Early Osteogenic-Induced Adipose-Derived Stem Cells and Canine Bone Regeneration Potential Analyzed Using Biodegradable Scaffolds.

The complex process of bone regeneration is influenced by factors such as inflammatory responses, tissue interactions, and progenitor cells. Currently, multiple traumas can interfere with fracture healing, causing the prolonging or failure of healing. In these cases, bone grafting is the most effective treatment. However, there are several drawbacks, such as morbidity at the donor site and availability of suitable materials. Advantages have been provided in this field by a variety of stem cell types. Adipose-derived stem cells (ASCs) show promise. In the radiological examination of this study, it was confirmed that the C/S group showed faster regeneration than the other groups, and Micro-CT also showed that the degree of bone formation in the defect area was highest in the C/S group. Compared to the control group, the change in cortical bone area in the defect area decreased in the sham group (0.874), while it slightly increased in the C/S group (1.027). An increase in relative vascularity indicates a decrease in overall bone density, but a weak depression filled with fibrous tissue was observed outside the compact bone. It was confirmed that newly formed cortical bone showed a slight difference in bone density compared to surrounding normal bone tissue due to increased distribution of cortical bone. In this study, we investigated the effect of bone regeneration by ADMSCs measured by radiation and pathological effects. These data can ultimately be applied to humans with important clinical applications in various bone diseases, regenerative, and early stages of formative differentiation.

Multiple time-dependent pathophysiological changes in a rabbit model of high-fat diet-induced hyperlipidemia.

High-fat diets (HFD) adversely affect organ systems. Several studies have examined HFD-related disorders in animals but only in a few organs and time points. Herein, we evaluated disease development with time-dependent HFD-induced pathological, cardiovascular, and morphological changes in rabbits with lipid metabolism similar to that in humans for 9 weeks. The body weights and waist ratio of the HFD group were higher than those in the control group. HFD significantly increased the total cholesterol, low-density lipoprotein, high-density lipoprotein, and phospholipid levels after 3 weeks. Liver enzyme levels increased with hepatomegaly, steatosis, and fibrosis after 3 or 6 weeks. RBCs and hemoglobin decreased, while platelets increased in the HFD group with atherosclerosis and inflammatory cell infiltration in the aorta after 6 weeks. Ejection fraction and fractional shortening values decreased in the HFD group after 9 weeks. Creatinine increased with glomerulosclerosis in the kidneys of the HFD groups after 3 weeks, indicating renal dysfunction. Lipid accumulation was found in the pancreas after 9 weeks. Lipid accumulation and hypertrophy were observed in the adrenal glands after 3 weeks. Overall, our findings provide global reference data on the time-dependent effects of HFD on the body and may serve as a guide for future HFD risk prevention.

The role of Sirtuin 2 in sustaining functional integrity of the liver.

AIM: Sirtuin 2 (SIRT2) is a NAD+-dependent deacetylase involved in various biological functions via deacetylation of proteins, including histone protein. Hepatic fat accumulation from aging and excess caloric intake contribute to development of non-alcoholic fatty liver disease. The study aim was to elucidate the role of SIRT2 in lipid metabolism homeostasis. MATERIALS AND METHODS: SIRT2+/+ (C57BL/6) and SIRT2-/- were randomly assigned to normal diet or high-fat diet (HFD) groups and fed for 6 weeks. Histological features of the livers were evaluated by hematoxylin and eosin and Masson's trichrome staining, and the levels of selected factors were determined by quantitative reverse transcription-polymerase chain reaction and western blot analysis. KEY FINDINGS: Although the SIRT2-/- mice were viable, their livers exhibited higher glycogen accumulation, and skeletal muscle showed features of increased metabolic demand. The SIRT2-/- mice attenuated HFD-induced weight gain, visceral adipose tissue formation, and fat accumulation in the liver in which the expressions of genes involved in metabolic substrate transport were modified. Additionally, the hepatocellular senescence and upregulated cell-cycle factors upon HFD intake in SIRT2-/- livers suggested a role of SIRT2 in gene expression during abnormal metabolism. Moreover, the fibrotic phenotype of liver tissue without fat accumulation and the increased expression of genes involved in liver fibrosis in the HFD-fed SIRT2-/- mice indicated that SIRT2 had a role in hepatocyte and hepatic stellate cell activation. SIGNIFICANCE: Our results indicated that SIRT2 has a critical role in regulating lipid metabolic homeostasis and in sustaining liver integrity by modulating related gene expression.

Effects of losartan and l-serine in a mouse liver fibrosis model.

Hepatic fibrosis is a common liver disease caused by excessive collagen deposition in the liver. Since liver transplantation is the only current treatment for cirrhosis with worsened fibrosis, a new strategy to develop anti-fibrosis drugs with no adverse effects is necessary. In recent studies, amino acids have been applied as a type of therapy in various fields. l-serine plays a major role in antioxidant production via the maintenance of nicotinamide adenine dinucleotide phosphate hydride production in the mitochondria. l-serine may reduce fibrotic lesions in a mouse model of chronic liver injury. This study used 27 six-week-old C57BL/6 mice and injected them three times a week for eight weeks with carbon tetrachloride (CCl4) (1.5 mg/kg, 10% v/v CCl4 in olive oil) to create a hepatic fibrosis mouse model. The mice, which weighed approximately 20-30 g, were randomly classified into four groups: 1) the olive oil group, which received intraperitoneal injection of olive oil (1.5 mg/kg, 3 times per week for 8 weeks); 2) the CCl4-only group; 3) the CCl4 + losartan (10 mg/kg, PO, 5 days on, weekend off for 8 weeks) group; and 4) the CCl4 + l-serine (100 g/L, free access for 8 weeks) group. Hematoxylin and eosin staining and Masson's trichrome staining showed reduced inflammatory cell deposition and collagen deposition in the liver tissue in the l-serine supplemented group. l-serine was found to reduce the spread of hepatic fibrosis and has potential use in clinical settings. Based on these histopathological observations, l-serine is a potential anti-fibrosis drug.

