Secondary bile acids in portal blood contribute to liver regeneration in a rat model of partial hepatectomy.

Gut metabolites via the portal vein affect several liver functions, including regeneration. Here, we investigated gut microbiota-derived metabolites in portal and peripheral serum during liver regeneration. We developed rat models of 70% partial hepatectomy (PHx) with and without prior gut microbiota modulation by three-week antibiotic (Abx) treatment. Sham without Abx were used as controls and compared to sham with Abx. Liver regeneration at day 2 following PHx was assessed by expression of proliferating cell nuclear antigen (PCNA) protein in liver tissues and cyclin genes in primary hepatocytes. High pressure liquid chromatography-mass spectrometry (HPLC-MS) based portal and peripheral venous serum metabolomics was performed to identify differentially altered metabolites (DAMs). Compared to controls, rat livers at day 2 post-PHx showed significant upregulation in the average number of PCNA-positive cells, which positively correlated with the expression of cell cycle genes in hepatocytes. In Abx-treated PHx, we observed reduced PCNA-positivity and downregulation in gene expression of various cyclins in hepatocytes compared to PHx. We identified 224 DAMs between controls vs PHx and 189 DAMs between Abx-treated PHx vs PHx in portal serum. Many common DAMs showed opposite expression trends in PHx vs controls and then Abx+PHx vs PHx in portal serum, such as sphingosine-1-phosphate and deoxycholic acid. In vitro studies with deoxycholic acid demonstrated that it enhanced the viability and proliferation of primary hepatocytes and hepatocyte organoids. The study underscores the critical role of deoxycholic acid in portal blood in enhancing hepatocyte proliferation and subsequently, liver regeneration.

Comparative Evaluation of Effect of TENS on Controlling Pain Associated with Orthodontic Separator Placement-A Clinical Study.

Aim: To assess and contrast the efficacy of transcutaneous electrical nerve stimulation (TENS) therapy and topical anesthetic gel in reducing the level of pain associated with the placement of orthodontic separators. Materials and Methods: A cohort of 40 individuals, aged between 16 and 20 years, who were in need of fixed orthodontic treatment, was chosen for the study. Elastomeric separators were positioned bilaterally adjacent to the first molars in all participants, who were subsequently separated into two distinct cohorts. Those in group I received topical anesthetic gel, while those in group II underwent TENS therapy. After the placement of separators, the assessment of pain was conducted using the Visual Analog Scale (VAS) at specific intervals: Immediately after placement, 6, 12, 24, and 48 hours. Result: TENS group showed a significantly lower VAS score compared to the local anesthetic gel group at immediate, 6 hours, and 12 hours. Conclusion: Following the placement of elastomeric separators, it was found that TENS was more successful at reducing pain.

A Review on Cone-Beam Computed Tomography and its Application in Dentistry.

Cone-beam computed tomography (CBCT), which has benefits over traditional radiography in this age of cutting-edge technology, has become increasingly popular in oral radiology. The utilization of CBCT is experiencing significant growth in several dental specialties, mainly for diagnostic purposes and treatment planning. Image acquisition and 3D imaging have helped the clinical use of cone-beam technology. The equipment has a user-friendly interface, minimum image distortion, and images compatible with many planning and simulation software applications. The present literature review aims to discuss CBCT and its various applications in dentistry.

Comparative Evaluation of Two Different Topical Anesthetic Agents against Pain Associated with Intraoral Injections in Pediatric Dental Patients.

Aim: This study aimed to examine the effectiveness of two topical anesthetics in minimizing the discomfort related to intraoral injections. Material and Method: For the study, 40 healthy kids between the ages of 6 and 10 were chosen. To the injection site, two topical anesthetic products were applied: lignocaine hydrochloride gel and benzocaine 20% gel. The effectiveness of the topical agents was evaluated using a four-point scale, which was previously used by Topçuoglu and colleagues to evaluate pain. Result: A significant difference between the lignocaine group (2.1 ± 0.5) and the benzocaine group (1.2 ± 0.6) was found on a four-point scale (P < 0.05). Conclusion: Benzocaine demonstrates a higher degree of efficacy in comparison with lignocaine. However, additional research is necessary to ascertain the result of this study.

