ALP Activity Research Articles

Enhancing osteoblast differentiation and bone repair: The priming effect of photobiomodulation on adipose stromal cells

Cellular therapy using adipose tissue–derived mesenchymal stromal cells (at-MSCs) has garnered attention for the treatment of bone defects. Therefore, preconditioning strategies to enhance the osteogenic potential of at-MSCs could optimize cell therapy outcomes, and photobiomodulation (PBM) therapy has emerged as an effective, noninvasive, and low-cost alternative. This study explored the impacts of PBM on at-MSCs differentiation and the subsequent repair of bone defects treated with cell injection. Rat at-MSCs were cultured and irradiated (at-MSCsPBM) following the PBM protocol (660 nm; 20 mW; 0.714 W/cm2; 0.14 J; 5 J/cm2). Cellular differentiation was assessed based on the expression of gene and protein markers. Reactive oxygen species (ROS) were detected using fluorescence. At-MSCsPBM were injected into 5-mm calvarial lesions, and bone formation was analyzed using micro-CT and histological evaluations. At-MSCs were used as control. Data were analyzed using the ANOVA or t-test. At-MSCsPBM exhibited high levels of gene and protein runt-related transcription factor-2 (Runx2) and alkaline phosphatase (Alp) expression. PBM increased ALP activity and significantly reduced ROS levels. In addition, PBM increased the expression of Wnt pathway–associated genes. In vivo, there was an increase in the morphometric parameters, including bone volume, percentage of bone volume, bone surface area, and trabecular number, in at-MSCsPBM-treated defects compared with those in the control. These findings suggest that PBM enhances the osteogenic potential of at-MSCs, thereby supporting the advancement of improved cellular therapies for bone regeneration.

New Therapeutic Rargets for Cynodon Dactylon in Atherosclerosis: Attenuation of TMAO Generation and Down-regulation of TLR-4 in Rats

Purpose: Atherosclerosis (AS) is a chronic disease with worldwide incidence. The current study was conducted to highlight the molecular pathogenesis of AS and equal effectiveness of cynodon dactylon’s ethanolic extract (CDE) on the AS-induced injuries. Methods: Atherosclerosis was induced experimentally in healthy male rats with a hypercholesterolemic diet (HCD) for 24 weeks. The HCD-received animals were either treated with normal saline or various doses of CDE and/or atorvastatin. After 24 weeks of the treatment period, the effects of HCD on lipid profile, oxidative/nitrosative stress status, inflammatory enzymes (LDH, ALP and CKMB) activities, trimethylamine oxide (TMAO) concentration in serum along with the expression of TLR-4 (qPCR) and CD31 (Immunohistochemistry) in coronary and aorta were analyzed Results: CDE was able to reduce the HCD-enhanced cholesterol and triglycerides. The HCDelevated activities of LDH, ALP and CK-MB were declined by CDE. The antioxidant potency of CDE was comparable with atorvastatin on HCD-induced oxidative/nitrosative stress. The serum concentration of TMAO was lowered (1.7-fold) by CDE in a dose-dependent manner, while CD31 expression in the endothelial of coronary arterials and aorta in CDE and atorvastatin-received animals was remarkably increased. The up-regulated expression of TLR-4 (2.5-fold) at mRNA level was regulated significantly (p < 0.05) and returned to normal level by CDE treatment. Conclusion: Results of the current study indicate that the up-regulation of TLR-4 as a main inflammatory gene and elevation of TMAO are involved in the pathophysiology of AS. Moreover, the protective effects of CDE on AS may be attributed to its anti-inflammatory and hypolipidemic properties.

3D printed polycaprolactone/gelatin/ordered mesoporous calcium magnesium silicate nanocomposite scaffold for bone tissue regeneration.

Tissue engineering scaffolds are three-dimensional structures that provide an appropriate environment for cellular attachment, proliferation, and differentiation. Depending on their specific purpose, these scaffolds must possess distinct features, including appropriate mechanical properties, porosity, desired degradation rate, and cell compatibility. This investigation aimed to fabricate a new nanocomposite scaffold using a 3D printing technique composed of poly(ε-caprolactone) (PCL)/Gelatin (GEL)/ordered mesoporous calcium-magnesium silicate (om-CMS) particles. Different weight ratios of om-CMS were added and optimized, and a series of scaffolds were constructed for comparison purposes, including PCL 50%/Gel 50%, PCL 50%/Gel 45%/om-CMS%5, and PCL 50%/Gel 40%/om-CMS%10. The optimized weight ratio of om-CMS was 10% without leaving behind negative effects on the filaments' structure. The scaffolds' physical and chemical properties were assessed using various techniques, and their degradation rate, bioactivity potential, cell viability, attachment, and ALP activity were evaluated in vitro. The results demonstrated that the PCL 50%/Gel 40%/om-CMS10% scaffold had promising potential for further studies in bone tissue regeneration.

