Adipose Stem Cell-Seeded Decellularized Porcine Pericardium: A Promising Functional Biomaterial to Synergistically Restore the Cardiac Functions Post-Myocardial Infarction.

Myocardial infarction (MI) is a serious cardiovascular disease as the leading cause of death globally. Hence, reconstruction of the cardiac tissue comes at the forefront of strategies adopted to restore heart functions following MI. In this investigation, we studied the capacity of rat adipose-derived mesenchymal stem cells (r-AdMSCs) and decellularized porcine pericardium (DPP) to restore heart functions in MI animals. MI was induced in four different groups, three of which were treated either using DPP (MI-DPP group), stem cells (MI-SC group), or both (MI-SC/DPP group). Cardiac functions of these groups and the Sham group were evaluated using echocardiography, the intraventricular pressure gradient (IVPG) on weeks 2 and 4, and intraventricular hemodynamics on week 4. On day 31, the animals were euthanized for histological analysis. Echocardiographic, IVPG and hemodynamic findings indicated that the three treatment strategies shared effectively in the regeneration process. However, the MI-SC/DPP group had a unique synergistic ability to restore heart functions superior to the other treatment protocols. Histology showed that the MI-SC/DPP group presented the lowest (p < 0.05) degeneration score and fibrosis % compared to the other groups. Conclusively, stem cell-seeded DPP is a promising platform for the delivery of stem cells and restoration of heart functions post-MI.

Evaluation of the efficacy of mitochondrial fission inhibitor (Mdivi-1) using non-alcoholic steatohepatitis (NASH) liver organoids.

Non-alcoholic steatohepatitis (NASH) is known to progress to cirrhosis and hepatocellular carcinoma in some patients. Although NASH is associated with abnormal mitochondrial function related to lipid metabolism, mechanisms for the development and effective treatments are still unclear. Therefore, new approaches to elucidate the pathophysiology are needed. In the previous study, we generated liver organoids from different stages of NASH model mice that could recapitulate the part of NASH pathology. In the present study, we investigated the relationship between mitochondrial function and NASH disease by comparing NASH liver organoids (NLO) and control liver organoids (CLO). Compared with CLO, mitochondrial and organoid morphology was abnormal in NLO, with increased expression of mitochondrial mitogen protein, DRP1, and mitochondria-derived reactive oxygen species (ROS) production. Treatment of NLO with a DPR1 inhibitor, Mdivi-1 resulted in the improvement of morphology and the decreased expression of fibrosis-related markers, Col1a1 and Acta2. In addition, treatment of NASH model mice with Mdivi-1 showed a decrease in fatty liver. Mdivi-1 treatment also prevented fibrosis and ROS production in the liver. These results indicate that NLO undergoes enhanced metabolism and abnormal mitochondrial morphology compared with CLO. It was also suggested that Mdivi-1 may be useful as a therapeutic agent to ameliorate NASH pathology.

Derivation of a new model of lung adenocarcinoma using canine lung cancer organoids for translational research in pulmonary medicine.

Canine primary lung cancer (cPLC) is a rare malignant tumor in dogs, and exhibits poor prognosis. Effective therapeutic drugs against cPLC have not been established yet. Also, cPLC resembles human lung cancer in histopathological characteristics and gene expression profiles and thus could be an important research model for this disease. Three-dimensional organoid culture is known to recapitulate the tissue dynamics in vivo. We, therefore, tried to generate cPLC organoids (cPLCO) for analyzing the profiles of cPLC. After samples from cPLC and the corresponding normal lung tissue were collected, cPLCO were successfully generated, which recapitulated the tissue architecture of cPLC, expressed lung adenocarcinoma marker (TTF1), and exhibited tumorigenesis in vivo. The sensitivity of cPLCO to anti-cancer drugs was different among strains. RNA-sequencing analysis showed significantly upregulated 11 genes in cPLCO compared with canine normal lung organoids (cNLO). Moreover, cPLCO were enriched with the MEK-signaling pathway compared with cNLO. The MEK inhibitor, trametinib decreased the viability of several strains of cPLCO and inhibited the growth of cPLC xenografts. Collectively, our established cPLCO model might be a useful tool for identifying novel biomarkers for cPLC and a new research model for dog and human lung cancer.

Comparison of Bovine- and Porcine-Derived Decellularized Biomaterials: Promising Platforms for Tissue Engineering Applications.