Investigation of the Surface Roughness and Hardness of Different Denture Teeth Materials: An In vitro Study.

Background: Surface roughness and hardness are key factors that influence the clinical performance and durability of denture teeth. Understanding variations in these properties among different denture teeth materials can assist in selecting the most suitable materials for optimal patient outcomes. This study aimed to investigate the surface roughness and hardness of four commonly used denture teeth materials: acrylic resin, composite resin, porcelain, and nanohybrid composite. Materials and Methods: Ten specimens were prepared for each denture teeth material, resulting in a total of 40 specimens. Surface roughness was assessed using a profilometer, and measurements were recorded in micrometers (µm). Hardness was determined using a Vickers hardness tester, and results were expressed as Vickers hardness numbers (VHN). The surface roughness and hardness data were analyzed using appropriate statistical tests (e.g., analysis of variance), with significance set at P < 0.05. Results: The results revealed significant differences in both surface roughness and hardness among the different denture teeth materials (P < 0.05). Acrylic resin exhibited the highest surface roughness (mean ± standard deviation: 3.45 ± 0.78 µm) and the lowest hardness (mean ± standard deviation: 45.6 ± 2.3 VHN). Composite resin demonstrated intermediate values of surface roughness (mean ± standard deviation: 1.87 ± 0.54 µm) and hardness (mean ± standard deviation: 65.2 ± 3.9 VHN). Porcelain demonstrated the smoothest surface (mean ± standard deviation: 0.94 ± 0.28 µm) and the highest hardness (mean ± standard deviation: 78.5 ± 4.1 VHN). Nanohybrid composite displayed surface roughness and hardness values similar to composite resin. Conclusion: This study demonstrated significant variations in surface roughness and hardness among the different denture teeth materials evaluated. Acrylic resin exhibited the roughest surface and lowest hardness, while porcelain demonstrated the smoothest surface and highest hardness. Composite resin and nanohybrid composite exhibited intermediate values. These findings provide valuable insights for prosthodontic practitioners in selecting denture teeth materials based on specific clinical requirements, aiming to achieve optimal aesthetics, reduced plaque accumulation, and improved wear resistance.

Liver Extracellular Matrix-Based Nanofiber Scaffolds for the Culture of Primary Hepatocytes and Drug Screening.

Immortalized liver cell lines and primary hepatocytes are currently used as in vitro models for hepatotoxic drug screening. However, a decline in the viability and functionality of hepatocytes with time is an important limitation of these culture models. Advancements in tissue engineering techniques have allowed us to overcome this challenge by designing suitable scaffolds for maintaining viable and functional primary hepatocytes for a longer period of time in culture. In the current study, we fabricated liver-specific nanofiber scaffolds with polylactic acid (PLA) along with a decellularized liver extracellular matrix (LEM) by the electrospinning technique. The fabricated hybrid PLA-LEM scaffolds were more hydrophilic and had better swelling properties than the PLA scaffolds. The hybrid scaffolds had a pore size of 38 ± 8 µm and supported primary rat hepatocyte cultures for 10 days. Increased viability (2-fold increase in the number of live cells) and functionality (5-fold increase in albumin secretion) were observed in primary hepatocytes cultured on the PLA-LEM scaffolds as compared to those on conventional collagen-coated plates on day 10 of culture. A significant increase in CYP1A2 enzyme activity was observed in hepatocytes cultured on PLA-LEM hybrid scaffolds in comparison to those on collagen upon induction with phenobarbital. Drugs like acetaminophen and rifampicin showed the highest toxicity in hepatocytes cultured on hybrid scaffolds. Also, the lethal dose of these drugs in rodents was accurately predicted as 1.6 g/kg and 594 mg/kg, respectively, from the corresponding IC50 values obtained from drug-treated hepatocytes on hybrid scaffolds. Thus, the fabricated liver-specific electrospun scaffolds maintained primary hepatocyte viability and functionality for an extended period in culture and served as an effective ex vivo drug screening platform to predict an accurate in vivo drug-induced hepatotoxicity.