Toxicological evaluation of a herbal tea made from the leaves of Setaria megaphylla, Ageratum conyzoides and Chromolaena odorata

The toxicological effect of an herbal tea made from leaf extracts of Ageratum conyzoides, Chromolaena odorata, and Setaria megaphylla, plants traditionally used in Nigeria for malaria treatment was studied. Hot aqueous extracts of the ternary combination (1:1:1 ratio) of the plants were administered in varying doses (0, 50, 100, and 200 mg/kg) to assess impact on hepatic, renal and haematologic function parameters, and liver and kidney tissue histology of forty male albino rats divided into four groups of ten animals each. The tea extracts were administered orally daily for 60 days. Biochemical analyses revealed that the extract significantly (p<0.05) reduced ALT (25.91 ± 2.35 to 6.84 ± 0.31 U/L), AST (36.39 ± 1.96 to 19.24 ± 1.19 U/L), and ALP (21.94 ± 3.51 to 11.06 ± 2.76 IU/L) activities, with no significant (p>0.05) changes in bilirubin, protein and albumin concentrations, suggesting non-hepatotoxic property. Among the kidney function, significant (p<0.05) dose-dependent increases was observed only in serum urea ranging from 23.09 ± 0.79 mg/dl (Control group) to 34.90 ± 0.52 mg/dl (200 mg/kg treated group). Increase in WBC count was observed indicative of a possible immune response, while other blood parameters remained stable. Histopathological examination confirmed that lower extract doses were relatively safe, whereas higher doses presented early signs of organ stress. These findings suggest that this herbal tea extract has a favourable safety profile at moderate doses, though higher doses may pose risk of organ toxicity, warranting further studies on dose optimization and elucidation of its folkloric therapeutic potential.

Toxicological evaluation of anti-cough herbal tea made from aqueous extracts of G. Kola, C. Citratus and B. pinnatum

Background: Herbal formulations currently contribute significantly to medical remedies with varying effects on health indices. Garcinia kola seeds, Cymbapogon citratus, and Bryophyllum pinnantum leaves are among the widely used in herbal formulations. Objectives: This study evaluated the toxicological effects of the ternary combination of these plant materials on some renal, hepatic, hematological and histological parameters of streptococci-infected white albino rats. Materials and Methods: Samples of Garcinia kola seeds, Cymbapogon citratus leaves, and Bryophyllum pinnantum leaves were sourced locally and authenticated by a taxonomist. Aqueous extract of the ternary combination comprising of 40% G. kola, 30% C. citratus, and 30% B. pinnantum was prepared and administered on albino rats. Thirty five white albino rats grouped into 7 groups comprising of normal control, treatment control, disease control, and test group were used for the evaluation after a 14 days treatment on infected and uninfected rats. The rats were sacrificed, and samples collected. Some renal, hepatic, histological, and hematological indices were analyzed. Results: Comparison of all hematological parameters (except for platelet count) between uninfected-untreated and unifected-treated with extract showed no significant (p>0.05) difference. Platelet count was significantly (p<0.05) raised in all groups treated with the extract. Comparison control and the extract-treated group showed no significant (p>0.05) difference in renal parameters indicating no adverse effects on renal function. However, comparison of parameters between control and the infected-treated group showed significantly elevated creatinine which alone may not be sufficient to support renal damage. Albumin, and total bilirubin were significantly (p<0.05) lower in the extract-treated group compared to the control group. In contrast, total protein and globulin were significantly (p<0.05) higher in the controls to the infected groups. ALT and AST were significantly (p<0.05) lower in the extract treated groups compared to the control group. Although comparison of ALP activity between uninfected-untreated and infected-treated group showed significant (p<0.05) elevation this alone may not be sufficient to support liver damage. The hematoxyline and eosine stained sections of the kidney and the liver tissues of the tea fed groups show normal kidney and liver histology. There was no significant change in the liver and kidney histology of the test and control groups. The extract did not cause adverse impact on these organs. Conclusion: Ternary combination of 40% G. kola, 30% C. citratus, and 30% B. pinnantum can effectively be used in treatment of cough without significant adverse effects on renal, hepatic, hematological and histological parameters. The ternary combination rather demonstrated significant hepato-protective feature.

