Systemic Dietary Hesperidin Modulation of Osteoclastogenesis,Bone Homeostasis and Periodontal Disease in Mice

This study aimed to evaluate the effects of hesperidin (HE) on in vitro osteoclastogenesis and dietary supplementation on mouse periodontal disease and femoral bone phenotype. RAW 264.7 cells were stimulated with RANKL in the presence or absence of HE (1, 100 or 500 µM) for 5 days, and evaluated by TRAP, TUNEL and Western Blot (WB) analyses. In vivo, C57BL/6 mice were given HE via oral gavage (125, 250 and 500 mg/kg) for 4 weeks. A sterile silk ligature was placed between the first and second right maxillary molars for 10 days and microcomputed tomography (μCT), histopathological and immunohistochemical evaluation were performed. Femoral bones subjected or not to dietary HE (500 mg/kg) for 6 and 12 weeks were evaluated using μCT. In vitro, HE 500 µM reduced formation of RANKL-stimulated TRAP-positive(+) multinucleated cells (500 µM) as well as c-Fos and NFATc1 protein expression (p < 0.05), markers of osteoclasts. In vivo, dietary HE 500 mg/kg increased the alveolar bone resorption in ligated teeth (p < 0.05) and resulted in a significant increase in TRAP+ cells (p < 0.05). Gingival inflammatory infiltrate was greater in the HE 500 mg/kg group even in the absence of ligature. In femurs, HE 500 mg/kg protected trabecular and cortical bone mass at 6 weeks of treatment. In conclusion, HE impaired in vitro osteoclastogenesis, but on the contrary, oral administration of a high concentration of dietary HE increased osteoclast numbers and promoted inflammation-induced alveolar bone loss. However, HE at 500 mg/kg can promote a bone-sparing effect on skeletal bone under physiological conditions.     

AT1 and AT2 Receptor Knockout Changed Osteonectin and Bone Density in Mice in Periodontal Inflammation Experimental Model

Background: The aim of this study was to evaluate the role of AT1 and AT2 receptors in a periodontal inflammation experimental model. Methods: Periodontal inflammation was induced by LPS/Porphyromonas gingivalis. Maxillae, femur, and vertebra were scanned using Micro-CT. Maxillae were analyzed histopathologically, immunohistochemically, and by RT-PCR. Results: The vertebra showed decreased BMD in AT1 H compared with WT H (p < 0.05). The femur showed increased Tb.Sp for AT1 H and AT2 H, p < 0.01 and p < 0.05, respectively. The Tb.N was decreased in the vertebra (WT H-AT1 H: p < 0.05; WT H-AT2 H: p < 0.05) and in the femur (WT H-AT1 H: p < 0.01; WT H-AT2 H: p < 0.05). AT1 PD increased linear bone loss (p < 0.05) and decreased osteoblast cells (p < 0.05). RANKL immunostaining was intense for AT1 PD and WT PD (p < 0.001). OPG was intense in the WT H, WT PD, and AT2 PD when compared to AT1 PD (p < 0.001). AT1 PD showed weak immunostaining for osteocalcin compared with WT H, WT PD, and AT2 PD (p < 0.001). AT1 H showed significantly stronger immunostaining for osteonectin in fibroblasts compared to AT2 H (p < 0.01). Conclusion: AT1 receptor knockout changed bone density, the quality and number of bone trabeculae, decreased the number of osteoblast cells, and increased osteonectin in fibroblasts.     

Early Bone Healing on Hydroxyapatite-Coated and Chemically-Modified Hydrophilic Implant Surfaces in an Ovine Model

Implant topography affects early peri-implant bone healing by changing the osteoconduction rate in the surrounding biological environment. Implant surfaces have been designed to promote faster and stronger bone formation for rapid and stable prosthesis loading. Early peri-implant bone healing has been observed with a sandblasted, acid-etched implant that was chemically modified to be hydrophilic (cmSLA). The present study investigates whether early peri-implant bone healing extends to a rough surface implant with a high crystalline hydroxyapatite surface (TSV MP-1 HA). Three implants were randomly placed in porous trabecular bone within both medial femoral condyles of 10 sheep. Early peri-implant bone stability was measured at 3- and 6-weeks healing time following implant insertion. Results indicated a similar implant stability quotient between the implants at insertion and over time. The significant increase over time of reverse torque values with respect to insertion torque (p < 0.001) did not differ between the implants. However, the bone-to-implant contact of TSV MP-1 HA was significantly higher than that of cmSLA implants at 6 weeks (p < 0.01). These data validate previous findings of a hydrophilic implant surface and extend the observation of early osseointegration to a rough surface implant in porous trabecular bone.     