Recombinant VEGF-C (Cys156Ser) improves mesenteric lymphatic drainage and gut immune surveillance in experimental cirrhosis.

Background & Aims: Lymphatic vessels (LVs) are crucial for maintaining abdominal fluid homoeostasis and immunity. In cirrhosis, mesenteric LVs (mLVs) are dilated and dysfunctional. Given the established role of vascular endothelial growth factor-C (VEGF-C) in improving LVs, we hypothesised that VEGF-C treatment could ameliorate the functions of mLVs in cirrhosis. Methods: In this study, we developed a nanoformulation comprising LV-specific growth factor, recombinant human VEGF-C (Cys156Ser) protein (E-VEGF-C) and delivered it orally in different models of rat cirrhosis to target mLVs. Cirrhotic rats were given nanoformulation without VEGF-C served as vehicles. Drainage of mLVs was analysed using tracer dye. Portal and systemic physiological assessments and computed tomography were performed to measure portal pressures and ascites. Gene expression and permeability of primary mesenteric lymphatic endothelial cells (LyECs) was studied. Immune cells in mesenteric lymph nodes (MLNs) were quantified by flow cytometry. Endogenous and exogenous gut bacterial translocation to MLNs was examined. Results: In cirrhotic rats, mLVs were dilated and leaky with impaired drainage. Treatment with E-VEGF-C induced proliferation of mLVs, reduced their diameter, and improved functional drainage. Ascites and portal pressures were significantly reduced in E-VEGF-C rats compared with vehicle rats. In MLNs of E-VEGF-C animals, CD8+CD134+ T cells were increased, whereas CD25+ regulatory T cells were decreased. Both endogenous and exogenous bacterial translocation were limited to MLNs in E-VEGF-C rats with reduced levels of endotoxins in ascites and blood in comparison with those in vehicle rats. E-VEGF-C treatment upregulated the expression of vascular endothelial-cadherin in LyECs and functionally improved the permeability of these cells. Conclusions: E-VEGF-C treatment ameliorates mesenteric lymph drainage and portal pressure and strengthens cytotoxic T-cell immunity in MLNs in experimental cirrhosis. It may thus serve as a promising therapy to manage ascites and reduce pathogenic gut bacterial translocation in cirrhosis. Impact and Implications: A human recombinant pro-lymphangiogenic growth factor, VEGF-C, was encapsulated in nanolipocarriers (E-VEGF-C) and orally delivered in different models of rat liver cirrhosis to facilitate its gut lymphatic vessel uptake. E-VEGF-C administration significantly increased mesenteric lymphatic vessel proliferation and improved lymph drainage, attenuating abdominal ascites and portal pressures in the animal models. E-VEGF-C treatment limited bacterial translocation to MLNs only with reduced gut bacterial load and ascitic endotoxins. E-VEGF-C therapy thus holds the potential to manage ascites and portal pressure and reduce gut bacterial translocation in patients with cirrhosis.

Primary Hepatocyte Isolation and Cultures: Technical Aspects, Challenges and Advancements.

Hepatocytes are differentiated cells that account for 80% of the hepatic volume and perform all major functions of the liver. In vivo, after an acute insult, adult hepatocytes retain their ability to proliferate and participate in liver regeneration. However, in vitro, prolonged culture and proliferation of viable and functional primary hepatocytes have remained the major and the most challenging goal of hepatocyte-based cell therapies and liver tissue engineering. The first functional cultures of rat primary hepatocytes between two layers of collagen gel, also termed as the "sandwich cultures", were reported in 1989. Since this study, several technical developments including choice of hydrogels, type of microenvironment, growth factors and culture conditions, mono or co-cultures of hepatocytes along with other supporting cell types have evolved for both rat and human primary hepatocytes in recent years. All these improvements have led to a substantial improvement in the number, life-span and hepatic functions of these cells in vitro for several downstream applications. In the current review, we highlight the details, limitations and prospects of different technical strategies being used in primary hepatocyte cultures. We discuss the use of newer biomaterials as scaffolds for efficient culture of primary hepatocytes. We also describe the derivation of mature hepatocytes from other cellular sources such as induced pluripotent stem cells, bone marrow stem cells and 3D liver organoids. Finally, we also explain the use of perfusion-based bioreactor systems and bioengineering strategies to support the long-term function of hepatocytes in 3D conditions.