Mechanistic Exploration of Smilax glabra Roxb. in Osteoarthritis: Insights from Network Pharmacology, Molecular Docking, and In Vitro Validation.

Background: Arthritis, a debilitating joint disease, remains a significant global health burden. This study uncovers the therapeutic potential of the medicinal plant Smilax glabra Roxb. (SGR) in attenuating progression of disease by modulating immune responses. Methods: Through computational approaches, key bioactive compounds in SGR were identified by using freely available databases: TCMSP, TCMID, HIT2.0, HERB, and INPUT in order to elucidate their underlying mechanisms of action. Therapeutic targets for the disease have been retrieved by TTD, GeneCard, and OMIM databases. The STRING database was used to analyze the protein-protein interactions (PPI) of intersecting genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to reveal the functional roles of genes. Mcule was used for molecular docking and binding affinity of compounds and targets were evaluated by DeepPurpose model. ALP activity, cell viability assay, TRAP staining were also performed. Results: A total of 14 active SGR compounds with 59 common targets for arthritis have been identified. These targets have a major role in controlling biological processes such as wound healing, oxygen responses, and chemical stimuli. Molecular docking by Mcule platform demonstrated that quercetin and β-sitosterol showed higher binding energy affinities with TNF, TP53, PTGS2, and JUN as compared to other targets. To explore the complex relationship between compounds and targets, pre-trained Davis and KIBA models were used to predict the affinity values of selected compounds. In MC3T3-E1 cells, ALP activity was significantly increased and bone marrow macrophages (BMM) showed a low number of TRAP-positive cells in SGR-treated cells. Conclusions: Our findings demonstrate that SGR effectively inhibits/regulates inflammatory responses, prevents cartilage degradation, promotes bone regeneration, and can be used as a promising candidate for the development of novel arthritis treatment.

Positive Regulation of Osteoblast Proliferation and Differentiation in MC3T3- E1 Cells by 7,3',4'-Trimethoxyflavone.

Increasing ratio of bone fragility, and susceptibility to fractures constitutes a major health problem worldwide. Therefore, we aimed to identify new compounds with a potential to increase proliferation and differentiation of osteoblasts. Cellular and molecular assays, such as ALP activity, alizarin staining, and flow cytometry were employed to check the effect of TMF on osteogenesis. Moreover, gene expression analysis of certain important genes and transcriptional factors was also performed. Our findings report for the first time that 7,3',4'-trimethoxyflavone is capable of enhancing proliferation, and differentiation in osteoblast cells. Results from flow cytometry analysis also indicated that TMF increases the number of cells in S-phase. Furthermore, treatment with TMF altered the expression of osteogenic genes, OCN and Axin-2 indicating possible activation of Wnt signaling pathway. Taken together, this study identified that 7,3',4'-trimethoxyflavone has the potential to enhance osteoblast proliferation and differentiation, possibly due to the activation of Wnt/β-catenin pathway. Thus, demonstrating TMF as naturally occurring agent to promote osteogenesis and prevention of bone fragility, and related disorders.

Enhancing alginate dialdehyde-gelatin (ADA-GEL) based hydrogels for biofabrication by addition of phytotherapeutics and mesoporous bioactive glass nanoparticles (MBGNs)

This study explores the 3D printing of alginate dialdehyde-gelatin (ADA-GEL) inks incorporating phytotherapeutic agents, such as ferulic acid (FA), and silicate mesoporous bioactive glass nanoparticles (MBGNs) at two different concentrations. 3D scaffolds with bioactive properties suitable for bone tissue engineering (TE) were obtained. The degradation and swelling behaviour of films and 3D printed scaffolds indicated an accelerated trend with increasing MBGN content, while FA appeared to stabilize the samples. Determination of the degree of crosslinking validated the increased stability of hydrogels due to the addition of FA and 0.1% (w/v) MBGNs. The incorporation of MBGNs not only improved the effective moduli and conferred bioactive properties through the formation of hydroxyapatite (HAp) on the surface of ADA-GEL-based samples but also enhanced VEGF-A expression of MC3T3-E1 cells. The beneficial impact of FA and low concentrations of MBGNs in ADA-GEL-based inks for 3D (bio)printing applications was corroborated through various printing experiments, resulting in higher printing resolution, as also confirmed by rheological measurements. Cytocompatibility investigations revealed enhanced MC3T3-E1 cell activity and viability. Furthermore, the presence of mineral phases, as confirmed by an in vitro biomineralization assay, and increased ALP activity after 21 days, attributed to the addition of FA and MBGNs, were demonstrated. Considering the acquired structural and biological properties, along with efficient drug delivery capability, enhanced biological activity, and improved 3D printability, the newly developed inks exhibit promising potential for biofabrication and bone TE.