The Effects of Nutrient Signaling Regulators in Combination with Phytocannabinoids on the Senescence-Associated Phenotype in Human Dermal Fibroblasts

Identifying effective anti-aging compounds is a cornerstone of modern longevity, aging, and skin-health research. There is considerable evidence of the effectiveness of nutrient signaling regulators such as metformin, resveratrol, and rapamycin in longevity and anti-aging studies; however, their potential protective role in skin aging is controversial. In light of the increasing appearance of phytocannabinoids in beauty products without rigorous research on their rejuvenation efficacy, we decided to investigate the potential role of phytocannabinoids in combination with nutrient signaling regulators in skin rejuvenation. Utilizing CCD-1064Sk skin fibroblasts, the effect of metformin, triacetylresveratrol, and rapamycin combined with phytocannabinoids on cellular viability, functional activity, metabolic function, and nuclear architecture was tested. We found triacetylresveratrol combined with cannabidiol increased the viability of skin fibroblasts (p < 0.0001), restored wound-healing functional activity (p < 0.001), reduced metabolic dysfunction, and ameliorated nuclear eccentricity and circularity in senescent fibroblasts (p < 0.01). Conversely, metformin with or without phytocannabinoids did not show any beneficial effects on functional activity, while rapamycin inhibited cell viability (p < 0.01) and the speed of wound healing (p < 0.001). Therefore, triacetylresveratrol and cannabidiol can be a valuable source of biologically active substances used in aging and more studies using animals to confirm the efficacy of cannabidiol combined with triacetylresveratrol should be performed.     

Increased Osteocyte Lacunae Density in the Hypermineralized Bone Matrix of Children with Osteogenesis Imperfecta Type I

Osteocytes are terminally differentiated osteoblasts embedded within the bone matrix and key orchestrators of bone metabolism. However, they are generally not characterized by conventional bone histomorphometry because of their location and the limited resolution of light microscopy. OI is characterized by disturbed bone homeostasis, matrix abnormalities and elevated bone matrix mineralization density. To gain further insights into osteocyte characteristics and bone metabolism in OI, we evaluated 2D osteocyte lacunae sections (OLS) based on quantitative backscattered electron imaging in transiliac bone biopsy samples from children with OI type I (n = 19) and age-matched controls (n = 24). The OLS characteristics were related to previously obtained, re-visited histomorphometric parameters. Moreover, we present pediatric bone mineralization density distribution reference data in OI type I (n = 19) and controls (n = 50) obtained with a field emission scanning electron microscope. Compared to controls, OI has highly increased OLS density in cortical and trabecular bone (+50.66%, +61.73%; both p < 0.001), whereas OLS area is slightly decreased in trabecular bone (−10.28%; p = 0.015). Correlation analyses show a low to moderate, positive association of OLS density with surface-based bone formation parameters and negative association with indices of osteoblast function. In conclusion, hyperosteocytosis of the hypermineralized OI bone matrix associates with abnormal bone cell metabolism and might further impact the mechanical competence of the bone tissue.     

Don’t Forget the Bones: Incidence and Risk Factors of Metabolic Bone Disease in a Cohort of Preterm Infants