Chemically Modified Dipeptide Based Hydrogel Supports Three-Dimensional Growth and Functions of Primary Hepatocytes.

A huge shortage of organ donors, particularly in the case of liver, has necessitated the development of alternative therapeutic strategies. Primary hepatocytes (pHCs) transplantation has made a considerable transition from bench to bedside, but the short-term viability and functionality of pHCs in in vitro limit their use for clinical applications. Different cell culture strategies are required to maintain the proliferation of pHCs for extended periods. Here, we described the formation of a hybrid scaffold based on a modified dipeptide for the culture of pHCs. First, the dipeptide (Dp), isoleucine-α,ß-dehydrophenylalanine (IΔF) was synthesized, purified, and fully characterized. IΔF readily formed a highly stable hydrogel, which was also characterized by CD, TEM, and thioflavin T assay. The addition of soluble liver extracellular matrix (sLEM) to the dipeptide readily formed a hybrid scaffold that was characterized by TEM, and its mechanical strength was determined by rheology experiments. The hybrid scaffold was translucent, biocompatible, and proteolytically stable and, with its mechanical strength, closely mimicked that of the native liver. LEM1-Dp matrix exhibited high biocompatibility in the readily available adherent liver cell line Huh7 and primary rat hepatocyte cells (pHCs). pHCs cultured on LEM1-Dp matrix also maintained significantly higher cell viability and an escalated expression of markers related to the hepatocytes such as albumin as compared to that observed in cells cultured on collagen type I (Col I)-coated substrate plate (col-TCTP). Z-stacking of confocal laser microscopy's volume view clearly indicated pHCs seeded on top of the hydrogel matrix migrated toward the Z direction showing 3D growth. Our results indicated that low molecular weight dipeptide hydrogel along with sLEM can resemble biomimetic 3D-like microenvironments for improved pHCs proliferation, differentiation, and function. This hybrid scaffold is also easy to scale up, which makes it suitable for several downstream applications of hepatocytes, including drug development, pHCs transplantation, and liver regeneration.

Immunonano-Lipocarrier-Mediated Liver Sinusoidal Endothelial Cell-Specific RUNX1 Inhibition Impedes Immune Cell Infiltration and Hepatic Inflammation in Murine Model of NASH.

BACKGROUND: Runt-related transcription factor (RUNX1) regulates inflammation in non-alcoholic steatohepatitis (NASH). METHODS: We performed in vivo targeted silencing of the RUNX1 gene in liver sinusoidal endothelial cells (LSECs) by using vegfr3 antibody tagged immunonano-lipocarriers encapsulated RUNX1 siRNA (RUNX1 siRNA) in murine models of methionine choline deficient (MCD) diet-induced NASH. MCD mice given nanolipocarriers-encapsulated negative siRNA were vehicle, and mice with standard diet were controls. RESULTS: Liver RUNX1 expression was increased in the LSECs of MCD mice in comparison to controls. RUNX1 protein expression was decreased by 40% in CD31-positive LSECs of RUNX1 siRNA mice in comparison to vehicle, resulting in the downregulation of adhesion molecules, ICAM1 expression, and VCAM1 expression in LSECs. There was a marked decrease in infiltrated T cells and myeloid cells along with reduced inflammatory cytokines in the liver of RUNX1 siRNA mice as compared to that observed in the vehicle. CONCLUSIONS: In vivo LSEC-specific silencing of RUNX1 using immunonano-lipocarriers encapsulated siRNA effectively reduces its expression of adhesion molecules, infiltrate on of immune cells in liver, and inflammation in NASH.

Non-matrigel scaffolds for organoid cultures.

Organoids are three-dimensional cell cultures mostly from tissue-resident or embryonic stem cells (one or multiple) on hydrogels along with defined growth factors. Currently, matrigel is the most commonly employed matrix for 3D organoid cultures. However, certain undesirable attributes of matrigel have paved the way for several other natural and synthetic hydrogel scaffolds for organoid cultures. In this review, we discuss the constraints of matrigel and describe other alternative scaffolds that have been used for organoid cultures. Given the potential of organoids in a plethora of therapeutic and pharmaceutical applications, it is indeed imperative to develop defined and customized hydrogels other than the matrigel.