Effect of climate, crop, and management on soil phosphatase activity in croplands: A global investigation and relationships with crop yield

Agricultural and livestock production cover more than a third of the Earth's land surface and are crucial to food supply. Soil extracellular enzymes play an important role in the transformation of elements and compounds in soil, particularly acid (ACP) and alkaline (ALP) phosphatases (both, APases). These enzymes have a vital role in releasing phosphorus (P) from organic matter. However, the effect of climate variables and agro-ecosystem management on APase activity in croplands remains unclear, as does its eventual relationship with agricultural productivity. Therefore, we compiled a global database of APase activity in croplands (between 1977 and 2022) and we analysed 5876 observations across 474 papers to study climate variables, crop family, and management effects on ACP and ALP activity, and their relationship with yield. ACP activity is reduced by higher temperatures (p<0.001) and lower rainfall (p=0.002). There was an interaction effect of temperature and precipitation on ALP activity (p=0.046), with the negative effect of temperature being stronger with high precipitation, and low precipitation showing low ALP activity levels at any temperature. The crop family greatly influenced APase activity (p<0.001). Management practices affected ACP and ALP activity differently; ACP activity was positively influenced by organic fertilization combined with, crop rotation or irrigation by an average of 15.6 % and 30.7 %, respectively. ALP activity was mainly positively influenced by the interaction of two different factors: organic or inorganic-organic fertilization and reduced or zero tillage. Further understanding of soil enzyme mechanisms would aid global food security and yield. As ACP activity doubles from 100.0 to 200.0 mg pNP kg−1h−1, the crop yield increases by more than two-fold, an outcome not demonstrated in croplands until now. These results could enhance yield potential through the promotion of APase activity, and the consideration of climate variables and agro-ecosystem management, which could ultimately improve cost-benefit ratios for sustainable crop growth.

ANXA2 promotes osteogenic differentiation and inhibits cellular senescence of periodontal ligament cells (PDLCs) in high glucose conditions.

Periodontal ligament cells (PDLCs) are a major component of the periodontal ligament and have an important role in the regeneration of periodontal tissue and maintenance of homeostasis. High glucose can affect the activity and function of PDLCs in a variety of ways; therefore, it is particularly important to find ways to alleviate the effects of high glucose on PDLCs. Annexin A2 (ANXA2) is a calcium- and phospholipid-binding protein involved in a variety of cellular functions and processes, including cellular cytokinesis, cytophagy, migration, and proliferation. The aim of this study was to exploring whether ANXA2 attenuates the deleterious effects of high glucose on PDLCs and promotes osteogenic differentiation capacity. Osteogenic differentiation potential, cellular senescence, oxidative stress, and cellular autophagy were detected. Culturing PDLCs with medium containing different glucose concentrations (CTRL, 8 mM, 10 mM, 25 mM, and 40mM) revealed that high glucose decreased the protein expression of ANXA2 (p<0.0001). In addition, high glucose decreased the osteogenic differentiation potential of PDLCs as evidenced by decreased calcium deposition (p=0.0003), lowered ALP activity (p=0.0010), and a decline in the expression of osteogenesis-related genes (p=0.0008). Moreover, β-Galactosidase staining and expression of p16, p21 and p53 genes showed that it increased cellular senescence in PDLCs (p<0.0001). Meanwhile high glucose increased oxidative stress in PDLCs as shown by ROS (p<0.0001). However, these damages caused by high glucose were inhibited after the addition of 1 µM recombinant ANXA2 (rANXA2), and we found that rANXA2 enhanced autophagy in PDLCs under high glucose conditions. Therefore, our present study demonstrates that alterations in ANXA2 under high glucose conditions may be a factor in the decreased osteogenic differentiation potential of PDLCs. Meanwhile, ANXA2 is associated with autophagy, oxidative stress, and cellular senescence under high glucose conditions.

Modulation of osteoblastogenesis by NRF2: NRF2 activation suppresses osteogenic differentiation and enhances mineralization in human bone marrow-derived mesenchymal stromal cells.