Metabolic bone disease of prematurity (MBD) is a condition of reduced bone mineral content (BMC) compared to that expected for gestational age (GA). Preterm birth interrupts the physiological process of calcium (Ca) and phosphorus (P) deposition that occurs mostly in the third trimester of pregnancy, leading to an inadequate bone mineralization during intrauterine life (IUL). After birth, an insufficient intake of Ca and P carries on this alteration, resulting in overt disease. If MBD is often a self-limited condition, in some cases it could hesitate the permanent alteration of bone structures with growth faltering and failure to wean off mechanical ventilation due to excessive chest wall compliance. Despite advances in neonatal intensive care, MBD is still frequent in preterm infants, with an incidence of 16–23% in very-low-birth-weight (VLBW, birth weight <1500 g) and 40–60% in extremely low-birth-weight (ELBW, birth weight <1000 g) infants. Several risk factors are associated with MBD (e.g., malabsorption syndrome, parenteral nutrition (PN), pulmonary bronchodysplasia (BPD), necrotizing enterocolitis (NEC), and some chronic medications). The aim of this study was to evaluate the rate of MBD in a cohort of VLBWI and the role of some risk factors. We enrolled 238 VLBWIs (107 male). 52 subjects were classified as increased risk (G1) and 186 as standard risk (G2) according to serum alkaline phosphatase (ALP) and phosphorus (P) levels. G1 subjects have lower GA (p < 0.01) and BW (p < 0.001). Moreover, they need longer PN support (p < 0.05) and invasive ventilation (p < 0.01). G1 presented a higher rate of BPD (p = 0.026). At linear regression analysis, BW and PN resulted as independent predictor of increased risk (p = 0.001, p = 0.040, respectively). Preventive strategies are fundamental to prevent chronic alteration in bone structures and to reduce the risk of short stature. Screening for MBD based on serum ALP could be helpful in clinical practice to identify subjects at increased risk.     

Blood Cell Responses Following Heavy Alcohol Consumption Coincide with Changes in Acute Phase Reactants of Inflammation,Indices of Hemolysis and Immune Responses to Ethanol Metabolites

Aberrations in blood cells are common among heavy alcohol drinkers. In order to shed further light on such responses, we compared blood cell status with markers of hemolysis, mediators of inflammation and immune responses to ethanol metabolites in alcohol-dependent patients at the time of admission for detoxification and after abstinence. Blood cell counts, indices of hemolysis (LDH, haptoglobin, bilirubin), calprotectin (a marker of neutrophil activation), suPAR, CD163, pro- and anti-inflammatory cytokines and autoantibodies against protein adducts with acetaldehyde, the first metabolite of ethanol, were measured from alcohol-dependent patients (73 men, 26 women, mean age 43.8 ± 10.4 years) at baseline and after 8 ± 1 days of abstinence. The assessments also included information on the quantities of alcohol drinking and assays for biomarkers of alcohol consumption (CDT), liver function (AST, ALT, ALP, GGT) and acute phase reactants of inflammation. At baseline, the patients showed elevated values of CDT and biomarkers of liver status, which decreased significantly during abstinence. A significant decrease also occurred in LDH, bilirubin, CD163 and IgA and IgM antibodies against acetaldehyde adducts, whereas a significant increase was noted in blood leukocytes, platelets, MCV and suPAR levels. The changes in blood leukocytes correlated with those in serum calprotectin (p < 0.001), haptoglobin (p < 0.001), IL-6 (p < 0.02) and suPAR (p < 0.02). The changes in MCV correlated with those in LDH (p < 0.02), MCH (p < 0.01), bilirubin (p < 0.001) and anti-adduct IgG (p < 0.01). The data indicates that ethanol-induced changes in blood leukocytes are related with acute phase reactants of inflammation and release of neutrophil calprotectin. The studies also highlight the role of hemolysis and immune responses to ethanol metabolites underlying erythrocyte abnormalities in alcohol abusers.     

β-Conglycinin Reduces the Tight Junction Occludin and ZO-1 Expression in IPEC-J2

Soybean allergy presents a health threat to humans and animals. The mechanism by which food/feed allergen β-conglycinin injures the intestinal barrier has not been well understood. In this study, the changes of epithelial permeability, integrity, metabolic activity, the tight junction (TJ) distribution and expression induced by β-conglycinin were evaluated using IPEC-J2 model. The results showed a significant decrease of trans-epithelial electrical resistance (TEER) (p < 0.001) and metabolic activity (p < 0.001) and a remarkable increase of alkaline phosphatase (AP) activity (p < 0.001) in a dose-dependent manner. The expression levels of tight junction occludin and ZO-1 were decreased (p < 0.05). The reduced fluorescence of targets and change of cellular morphology were recorded. The tight junction occludin and ZO-1 mRNA expression linearly declined with increasing β-conglycinin (p < 0.001).     