Cirrhotic Endothelial Progenitor Cells Enhance Liver Angiogenesis and Fibrosis and Aggravate Portal Hypertension in Bile Duct-Ligated Cirrhotic Rats.

BACKGROUND: Circulating cirrhotic endothelial progenitor cells (EPC) interact with both liver sinusoidal endothelial cells (LSEC) and hepatic stellate cells (HSC) and promote angiogenesis in vitro. This study evaluated the effect of cirrhotic and control EPCs on hepatic angiogenesis, microcirculation, and fibrosis in vivo in rat models of cirrhosis. METHODOLOGY: Animal models of cirrhosis were prepared by bile duct ligation (BDL). Circulating EPCs isolated from healthy human and cirrhotic blood were characterized by flow cytometry, cultured and administered through the tail vein in BDL rats after 2 weeks of ligation. The cells were given thrice a week for 2 weeks. The untreated group of BDL rats received only saline. Fibrosis was evaluated by Masson's trichrome staining. Dedifferentiated LSECs were identified by the expression of CD31, and activated HSCs were marked as alpha-SMA-positive cells and were studied by immunohistochemistry and western blotting in saline-, healthy EPC-, and cirrhotic EPC-treated rats. In vivo, hepatic and systemic hemodynamic parameters were evaluated. Liver functions were evaluated. RESULTS: In comparison to controls, BDL rats revealed an increase of fibrosis and angiogenesis. Among the treated rats, cirrhotic EPC-treated rats had increased fibrosis grade as compared to healthy EPC-treated and saline-treated rats. There was an increase of both fibrosis and angiogenesis markers, alpha-SMA and CD31 in cirrhotic EPC-treated rats as compared to healthy EPC-treated and saline-treated rats in immunohistochemistry and western blot studies. Cirrhotic EPC-treated BDL rats had high portal pressure and portal blood flow with significantly elevated hepatic vascular resistance in comparison with healthy EPC- and saline-treated BDL animals, without significant differences in mean arterial pressure. Cirrhotic EPC-treated BDL rats also showed a substantial increase in the hepatic expression of angiogenic receptors, VEGFR2 and CXCR4 in comparison with saline-treated rats. CONCLUSION: The study suggests that transplantation of cirrhotic EPCs enhances LSEC differentiation and angiogenesis, activates HSCs and worsens fibrosis, thus resulting in hepatic hemodynamic derangements in BDL-induced cirrhosis.

Endothelial progenitor cells from aged subjects display decreased expression of sirtuin 1, angiogenic functions, and increased senescence.

Studies have demonstrated that aging is associated with a substantial decline in numbers and angiogenic activity of endothelial progenitor cells (EPCs). In view of senescence being an important regulator of age-related cell survival and function, in the current study, we correlated EPCs numbers and functions with their senescence status and mechanisms in young and elderly subjects. Healthy young subjects (n = 30, below 60 y) and old subjects (n = 30, equal to or above 60 y) participated in the study. Subjects had no significant disease or risk factors of disease and aging was the only risk factor in the aged subjects. Enumeration of CD34-vegfr2 dual positive EPCs was performed. The ex vivo culture of EPCs was done to study colony formation, migration, and senescence-associated beta-galactosidase activity. The expression of cell cycle and senescence regulatory proteins including, p53, p21, and sirtuin 1 (SIRT1), a deacetylase protein was studied in cultured EPCs by RT-PCR and immunofluorescence staining. In vivo proliferation, ex vivo colonies, migration, and secretory ability of EPCs was significantly higher in young subjects than that in elderly subjects. EPCs in old subjects showed enhanced senescence and decreased expression of SIRT1 in comparison to that observed in young subjects. An inhibition of SIRT1 in EPCs of young subjects led to significant increase in senescence and reduction of cell differentiation. The study suggests that EPCs have decreased proliferation and functions in aged subjects due to increased senescence which may be attributable to decreased expression of SIRT1.