Mesenchymal stromal stem cells (MSCs) or skeletal stem cells (SSCs) play a major role in tissue repair due to their robust ability to differentiate into osteoblasts, chondrocytes, and adipocytes. Complex cell signaling cascades tightly regulate this differentiation. In osteogenic differentiation, Runt-related transcription factor 2 (RUNX2) and ALP activity are essential. Furthermore, during the latter stages of osteogenic differentiation, mineral formation mediated by the osteoblast occurs with the secretion of a collagenous extracellular matrix and calcium deposition. Activation of nuclear factor erythroid 2-related factor 2 (NRF2), an important transcription factor against oxidative stress, inhibits osteogenic differentiation and mineralization via modulation of RUNX2 function; however, the exact role of NRF2 in osteoblastogenesis remains unclear. Here, we demonstrate that NRF2 activation in human bone marrow-derived stromal cells (HBMSCs) suppressed osteogenic differentiation. NRF2 activation increased the expression of STRO-1 and KITLG (stem cell markers), indicating NRF2 protects HBMSCs stemness against osteogenic differentiation. In contrast, NRF2 activation enhanced mineralization, which is typically linked to osteogenic differentiation. We determined that these divergent results were due in part to the modulation of cellular calcium flux genes by NRF2 activation. The current findings demonstrate a dual role for NRF2 as a HBMSC maintenance factor as well as a central factor in mineralization, with implications therein for elucidation of bone formation and cellular Ca2+ kinetics, dystrophic calcification and, potentially, application in the modulation of bone formation.

REVITALIZATION OF CRITICAL-SIZED CALVARIAL DEFECTS IN RATS USING HEPARIN-CONJUGATED FIBRIN HYDROGEL WITH BMP-2 AND ADIPOSE-DERIVED PERICYTES

Massive bone defects present challenges in orthopedic surgery, requiring innovative solutions. Hydrogels offer promise due to their resemblance to the extracellular matrix. Adipose tissue-derived pericytes (ADPs) and osteoinductive proteins like BMP-2 show potential for bone tissue engineering. Combining them via heparin-conjugated fibrin hydrogel aims to enhance healing in critical-sized calvarial defects. The study investigated the use of a heparin-conjugated fibrin (HCF) hydrogel containing BMP-2 and adipose-derived pericytes for repairing critical-sized calvarial defects in rats. ADPs were cultured from rat adipose tissue, and the HCF hydrogel was prepared accordingly. In vitro experiments assessed BMP-2 release kinetics and bioactivity, while mineralization assays were conducted on cultured ADPs. In vivo experiments involved surgically creating calvarial defects in rats and implanting HCF gel alone or with BMP-2, pericytes, or both. Micro-CT imaging and histological analysis were performed post-implantation to evaluate bone formation. In our study, we prepared the HCF hydrogel by combining heparin-conjugated fibrinogen, human fibrinogen, human thrombin, aprotinin, and calcium chloride. Gelation occurred within 3 minutes at room temperature. SEM analysis revealed an open interconnected pore morphology and macroporous structure, facilitating cell attachment and proliferation. ELISA showed sustained release of BMP-2 from the HCF hydrogel over 28 days. Bioactivity assays demonstrated the ability of released BMP-2 to enhance ALP activity in rat neonatal calvarial osteoblasts. Additionally, BMP-2 induced osteogenic differentiation of rat ADPs, as evidenced by increased ALP activity, osteocalcin expression, and calcium deposition. Implantation of HCF hydrogels containing BMP-2 and/or ADPs into rat calvarial defects significantly promoted bone tissue regeneration compared to controls, with the greatest effect observed in the group receiving both BMP-2 and ADPs. Micro-CT and histological analysis confirmed substantial bone formation and defect closure after three months. In conclusion, our in vitro findings demonstrated that the HCF hydrogel provided sustained release of BMP-2, retaining its bioactivity and promoting osteogenesis in rat calvarial osteoblasts and ADPs. In vivo results showed that combining allogeneic ADPs with BMP-2 enhanced bone regeneration in critical-sized calvarial defects, indicating the potential of this approach for large bone defect restoration.