Saliva and Plasma Neutralizing Activity Induced by the Administration of a Third bnt162b2 Vaccine Dose

The BNT162b2 vaccine induces neutralizing activity (NA) in serum, but no data are available on whether a third-dose activates specific-immunity within the oral mucosa, representing the primary route of viral-entry. To carefully address this issue, we investigated if such immunity is boosted by SARS-CoV-2-infection; how long it is maintained over-time; and if it protects against the SARS-CoV-2 lineage B.1 (EU) and the emerging Delta and Omicron variants. NA was measured in plasma and saliva samples from: uninfected SARS-CoV-2-Vaccinated (SV), subjects infected prior to vaccination (SIV), and subjects who were infected after the second (SIV2) or the third (SIV3) vaccine dose. Samples were collected immediately before (T0), 15 days (T1), and 90 days (T2) post third-dose administration (SV and SIV), or 15 days post-infection (SIV2 and SIV3). In all the enrolled groups, NA in plasma and saliva: (i) was higher against EU compared to the other variants at all time-points (SV: T0 and T1, EU vs. both Delta and Omicron p < 0.001; T2 p < 0.01) (SIV: T0, EU vs. Delta p < 0.05; EU vs. Omi p < 0.01; T1 and T2 EU vs. Delta p < 0.01; EU vs. Omi p < 0.001); (ii) was boosted by the administration of the third dose; iii) declined over-time, albeit being detectable in almost all subjects at T2. The monitoring of NA over time will be important in clarifying if different NA levels may influence either acquisition or course of infection to properly plan the timing of a fourth vaccine dose administration.     

Effect of Long-Term Dietary Arginyl-Fructose (AF) on Hyperglycemia and HbA1c in Diabetic db/db Mice

We have previously reported that Amadori compounds exert anti-diabetic effects by lowering sucrose-induced hyperglycemia in normal Sprague-Dawley rats. In the present study we extended our recent findings to evaluate whether α-glucosidase inhibitor arginyl-fructose (AF) lowers blood glucose level in diabetic db/db mice, a genetic model for type 2 diabetes. The db/db mice were randomly assigned to high-carbohydrate diets (66.1% corn starch) with and without AF (4% in the diet) for 6 weeks. Changes in body weight, blood glucose level, and food intake were measured daily for 42 days. Dietary supplementation of AF resulted in a significant decrease of blood glucose level (p < 0.001) and body weight (p < 0.001). The level of HbA1c, a better indicator of plasma glucose concentration over prolonged periods of time, was also significantly decreased for 6-week period (p < 0.001). Dietary treatment of acarbose^® (0.04% in diet), a positive control, also significantly alleviated the level of blood glucose, HbA1c, and body weight. These results indicate that AF Maillard reaction product improves postprandial hyperglycemia by suppressing glucose absorption as well as decreasing HbA1c level.     

PCSK9 Affects Astrocyte Cholesterol Metabolism and Reduces Neuron Cholesterol Supplying In Vitro: Potential Implications in Alzheimer’s Disease

The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) involvement in Alzheimer’s disease (AD) is poorly investigated. We evaluated the in vitro PCSK9 modulation of astrocyte cholesterol metabolism and neuronal cholesterol supplying, which is fundamental for neuronal functions. Moreover, we investigated PCSK9 neurotoxic effects. In human astrocytoma cells, PCSK9 reduced cholesterol content (−20%; p < 0.05), with a greater effect in presence of beta amyloid peptide (Aβ) (−37%; p < 0.01). PCSK9 increased cholesterol synthesis and reduced the uptake of apoE-HDL-derived cholesterol (−36%; p < 0.0001), as well as the LDL receptor (LDLR) and the apoE receptor 2 (ApoER2) expression (−66% and −31%, respectively; p < 0.01). PCSK9 did not modulate ABCA1- and ABCG1-cholesterol efflux, ABCA1 levels, or membrane cholesterol. Conversely, ABCA1 expression and activity, as well as membrane cholesterol, were reduced by Aβ (p < 0.05). In human neuronal cells, PCSK9 reduced apoE-HDL-derived cholesterol uptake (−41%; p < 0.001) and LDLR/apoER2 expression (p < 0.05). Reduced cholesterol internalization occurred also in PCSK9-overexpressing neurons exposed to an astrocyte-conditioned medium (−39%; p < 0.001). PCSK9 reduced neuronal cholesterol content overall (−29%; p < 0.05) and increased the Aβ-induced neurotoxicity (p < 0.0001). Our data revealed an interfering effect of PCSK9, in cooperation with Aβ, on brain cholesterol metabolism leading to neuronal cholesterol reduction, a potentially deleterious effect. PCSK9 also exerted a neurotoxic effect, and thus represents a potential pharmacological target in AD.     