Animal-derived xenogeneic biomaterials utilized in different surgeries are promising for various applications in tissue engineering. However, tissue decellularization is necessary to attain a bioactive extracellular matrix (ECM) that can be safely transplanted. The main objective of the present study is to assess the structural integrity, biocompatibility, and potential use of various acellular biomaterials for tissue engineering applications. Hence, a bovine pericardium (BP), porcine pericardium (PP), and porcine tunica vaginalis (PTV) were decellularized using a Trypsin, Triton X (TX), and sodium dodecyl sulfate (SDS) (Trypsin + TX + SDS) protocol. The results reveal effective elimination of the cellular antigens with preservation of the ECM integrity confirmed via staining and electron microscopy. The elasticity of the decellularized PP (DPP) was markedly (p < 0.0001) increased. The tensile strength of DBP, and DPP was not affected after decellularization. All decellularized tissues were biocompatible with persistent growth of the adipose stem cells over 30 days. The staining confirmed cell adherence either to the peripheries of the materials or within their matrices. Moreover, the in vivo investigation confirmed the biocompatibility and degradability of the decellularized scaffolds. Conclusively, Trypsin + TX + SDS is a successful new protocol for tissue decellularization. Moreover, decellularized pericardia and tunica vaginalis are promising scaffolds for the engineering of different tissues with higher potential for the use of DPP in cardiovascular applications and DBP and DPTV in the reconstruction of higher-stress-bearing abdominal walls.

Anti-cancer activity of Chaga mushroom (Inonotus obliquus) against dog bladder cancer organoids.

Despite its disadvantages, chemotherapy is still commonly used for the treatment of bladder cancer (BC). Developing natural supplements that can target cancer stem cells (CSCs) which cause drug resistance and distant metastasis is necessary. Chaga mushrooms are popular to have several health-promoting and anti-cancer potentials. Organoid culture can recapitulate tumor heterogeneity, epithelial environment, and genetic and molecular imprints of the original tissues. In the previous study, we generated dog bladder cancer organoids (DBCO) as a novel experimental model of muscle-invasive BCO. Therefore, the present study aimed to examine the anti-tumor potentials of Chaga mushroom extract (Chaga) against DBCO. Four strains of DBCO were used in the present study. Treatment with Chaga inhibited the cell viability of DBCO in a concentration-dependent way. Treatment of DBCO with Chaga has significantly arrested its cell cycle and induced apoptosis. Expression of bladder CSC markers, CD44, C-MYC, SOX2, and YAP1, declined in the Chaga-treated DBCO. Also, Chaga inhibited the phosphorylation of ERK in DBCO. Expression of downstream signals of ERK, C-MYC, and Cyclins (Cyclin-A2, Cyclin-D1, Cyclin-E1, and CDK4) was also inhibited by Chaga in DBCO. Interestingly, the combinational treatment of DBCO with Chaga and anti-cancer drugs, vinblastine, mitoxantrone, or carboplatin, showed a potentiating activity. In vivo, Chaga administration decreased tumor growth and weight of DBCO-derived xenograft in mice with the induction of necrotic lesions. In conclusion, Chaga diminished the cell viability of DBCO by inhibiting proliferation-related signals and stemness conditions as well as by arresting the cell cycle. Collectively, these data suggest the value of Chaga as a promising natural supplement that could potentiate the effect of adjuvant chemotherapy, lower its adverse effects, and thus, limit the recurrence and metastasis of BC.

Establishment of an experimental model of canine malignant mesothelioma organoid culture using a three-dimensional culture method.

Canine malignant mesothelioma (cMM) is a rare and drug-resistant malignant tumor. Due to few patients and experimental models, there have not been enough studies to demonstrate the pathogenesis of the disease and novel effective treatment for cMM. Since cMM resembles human MM (hMM) in histopathological characteristics, it is also considered a promising research model of hMM. Compared with conventional 2-dimensional (2D) culture methods, 3-dimensional (3D) organoid culture can recapitulate the properties of original tumor tissues. However, cMM organoids have never been developed. In the present study, we for the first time generated cMM organoids using the pleural effusion samples. Organoids from individual MM dogs were successfully generated. They exhibited the characteristics of MM and expressed mesothelial cell markers, such as WT-1 and mesothelin. The sensitivity to anti-cancer drugs was different in each strain of cMM organoids. RNA sequencing analysis showed cell adhesion molecule pathways were specifically upregulated in cMM organoids compared with their corresponding 2D cultured cells. Among these genes, the expression level of E-cadherin was drastically higher in the organoids than that in the 2D cells. In conclusion, our established cMM organoids might become a new experimental tool to provide new insights into canine and human MM therapy.

Establishment of a direct 2.5D organoid culture model using companion animal cancer tissues.