Nogo-A Is Critical for Pro-Inflammatory Gene Regulation in Myocytes and Macrophages.

Nogo-A (Rtn 4A), a member of the reticulon 4 (Rtn4) protein family, is a neurite outgrowth inhibitor protein that is primarily expressed in the central nervous system (CNS). However, previous studies revealed that Nogo-A was upregulated in skeletal muscles of Amyotrophic lateral sclerosis (ALS) patients. Additionally, experiments showed that endoplasmic reticulum (ER) stress marker, C/EBP homologous protein (CHOP), was upregulated in gastrocnemius muscle of a murine model of ALS. We therefore hypothesized that Nogo-A might relate to skeletal muscle diseases. According to our knocking down and overexpression results in muscle cell line (C2C12), we have found that upregulation of Nogo-A resulted in upregulation of CHOP, pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, while downregulation of Nogo-A led to downregulation of CHOP, IL-6 and TNF-α. Immunofluorescence results showed that Nogo-A and CHOP were expressed by myofibers as well as tissue macrophages. Since resident macrophages share similar functions as bone marrow-derived macrophages (BMDM), we therefore, isolated macrophages from bone marrow to study the role of Nogo-A in activation of these cells. Lipopolysaccharide (LPS)-stimulated BMDM in Nogo-KO mice showed low mRNA expression of CHOP, IL-6 and TNF-α compared to BMDM in wild type (WT) mice. Interestingly, Nogo knockout (KO) BMDM exhibited lower migratory activity and phagocytic ability compared with WT BMDM after LPS treatment. In addition, mice experiments data revealed that upregulation of Nogo-A in notexin- and tunicamycin-treated muscles was associated with upregulation of CHOP, IL-6 and TNF-α in WT group, while in Nogo-KO group resulted in low expression level of CHOP, IL-6 and TNF-α. Furthermore, upregulation of Nogo-A in dystrophin-deficient (mdx) murine model, myopathy and Duchenne muscle dystrophy (DMD) clinical biopsies was associated with upregulation of CHOP, IL-6 and TNF-α. To the best of our knowledge, this is the first study to demonstrate Nogo-A as a regulator of inflammation in diseased muscle and bone marrow macrophages and that deletion of Nogo-A alleviates muscle inflammation and it can be utilized as a therapeutic target for improving muscle diseases.

Nogo-A regulates myogenesis via interacting with Filamin-C.

Among the three isoforms encoded by Rtn4, Nogo-A has been intensely investigated as a central nervous system inhibitor. Although Nogo-A expression is increased in muscles of patients with amyotrophic lateral sclerosis, its role in muscle homeostasis and regeneration is not well elucidated. In this study, we discovered a significant increase in Nogo-A expression in various muscle-related pathological conditions. Nogo-/- mice displayed dystrophic muscle structure, dysregulated muscle regeneration following injury, and altered gene expression involving lipid storage and muscle cell differentiation. We hypothesized that increased Nogo-A levels might regulate muscle regeneration. Differentiating myoblasts exhibited Nogo-A upregulation and silencing Nogo-A abrogated myoblast differentiation. Nogo-A interacted with filamin-C, suggesting a role for Nogo-A in cytoskeletal arrangement during myogenesis. In conclusion, Nogo-A maintains muscle homeostasis and integrity, and pathologically altered Nogo-A expression mediates muscle regeneration, suggesting Nogo-A as a novel target for the treatment of myopathies in clinical settings.

Differentiation of equine induced pluripotent stem cells into mesenchymal lineage for therapeutic use.

In previous work, we established an equine induced pluripotent stem cell line (E-iPSCs) from equine adipose-derived stem cells (ASCs) using a lentiviral vector encoding four transcription factors: Oct4, Sox2, Klf4, and c-Myc. In the current study, we attempted to differentiate these established E-iPSCs into mesenchymal stem cells (MSCs) by serial passaging using MSC-defined media for stem cell expansion. Differentiation of the MSCs was confirmed by analyzing expression levels of the MSC surface markers CD44 and CD29, and the pluripotency markers Nanog and Oct4. Results indicated that the E-iPSC-derived MSCs (E-iPSC-MSCs) retained the characteristics of MSCs, including the ability to differentiate into chondrogenic, osteogenic, or myogenic lineages. E-iPSC-MSCs were rendered suitable for therapeutic use by inhibiting immune rejection through exposure to transforming growth factor beta 2 (TGF-ß2) in culture, which down-regulated the expression of major histocompatibility complex class I (MHC class I) proteins that cause immune rejection if they are incompatible with the MHC antigen of the recipient. We reported 16 cases of E-iPSC-MSC transplantations into injured horses with generally positive effects, such as reduced lameness and fraction lines. Our findings indicate that E-iPSC-MSCs can demonstrate MSC characteristics and be safely and practically used in the treatment of musculoskeletal injuries in horses.