Cellulose nano-dispersions enhanced by ultrasound assisted chemical modification drive osteoblast proliferation and differentiation in PVA/HA bone tissue engineering scaffolds

To develop a biological bone tissue scaffold with uniform pore size and good cell adhesion was both challenging and imperative. We prepared modified cellulose nanocrystals (CNCs) dispersants (K-PCNCs) by ultrasound-assisted alkylation modification. Subsequently, nano-hydroxyapatite (HC-K) was synthesized using K-PCNCs as a dispersant and composited with polyvinyl alcohol (PVA) to prepare the scaffold using the ice template method. The results showed that the water contact angle and degree of substitution (135°, 1.53) of the K-PCNCs were highest when the ultrasound power was 450 W and the time was 2 h. The dispersion of K-PCNCs prepared under this condition was optimal. SEM showed that the pore distribution of the composite scaffolds was more homogeneous than the PVA scaffold. The porosity, equilibrium swelling rate, and mechanical properties of the composite scaffolds increased and then decreased with the increase of HC-K content, and reached the maximum values (56.1 %, 807.7 %, and 0.085 ± 0.004 MPa) at 9 % (w/w) of HC-K content. Cell experiments confirmed scaffold has good cytocompatibility and mineralization capacity. The ALP activity reached 1.71 ± 0.25 (ALP activity/mg protein). In conclusion, the scaffolds we developed have good biocompatibility and mechanical properties and have great potential in promoting bone defect repair.

Si and Zn dual ions upregulate the osteogenic differentiation of mBMSCs: mRNA transcriptomic sequencing analysis

Both silicon (Si) and zinc (Zn) ions are essential elements to bone health and their mechanisms for promoting osteogenesis have aroused the extensive attention of researchers. Thereinto, the mechanism by which dual ions promote osteogenic differentiation remains to be elucidated. Herein, the effects of Si and Zn ions on the cytological behaviors of mBMSCs were firstly studied. Then, the molecular mechanism of Si-Zn dual ions regulating the osteogenic differentiation of mBMSCs was investigated via transcriptome sequencing technology. In the single-ion system, Si ion at the concentration of 1.5 mM (Si-1.5) had better comprehensive effects of cell proliferation, ALP activity and osteogenesis-related gene expression levels (ALP, Runx2, OCN, Col-I and BSP); Zn ion at the concentration of 50 μM (Zn-50) demonstrated better combining effects of cell proliferation, ALP activity and same osteogenic genes expression levels. In the dual-ion system, the Si (1.5 mM)-Zn (50 μM) group (Si1.5-Zn50) synthetically enhanced ALP activity and osteogenesis genes compared with single-ion groups. Analysis of the transcriptome sequencing results showed that Si ion had a certain effect on promoting the osteogenic differentiation of mBMSCs; Zn ion had a stronger effect of contributing to a better osteogenic differentiation of mBMSCs than that of Si ion; the Si-Zn dual ions had a synergistic enhancement on conducting to the osteogenic differentiation of mBMSCs compared to single ion (Si or Zn). This study offers a blueprint for exploring the regulation mechanism of osteogenic differentiation by dual ions.Graphical

Incorporation of carboxymethyl chitosan (CMCS) for the modulation of physio-chemical characteristics and cell proliferation environment of the composite hydrogel microspheres

Hydrogels have excellent swelling properties and have been widely applied in tissue engineering because of their similarity to the extracellular matrix (ECM). Sodium alginate (SA) and carboxymethyl chitosan (CMCS) were prepared into hydrogel microspheres with Ca2+crosslinking in our study. The morphology, inner structure, mechanical properties, water content, swelling rate and BMP-2 loading and releasing properties were characterized. Our results showed that the composite SA /CMCS hydrogel microspheres were translucent and spherical in shape with uniform particle size. The incorporation of CMCS further increased the diameters of the microspheres, internal pore structure, water content, and mechanical properties of the SA/CMCS hydrogel microspheres. At the same SA concentration, with the increase of CMSC concentration, the diameter of microspheres could be increased by about 0.4 mm, the water content can be increased about 1%-2%. As for the mechanical properties, the compressive strength can be increased by 0.04-0.1 MPa, and the modulus of elasticity can be increased by 0.1-0.15 MPa. BMP-2 was chosen as a model agent and it could be loaded into SA/CMCS microspheres, and the incorporation of CMCS increased BMP-2 loading. The encapsulated BMP-2 was sustainably releasedin vitro. The leaching solutions of the SA/CMCS hydrogel microspheres exhibited good cytocompatibility and could increase ALP activity, ALP expression, and biomineralization on MC3T3-E1 cells. After 7 d of co-culture, ALP activities in S2.5C2 and S2.5C3 groups was increased by 50% and 45% compared with that of the control group. When embedded in the SA/CMCS microspheres, the MC3T3-E1 cells were evenly distributed inside the hydrogel microspheres and remained viable. Transcriptomic studies showed that incorporation of CMCS induced upregulation of 1141 differentially expressed genes (DEGs) and downregulation of 1614 DEGs compared with SA microspheres. The most significantly enriched pathways were the Wnt and MAPK signaling pathways induced by the incorporation of CMCS and BMP-2. In conclusion, our results indicated that the physiochemical characteristics of the SA hydrogel microspheres could be greatly modulated by CMCS to better mimic the ECM microenvironment and induce osteo-inductive activities of MC3T3-E1 cells.

