-ischemic dilated cardiomyopathy (NI-DCM) patients, which might impact their prognostic performance in NI-DCM patients. Therefore, we aimed to assess the association of inflammation-related biomarkers with heart failure severity parameters and adverse cardiac events in a pure NI-DCM patient cohort. Fifty-seven patients with NI-DCM underwent endomyocardial biopsy. Biopsies were evaluated by immunohistochemistry for CD3+, CD45ro+, CD68+, CD4+, CD54+, and HLA-DR+ cells. Blood samples were tested for high-sensitivity C-reactive protein (hs-CRP), interleukin-6, tumor necrosis factor-α (TNF-α), soluble urokinase-type plasminogen activator receptor and adiponectin. During a five-year follow-up, twenty-seven patients experienced at least one composite adverse cardiac event: left ventricle assist device implantation, heart transplantation or death. Interleukin-6, TNF-α and adiponectin correlated with heart failure severity parameters. Patients with higher levels of interleukin-6, TNF-α, adiponectin or hs-CRP, or a higher number of CD3+ or CD45ro+ cells, had lower survival rates. Interleukin-6, adiponectin, and CD45ro+ cells were independently associated with poor clinical outcomes. All patients who had interleukin-6, TNF-α and adiponectin concentrations above the threshold experienced an adverse cardiac event. Therefore, a combination of these cytokines can identify high-risk NI-DCM patients.

In vitro comparison of 3D printed polylactic acid/hydroxyapatite and polylactic acid/bioglass composite scaffolds: Insights into materials for bone regeneration.

3D printing of polylactic acid (PLA) and hydroxyapatite (HA) or bioglass (BG) bioceramics composites is the most promising technique for artificial bone construction. However, HA and BG have different chemical composition as well as different bone regeneration inducing mechanisms. Thus, it is important to compare differentiation processes induced by 3D printed PLA + HA and PLA + BG scaffolds in order to evaluate the strongest osteoconductive and osteoinductive properties possessing bioceramics. In this study, we analysed porous PLA + HA (10%) and PLA + BG (10%) composites' effect on rat's dental pulp stem cells fate in vitro. Obtained results indicated, that PLA + BG scaffolds lead to weaker cell adhesion and proliferation than PLA + HA. Nevertheless, osteoinductive and other biofriendly properties were more pronounced by PLA + BG composites. Overall, the results showed a strong advantage of bioceramic BG against HA, thus, 3D printed PLA + BG composite scaffolds could be a perspective component for patient-specific, cheaper and faster artificial bone tissue production.

The effect of larger than cell diameter polylactic acid surface patterns on osteogenic differentiation of rat dental pulp stem cells.

Topography of the scaffold is one of the most important factors defining the quality of artificial bone. However, the production of precise micro- and nano-structured scaffolds, which is known to enhance osteogenic differentiation, is expensive and time-consuming. Meanwhile, little is known about macro-patterns (larger than cell diameter) effect on cell fate, while this kind of structures would significantly facilitate the manufacturing of artificial skeleton. Therefore, this research is focused on polylactic acid scaffold's macro-pattern impact on rat's dental pulp stem cells (DPSCs) morphology, proliferation, and osteogenic differentiation. For this study, two types of scaffolds were 3D printed: wavy and porous. Wavy scaffolds consisted of 188 µm wide joined threads, meaning that cells might have been curved on the filament as well as compressed in the groove. Porous scaffolds were designed to avoid groove formation and consisted of 500 µm threads, arranged in the woodpile manner, forming 300 µm diameter pores. We found that both macro-surfaces influenced DPSC morphology compared to control. As a consequence, enhanced DPSC proliferation and increased osteogenic differentiation potential was registered in cells grown on these scaffolds. Finally, our results showed that the construction of an artificial bone did not necessarily require the precise structuring of the scaffold, because both types of macro-topographic PLA scaffolds were sufficient enough to induce spontaneous DPSC osteogenic differentiation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 174-186, 2019.

A Novel Murine Model of Parvovirus Associated Dilated Cardiomyopathy Induced by Immunization with VP1-Unique Region of Parvovirus B19.

Background. Parvovirus B19 (B19V) is a common finding in endomyocardial biopsy specimens from myocarditis and dilated cardiomyopathy patients. However, current understanding of how B19V is contributing to cardiac damage is rather limited due to the lack of appropriate mice models. In this work we demonstrate that immunization of BALB/c mice with the major immunogenic determinant of B19V located in the unique sequence of capsid protein VP1 (VP1u) is an adequate model to study B19V associated heart damage. Methods and Results. We immunized mice in the experimental group with recombinant VP1u; immunization with cardiac myosin derived peptide served as a positive reference and phosphate buffered saline served as negative control. Cardiac function and dimensions were followed echocardiographically 69 days after immunization. Progressive dilatation of left ventricle and decline of ejection fraction were observed in VP1u- and myosin-immunized mice. Histologically, severe cardiac fibrosis and accumulation of heart failure cells in lungs were observed 69 days after immunization. Transcriptomic profiling revealed ongoing cardiac remodeling and immune process in VP1u- and myosin-immunized mice. Conclusions. Immunization of BALB/c mice with VP1u induces dilated cardiomyopathy in BALB/c mice and it could be used as a model to study clinically relevant B19V associated cardiac damage.