Tear Proteome Revealed Association of S100A Family Proteins and Mesothelin with Thrombosis in Elderly Patients with Retinal Vein Occlusion

Tear samples collected from patients with central retinal vein occlusion (CRVO; n = 28) and healthy volunteers (n = 29) were analyzed using a proteomic label-free absolute quantitative approach. A large proportion (458 proteins with a frequency > 0.6) of tear proteomes was found to be shared between the study groups. Comparative proteomic analysis revealed 29 proteins (p < 0.05) significantly differed between CRVO patients and the control group. Among them, S100A6 (log (2) FC = 1.11, p < 0.001), S100A8 (log (2) FC = 2.45, p < 0.001), S100A9 (log2 (FC) = 2.08, p < 0.001), and mesothelin ((log2 (FC) = 0.82, p < 0.001) were the most abundantly represented upregulated proteins, and β2-microglobulin was the most downregulated protein (log2 (FC) = −2.13, p < 0.001). The selected up- and downregulated proteins were gathered to customize a map of CRVO-related critical protein interactions with quantitative properties. The customized map (FDR < 0.01) revealed inflammation, impairment of retinal hemostasis, and immune response as the main set of processes associated with CRVO ischemic condition. The semantic analysis displayed the prevalence of core biological processes covering dysregulation of mitochondrial organization and utilization of improperly or topologically incorrect folded proteins as a consequence of oxidative stress, and escalating of the ischemic condition caused by the local retinal hemostasis dysregulation. The most significantly different proteins (S100A6, S100A8, S100A9, MSLN, and β2-microglobulin) were applied for the ROC analysis, and their AUC varied from 0.772 to 0.952, suggesting probable association with the CRVO.     

Immunosenescence in Aging-Related Vascular Dysfunction

The immunosenescence-related disproportion in T lymphocytes may have important consequences for endothelial dysfunction, which is a key event in vascular aging. The study was designed to assess the prognostic values of the inflammatory-immune profile to better predict and prevent vascular diseases associated with old age. Eighty individuals aged 70.9 ± 5.3 years were allocated to a low- (LGI) or high-grade inflammation (HGI) group based on CRP (<3 or ≥3 mg/L) as a conventional risk marker of cardiovascular diseases. Significant changes in inflammatory and endothelium-specific variables IL-1β, IL-6, TNFα, oxLDL, H₂O₂, NO, 3-nitrotyrosine, and endothelial progenitor cells (OR 7.61, 95% CI 2.56–29.05, p < 0.0001), confirmed their interplay in vascular inflammation. The flow-cytometry analysis demonstrated a high disproportion in T lymphocytes CD4⁺ and CD8⁺ between LGI and HGI groups. CRP was <3 mg/mL for the CD4/CD8 ratio within the reference values ≥ 1 or ≤2.5, unlike for the CD4/CD8 ratio < 1 and >2.5. The odds ratios for the distribution of CD4⁺ (OR 5.98, 95% CI 0.001–0.008, p < 0.001), CD8⁺ (OR 0.23, 95% CI 0.08–0.59, p < 0.01), and CD8CD45RO⁺ T naïve cells (OR 0.27, 95% CI 0.097–0.695, p < 0.01) and CD4/CD8 (OR 5.69, 95% CI 2.07–17.32, p < 0.001) indicated a potential diagnostic value of T lymphocytes for clinical prognosis in aging-related vascular dysfunction.     

Human Herpesvirus-6 and -7 in the Brain Microenvironment of Persons with Neurological Pathology and Healthy People

During persistent human beta-herpesvirus (HHV) infection, clinical manifestations may not appear. However, the lifelong influence of HHV is often associated with pathological changes in the central nervous system. Herein, we evaluated possible associations between immunoexpression of HHV-6, -7, and cellular immune response across different brain regions. The study aimed to explore HHV-6, -7 infection within the cortical lobes in cases of unspecified encephalopathy (UEP) and nonpathological conditions. We confirmed the presence of viral DNA by nPCR and viral antigens by immunohistochemistry. Overall, we have shown a significant increase (p < 0.001) of HHV antigen expression, especially HHV-7 in the temporal gray matter. Although HHV-infected neurons were found notably in the case of HHV-7, our observations suggest that higher (p < 0.001) cell tropism is associated with glial and endothelial cells in both UEP group and controls. HHV-6, predominantly detected in oligodendrocytes (p < 0.001), and HHV-7, predominantly detected in both astrocytes and oligodendrocytes (p < 0.001), exhibit varying effects on neural homeostasis. This indicates a high number (p < 0.001) of activated microglia observed in the temporal lobe in the UEP group. The question remains of whether human HHV contributes to neurological diseases or are markers for some aspect of the disease process.     