Like humans, cancer affects companion animals with similar genetic risks and incident rates. To improve treatment strategies for pet cancers, new research models are necessary. Patient-derived 3D organoid culture models are valuable and ensure the development of new effective therapies. In the previous study, we established a 3D organoid-derived 2.5D organoid culture model that recapitulated some characteristics of their parental 3D organoids. In the present study, we aimed to generate a 2.5D organoid culture model directly from cancer-diseased dogs and cats using special 2.5D media. The primary cultured cells in 2.5D media (direct 2.5D organoids) showed better attachment, growth, marker expression, and faster proliferation speed than those cultured in normal Dulbecco's Modified Eagle Medium media. The direct 2.5D organoids showed expression of each specific marker to their original cancer tissues and exhibited tumorigenesis in vivo. Moreover, the direct 2.5D organoids exhibited concentration-dependent responses to anti-cancer drugs, and different sensitivity profiles were shown among the strains. Our data suggest that the direct 2.5D organoid culture model might become a useful tool beyond 2D cell lines to study cancer biology in companion animals and could provide new platforms for screening the anti-cancer drugs.

Establishment of an experimental model of normal dog bladder organoid using a three-dimensional culture method.

Dog bladder cancer (BC) is mostly muscle-invasive (MI) with poor prognosis, and its pathogenesis is close to human MIBC. Three-dimensional (3D) organoid culture ensures novel knowledge on cancer diseases including BC. Recently, we have established dog BC organoids (BCO) using their urine samples. BCO recapitulated the epithelial structures, characteristics, and drug sensitivity of BC-diseased dogs. However, organoids from dog normal bladder epithelium are not established yet. Therefore, the present study aimed to establish dog normal bladder organoids (NBO) for further understanding the pathogenesis of dog BC and human MIBC. The established NBO underwent various analyzes including cell marker expressions, histopathological structures, cancer-related gene expression patterns, and drug sensitivity. NBO could be produced non-invasively with a continuous culturing and recapitulated the structures and characteristics of the dog's normal bladder mucosal tissues. Different drug sensitivities were observed in each NBO. The analysis of RNA sequencing revealed that several novel genes were changed in NBO compared with BCO. NBO showed a higher expression of p53 and E-cadherin, but a lower expression of MDM2 and Twist1 compared with BCO. These results suggest that NBO could be a promising experimental 3D model for studying the developmental mechanisms of dog BC and human MIBC.

Anti-cancer activity of amorphous curcumin preparation in patient-derived colorectal cancer organoids.

Despite its adverse effects, chemotherapy is generally used for the treatment of colorectal cancer (CRC). Development of supplement preparations targeting cancer stem cells (CSCs) that cause distant metastasis and drug resistance is required. Although curcumin is known to have anti-tumor, hepatoprotective, and hypoglycemic-like actions, its low water solubility, oral absorption, and bioavailability impede its therapeutic uses. Patient-derived organoid cultures can recapitulate heterogeneity, epithelial structures, and molecular imprints of their parental tissues. In the present study, anti-carcinogenic properties of amorphous curcumin (AC), a compound with improved solubility and bioavailability, were evaluated against human CRC organoids. Treatment with AC inhibited the cell viability of CRC organoids in a concentration-dependent manner. AC arrested the cell cycle of CRC organoids and induced apoptosis. AC inhibited phosphorylation of ERK. Expression of downstream signals of ERK, namely c-MYC and cyclin-D1, were inhibited. Expressions of CSC markers, CD44, LGR5, and CD133, were declined in the AC-treated CRC organoids. The combinational treatment of CRC organoids with AC and anti-cancer drugs, oxaliplatin, 5-FU, or irinotecan showed a synergistic activity. In vivo, AC decreased the tumor growth of CRC organoids in mice with the induction of necrotic lesions. In conclusion, AC diminished the cell viability of CRC organoids through the inhibition of proliferation-related signals and CSC marker expression in addition to arresting the cell cycle. Collectively, these data suggest the value of AC as a promising supplement that could be used in combination with anti-cancer drugs to prevent the recurrence and metastasis of CRC.

Incidence and risk factors for cardiac implantable electronic device infection in current clinical settings in a Japanese population: A 20-year single-center observational study.