Role of sodium-dependent vitamin C transporter 2 in human periodontal ligament fibroblasts.

Ascorbic acid (AA) is a water-soluble vitamin that has antioxidant properties and regulates homeostasis of connective tissue through controlling various enzymatic activities. Two cell surface glycoproteins, sodium-dependent vitamin C transporter (SVCT) 1 and SVCT2, are known as ascorbate transporters. The purpose of this study was to investigate the expression pattern and functions of SVCTs in periodontal ligament (PDL) and PDL fibroblast (PDLF). Gene expression was examined using real-time polymerase chain reaction (PCR) and reverse transcription PCR. SVCT2 expression was determined by immunofluorescence staining, western blot and flow cytometry. ALP activity and collagen production were examined using ALP staining and collagen staining. Short interfering RNA was used to knock down the gene level of SVCT2. Change of comprehensive gene expression under SVCT2 knockdown condition was examined by RNA-sequencing analysis. Real-time PCR, fluorescent immunostaining, western blot and flowy cytometry showed that SVCT2 was expressed in PDLF and PDL. ALP activity, collagen production, and SVCT2 expression were enhanced upon AA stimulation in PDLF. The enhancement of ALP activity, collagen production, and SVCT2 expression by AA was abolished under SVCT2 knockdown condition. RNA-sequencing revealed that gene expression of CLDN4, Cyclin E2, CAMK4, MSH5, DMC1, and Nidgen2 were changed by SVCT2 knockdown. Among them, the expression of MSH5 and DMC1, which are related to DNA damage sensor activity, was enhanced by AA, suggesting the new molecular target of AA in PDLF. Our study reveals the SVCT2 expression in PDL and the pivotal role of SVCT2 in mediating AA-induced enhancements of ALP activity and collagen production in PDLF. Additionally, we identify alterations in gene expression profiles, highlighting potential molecular targets influenced by AA through SVCT2. These findings deepen our understanding of periodontal tissue homeostasis mechanisms and suggest promising intervention targeting AA metabolism.

Nutritional and safety assessment of aqueous extract of nutmeg seeds, Myristica fragrans

Nutmeg, Myristica fragrans is used as a spice or medicinal plant to manage ailments. This study evaluated the nutritional and safety assessment of an aqueous extract of Myristica fragrans in Wistar rats. Thirty male Wistar rats weighing 180 - 240 g were randomly distributed into five groups: Control received distilled water, while 100, 200, 300, and 400 mg/kg body weight represented the treatment groups, respectively. The extract was orally administered to the animals for 28 days and then euthanized under anesthesia. Blood, serum, and liver homogenates from rats were used for biochemical analysis. The phytate and tannin contents were 0.38 mg/100 g and 0.15 mg/100 g, respectively. The calculated phytic acid to zinc ratio was 0.12. The respective percentage proximate components of the extract include moisture, ash, fat, protein, and carbohydrates: 10.61%, 45.42%, 5.95%, 1.81%, 13.78%, and 22.43%, respectively. Sodium, potassium, calcium, iron, magnesium, zinc, manganese, and lead were present in the extract. There was a significant increase (p&lt;0.05) in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and acid phosphatase (ALP) activity in the groups treated with the extract compared to the control groups. Similarly, there was a significant increase (p&lt;0.05) in serum total bilirubin and albumin concentrations in the extract treatment groups compared to control groups. On the contrary, there was a significant decrease (p&lt;0.05) in the liver AST and ALP activity in the treatment groups compared to the control. The liver total protein and albumin concentrations showed a significant decrease (p&lt;0.05) in the extract treatment groups compared to the control. There was a significant decrease (p&lt;0.05) in the pack cell volume (PCV), haemoglobin (Hb), red blood cell (RBC), Mean cell volume (MCV), and platelet (PLT) levels in the animals in the extract treatment groups compared to the control group. Myristica fragrans is abundant in essential nutrients crucial for maintaining optimal physiological functions within the body, however, it is important to avoid excessive dosage or prolonged consumption which might be toxic to liver cells. Key words: Myristica, animals, tannins, phytic acid, chemical and drug Induced liver injury, minerals