5-ALA Fluorescence Image Guided Resection of Glioblastoma Multiforme: A Meta-Analysis of the Literature

Background: Glioblastoma multiforme (GBM) is one of the most deadly cancers in humans. Despite recent advances in anti-cancer therapies, most patients with GBM die from local disease progression. Fluorescence image guided surgical resection (FIGR) was recently advocated to enhance local control of GBM. This is meta-analyses of 5-aminolevulinic (5-ALA) induced FIGR. Materials: Review of the literature produced 503 potential publications; only 20 of these fulfilled the inclusion criteria of this analysis, including a total of 565 patients treated with 5-ALA-FIGR reporting on its outcomes and 800 histological samples reporting 5-ALA-FIGR sensitivity and specificity. Results: The mean gross total resection (GTR) rate was 75.4% (95% CI: 67.4–83.5, p < 0.001). The mean time to tumor progression (TTP) was 8.1 months (95% CI: 4.7–12, p < 0.001). The mean overall survival gain reported was 6.2 months (95% CI: −1–13, p < 0.001). The specificity was 88.9% (95% CI: 83.9–93.9, p < 0.001) and the sensitivity was 82.6% (95% CI: 73.9–91.9, p < 0.001). Conclusion: 5-ALA-FIGR in GBM is highly sensitive and specific, and imparts significant benefits to patients in terms of improved GTR and TTP.     

Relationship between IgA Nephropathy and Porphyromonas gingivalis; Red Complex of Periodontopathic Bacterial Species

IgA nephropathy (IgAN) has been considered to have a relationship with infection in the tonsil, because IgAN patients often manifest macro hematuria just after tonsillitis. In terms of oral-area infection, the red complex of periodontal bacteria (Porphyromonas gingivalis (P. gingivalis), Treponema denticol (T. denticola) and Tannerella forsythia (T. forsythia)) is important, but the relationship between these bacteria and IgAN remains unknown. In this study, the prevalence of the red complex of periodontal bacteria in tonsil was compared between IgAN and tonsillitis patients. The pathogenicity of IgAN induced by P. gingivalis was confirmed by the mice model treated with this bacterium. The prevalence of P. gingivalis and T. forsythia in IgAN patients was significantly higher than that in tonsillitis patients (p < 0.001 and p < 0.05, respectively). A total of 92% of tonsillitis patients were free from red complex bacteria, while only 48% of IgAN patients had any of these bacteria. Nasal administration of P. gingivalis in mice caused mesangial proliferation (p < 0.05 at days 28a nd 42; p < 0.01 at days 14 and 56) and IgA deposition (p < 0.001 at day 42 and 56 after administration). Scanning-electron-microscopic observation revealed that a high-density Electron-Dense Deposit was widely distributed in the mesangial region in the mice kidneys treated with P. gingivalis. These findings suggest that P. gingivalis is involved in the pathogenesis of IgAN.     

Magnesium Modified β-Tricalcium Phosphate Induces Cell Osteogenic Differentiation In Vitro and Bone Regeneration In Vivo

In vitro, in vivo, and clinical studies have shown how the physicochemical and biological properties of β-tricalcium phosphate (β-TCP) work in bone regeneration. This study aimed to improve the properties of β-TCP by achieving optimum surface and bulk β-TCP chemical/physical properties through the hydrothermal addition of magnesium (Mg) and to later establish the biocompatibility of β-TCP/Mg for bone grafting and tissue engineering treatments. Multiple in vitro and in vivo analyses were used to complete β-TCP/Mg physicochemical and biological characterization. The addition of MgO brought about a modest rise in the number of β-TCP surface particles, indicating improvements in alkaline phosphatase (ALP) activity on day 21 (p < 0.05) and in the WST-1assay on all days (p < 0.05), with a corresponding increase in the upregulation of ALP and bone sialoprotein. SEM analyses stated that the surfaces of the β-TCP particles were not altered after the addition of Mg. Micro-CT and histomorphometric analysis from rabbit calvaria critical defects resulted in β-TCP/Mg managing to reform more new bone than the control defects and β-TCP control at 2, 6, and 8 weeks (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001). The hydrothermal addition of MgO to the β-TCP surfaces ameliorated its biocompatibility without altering its surface roughness resulting from the elemental composition while enhancing cell viability and proliferation, inducing more bone regeneration by osteoconduction in vivo and osteoblastic differentiation in vitro.     