BACKGROUND: The incidence of cardiac implantable electronic device (CIED) infection is increasing worldwide. However, data regarding this phenomenon in Japan and information on factors associated with developing CIED infection are limited. Our aim was to compare the incidence of CIED infection between pre-current (past 10-20 years) and current (past 10 years) clinical settings and to investigate risk factors for CIED infection in current clinical settings in a Japanese population. METHODS: This observational study included 1749 patients (age 77 ± 12 years, 824 males) who underwent a CIED-related procedure between August 1999 and July 2019 at our institution. We defined the pre-current and current clinical setting periods as August 1999-July 2009 (period I) and August 2009-July 2019 (period II), respectively. We compared the incidence rate of CIED infection between periods and evaluated the risk factors for CIED infection in period II by multivariate analysis. RESULTS: A CIED infection was identified in 0.7% (5/709 patients) and 1.7% (17/1040) of patients in periods I and II, respectively. Notably, the rate of late (>6 months since last procedure) CIED infection was significantly increased in period II (1.3% vs. 0.1%, p < 0.01), despite the rate of early infection (≤6 months) being comparable (0.4% vs. 0.6%, p = 0.58). On multiple logistic regression, revision [odds ratio (95% confidence interval): 5.2 (1.6-16.3), p = 0.005] and age [0.96, (0.93-0.99), p = 0.007] were identified as independent risk factors for CIED infection in period II. CONCLUSIONS: Our findings suggest that the increasing incidence of CIED infection in current clinical settings was due to an increase in late CIED infection. Furthermore, revision and younger age were identified as independent risk factors for CIED infection in current clinical settings. Our data indicate that clinicians should consider whether the merit of a procedure can overcome the risk of infection when planning revision or implantation in younger patients.

Oral absorption profiles of sulfonamides in Shiba goats: a comparison among sulfadimidine, sulfadiazine and sulfanilamide.

The oral pharmacokinetics of three sulfonamides, sulfadimidine (pKa 7.5), sulfadiazine (pKa 6.5) and sulfanilamide (pKa 10.5), with different rates of unionization in rumen juice, were compared in Shiba goats to clarify the relationship between drug absorption profiles after their oral administration as well as their degree of unionization in the rumen. Sulfonamides were administered either into the left jugular vein or orally to five male goats at doses of 10 mg/kg body weight, using a crossover design with at least a 3-week washout period. The Tmax of sulfadimidine, sulfadiazine and sulfanilamide reached 2.0 ± 1.2, 6.0 ± 0.0, and 7.8 ± 1.6 hr, respectively, after their oral administration, and this was followed by their slow elimination due to a slow rate of drug absorption from the gastrointestinal tract. The MAT and t1/2ka of sulfadiazine (13.2 ± 2.0 and 10.9 ± 1.08 hr) were significantly longer than those of sulfanilamide (9.09 ± 1.67 and 7.46 ± 1.70 hr) and sulfadimidine (7.52 ± 0.85 and 5.17 ± 0.66 hr). These results suggest that the absorption rates of highly unionized drugs (such as sulfanilamide and sulfadimidine) from the forestomach of goats may be markedly higher than less unionized ones (such as sulfadiazine). The mean oral bioavailability of sulfadiazine was high (83.9 ± 17.0%), whereas those of sulfadimidine and sulfanilamide were low (44.9 ± 16.4% and 49.2 ± 2.11%, respectively).

Four-peptide-nanotube bundle formation by self-assembling of cyclic tetra-ß-peptide using G-quartet motif.

A hydrophilic cyclic tetra-ß-peptide having guanine at the side chain (CPG) was synthesized to construct a four-peptide-nanotube bundle with the formation of guanine quadruplex (G-quartet). CPG was so soluble in water that molecular association of CPG was very limited in water. However, in the presence of potassium ion, circular dichroism spectral change showed initial molecular association due to the G-quartet formation followed by a slow growth into peptide nanotubes. Transmission electron microscope observation and electron diffraction analysis revealed that CPG self-assembled into a four-peptide-nanotube bundle with a uniform diameter of 7-8 nm.

Peptide nanotube composed of cyclic tetra-ß-peptide having polydiacetylene.

A cyclic tetra-ß-peptide composed of three ß-alanine residues and a L-ß-homolysine residue having 10,12-pentacosadiynoic amide at the side chain was synthesized. The cyclic tetra-ß-peptide formed peptide nanotubes in CDCl(3) . The diacetylene units along the peptide nanotubes were polymerized by UV irradiation to obtain peptide nanotubes with polydiacetylene along the nanotube. TEM and AFM observations of the polymerized cyclic tetra-ß-peptides confirmed that the peptide nanotube structure was preserved after polymerization.

Nanofiber formation of amphiphilic cyclic tri-beta-peptide.

A novel amphiphilic cyclic peptide composed of two beta-glucosamino acids and one trans-2-aminocyclohexylcarboxylic acid was synthesized and investigated on assembly formation. The cyclic tri-beta-peptide was self-assembled into rodlike crystals or nanofibers depending on preparative conditions. The rodlike crystals showed a layer spacing of 4.8 A along the long axis, and columnar spacings of 10.8 and 21.5 A by electron diffraction analysis along the short axis. The former confirms the columnar structure upon molecular stacking, and the latter indicates triple bundle formation of the columnar assemblies. Fourier transform infrared (FT-IR) measurement of the fibrous assembly showed formation of homogeneous hydrogen bonds among amide groups, also supporting the molecular stacking of cyclic beta-peptides. Straight nanofibers with uniform diameter were also uniquely obtained.