Metformin enhances osteogenic differentiation of BMSC by modulating macrophage M2 polarization

Bone marrow-derived mesenchymal stem cells (BMSCs) are capable of developing into osteoblastic cell lines in vitro and regenerating bone tissue in vivo, and they are considered to be a reliable source for bone regenerative medicine. In recent years, studies have shown that the immune microenvironment is important for osteogenesis, in which macrophages are an important component of innate immunity and coordinate with stem cells. Metformin (Met), a hypoglycemic drug that exerts a powerful effect on metabolic signaling, has been shown to modulate inflammatory responses and osteogenic activity. However, whether metformin modulates macrophage polarization and subsequently affects osteogenesis remains to be elucidated. In the present study, we investigated the potential immunomodulatory capacity of metformin on macrophage inflammatory responses and phenotypic switching, and the subsequent effects on osteogenic differentiation of BMSCs. Flow cytometry and qPCR were used to study the effects of metformin on macrophage phenotypic regulation. qPCR, ALP, ARS and calcium content measurement and ALP activity assay were used to determine the effects of macrophage-secreted activators on the osteogenic differentiation of BMSCs. Our study demonstrates that metformin can improve the immune microenvironment by modulating macrophage polarization towards an anti-inflammatory phenotype, promoting an increase in a range of anti-inflammatory factors and inhibiting pro-inflammatory factors. This was characterized by increased expression of IL-10 and CD206, Arg-1 and decreased expression of IL-1β, TNF-α, IL-6 and iNOS. In addition, metformin-modulated macrophage-conditioned medium promoted osteogenic differentiation of BMSCs, increased the expression levels of genes (ALP, Runx-2, OCN, and Col-1), enhanced ALP activity, and significantly formed mineralized nodules. In conclusion, our new study elucidates that metformin can promote osteogenic differentiation of BMSCs by modulating macrophage phenotype and thereby.

Haematological and biochemical studies on Justicia secunda infusion used in treatment of anaemia

The effect of ethanolic and infusion of Justicia secunda leaves in the animal organs was investigated using standard analytical protocols. A total of 40 albino rats divided into four groups of five rats each comprising one normal groups and three groups treated with 125, 250 and 500mg/kg body weight respectively. The infusion of J. secunda leaves was prepared using conventional methods. The hematological, biochemical and histological studies were also carried out as reported in previous literatures. The findings confirmed that Justicia secunda leaves are not toxic at the doses administered, there were significant concentrations of essential minerals such as sodium, potassium, calcium, iron, phosphorus, and sulfur. The hematological study conducted on the infusion of the leaves revealed several positive effects on blood parameters in experimental rats. The extract significantly P&lt;0.05 elevated hemoglobin and platelet count, particularly at higher doses, compared to the control group. The liver function tests showed no significant alterations in AST, ALT, TP, and ALB levels; however, the infusion resulted in some alterations in ALP activity and total bilirubin concentration. The histopathological examination of liver tissues showed mild congestion and degenerative changes in higher dose groups, indicating potential dose-dependent hepatomegaly and stress on hepatic architecture.

MgO-enhanced β-TCP promotes osteogenesis in both in vitro and in vivo rat models

Allogeneic bone grafts are used to treat bone defects in orthopedic surgery, but the osteogenic potential of artificial bones remains a challenge. In this study, we developed a β-tricalcium phosphate (β-TCP) formulation containing MgO, ZnO, SrO, and SiO2 and compared its bone-forming ability with that of β-TCP without biological elements. We prepared β-TCP discs with 60% porosity containing 1.0 wt% of these biological elements. β-TCP scaffolds were loaded with bone marrow-derived mesenchymal stem cells (BMSC) from 7-week-old male rats and cultured for 2 weeks. ALP activity and mRNA expression of osteogenic markers were evaluated. In addition, scaffolds were implanted subcutaneously in rats and analyzed after 7 weeks. In vitro, the MgO group showed lower Ca concentrations and higher osteogenic marker expression compared to controls. In vivo, the MgO group showed higher ALP activity compared to controls, and RT-qPCR analysis showed significant expression of BMP2 and VEGF. Histopathology, fluorescent immunostaining, and micro-CT also showed relatively better bone formation in the MgO group. β-TCP with MgO may enhance bone morphology in vitro and in vivo and improve the prognosis of patients with substantial and refractory bone defects.