Organotypic Culture of Testicular Tissue from Infant Boys with Cryptorchidism

Organotypic culture of human fetal testis has achieved fertilization-competent spermatids followed by blastocysts development. This study focuses on whether the organotypic culture of testicular tissue from infant boys with cryptorchidism could support the development of spermatogonia and somatic cells. Frozen-thawed tissues were cultured in two different media, with or without retinoic acid (RA), for 60 days and evaluated by tissue morphology and immunostaining using germ and somatic cell markers. During the 60-day culture, spermatocytes stained by boule-like RNA-binding protein (BOLL) were induced in biopsies cultured with RA. Increased AR expression (p < 0.001) and decreased AMH expression (p < 0.001) in Sertoli cells indicated advancement of Sertoli cell maturity. An increased number of SOX9-positive Sertoli cells (p < 0.05) was observed, while the percentage of tubules with spermatogonia was reduced (p < 0.001). More tubules with alpha-smooth muscle actin (ACTA, peritubular myoid cells (PTMCs) marker) were observed in an RA-absent medium (p = 0.02). CYP17A1/STAR-positive Leydig cells demonstrated sustained steroidogenic function. Our culture conditions support the initiation of spermatocytes and enhanced maturation of Sertoli cells and PTMCs within infant testicular tissues. This study may be a basis for future studies focusing on maintaining and increasing the number of spermatogonia and identifying different factors and hormones, further advancing in vitro spermatogenesis.     

Decursinol Angelate Arrest Melanoma Cell Proliferation by Initiating Cell Death and Tumor Shrinkage via Induction of Apoptosis

Melanoma is known to aggressively metastasize and is one of the prominent causes of skin cancer mortality. This study was designed to assess the molecular mechanism of decursinol angelate (DA) against murine melanoma cell line (B16F10 cells). Treatment of DA resulted in growth inhibition and cell cycle arrest at G0/G1 (p < 0.001) phase, evaluated through immunoblotting. Moreover, autophagy-related proteins such as ATG-5 (p < 0.0001), ATG-7 (p < 0.0001), beclin-1 (p < 0.0001) and transition of LC3-I to LC3-II (p < 0.0001) were markedly decreased, indicating autophagosome inhibition. Additionally, DA treatment triggered apoptotic events which were corroborated by the occurrence of distorted nuclei, elevated reactive oxygen species (ROS) levels and reduction in the mitochondrial membrane potential. Subsequently, there was an increase in the expression of pro-apoptotic protein Bax in a dose-dependent manner, with the corresponding downregulation of Bcl-2 expression and cytochrome C expression following 24 h DA treatment in A375.SM and B16F10 cells. We substantiated our results for apoptotic occurrence through flow cytometry in B16F10 cells. Furthermore, we treated B16F10 cells with N-acetyl-L-cysteine (NAC). NAC treatment upregulated ATG-5 (p < 0.0001), beclin-1 (p < 0.0001) and LC3-I to LC3-II (p < 0.0001) conversion, which was inhibited in the DA treatment group. We also noticed a systematic upregulation of important markers for progression of G1 cell phase such as CDK-2 (p < 0.029), CDK-4 (p < 0.036), cyclin D1 (p < 0.0003) and cyclin E (p < 0.020) upon NAC treatment. In addition, we also observed a significant fold reduction (p < 0.05) in ROS fluorescent intensity and the expression of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase-9 (p > 0.010) and cleaved caspase-3 (p < 0.0001). NAC treatment was able to ameliorate DA-induced apoptosis and cell cycle arrest to support our finding. Our in vivo xenograft model also revealed similar findings, such as downregulation of CDK-2 (p < 0.0001) and CDK-4 (p < 0.0142) and upregulation of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase 3 (p < 0.0001) and cleaved caspase 9 (p < 0.0001). In summary, our study revealed that DA is an effective treatment against B16F10 melanoma cells and xenograft mice model.