Increased Alkaline Phosphatase Activity Research Articles

Enhancing Bioactivity of Titanium-Based Materials Through Chitosan Based Coating and Calcitriol Functionalization.

Titanium (Ti)-based materials are favored for hard tissue applications, yet their bioinertness limits their success. This study hypothesizes that functionalizing Ti materials with chitosan nano/microspheres and calcitriol (VD) will enhance their bioactivity by improving cellular activities and mineralization. To test this, chitosan particles were applied uniformly onto Ti surfaces using electrophoretic deposition (EPD) at 20V for 3 minutes. VD was then loaded onto the coated surfaces, and the release profile of VD was monitored. Human fetal osteoblastic cells (hFOB) were cultured on the VD-loaded Ti surfaces. Cellular activities such as proliferation, Alkaline phosphatase (ALP) activity, osteogenic gene expression (runt-related transcription factor 2 (Runx2), collagen type 1 (Col I), osteocalcin ( OCn), osteopontin (OP)), and mineralization were assessed. Von Kossa staining was performed to analyze mineralization, and the expression of cell adhesion proteins (N-cadherin (NC), integrin alpha V (IaV), integrin beta 3, (Ib3)) was measured. The results showed that approximately 50% of the VD released over 50 hours. The chitosan coating increased surface roughness three-fold, and this, combined with VD release, resulted in reduced cell proliferation but increased ALP activity, suggesting enhanced differentiation. VD-functionalized Ti surfaces showed statistically significant differences in osteogenic gene expressions, particularly on rougher surfaces. Additionally, the expression of cell adhesion proteins (NC, IaV, Ib3) was upregulated on VD-containing coated surfaces. Von Kossa analysis revealed that surface roughness significantly enhanced mineralization, particularly on VD-free surfaces by day 7, while mineralization on VD-containing bare surfaces started on day 14. These findings demonstrate that VD-loaded chitosan coatings significantly enhance the biocompatibility and bioactivity of Ti-based materials, highlighting their potential for applications in bone regeneration.

Polyphenol extract from Tagetes erecta L. flowers stimulates osteogenesis via β-catenin activation.

Osteoporosis, a prevalent bone disorder, results in reduced bone mineral density and mass. With minimal side effects, medicinal plant-based natural remedies are increasingly explored for osteoporosis. However, the osteogenic potential of Tagetes erecta L. flower, traditionally used for cardiovascular and renal diseases, has not yet been studied. This study investigates the osteogenic effects of the polyphenol-enriched extract from T. erecta L. flowers (TE) and its main components on osteoblast differentiation, with an emphasis on anti-osteoporotic activity. The osteogenic activity of TE was assessed in MC3T3-E1 preosteoblast cells, analyzing osteogenic alkaline phosphatase (ALP) activity via a colorimetric assay and mineralization through Alizarin Red S staining over 14 d. Expression levels of osteogenic markers-transcription factor osterix (SP7), runt-related transcription factor 2 (RUNX2), and ALP-were quantified through quantitative reverse transcription-polymerase chain reaction and western blotting. In vivo effects were evaluated using zebrafish larvae for bone formation and anti-osteoporotic properties. Vertebral development was visualized by staining mineralized structures with calcein or Alizarin Red S. Prednisolone (PDS) was administered to zebrafish larvae to model osteoporosis. Furthermore, molecular docking simulations were conducted to assess the binding affinity of TE components to the ATP-binding pocket of glycogen synthase kinase-3β (GSK-3β), and their inhibitory potential on GSK-3β kinase activity was quantified by in vitro kinase assays. Cellular thermal shift assay (CETSA) was performed to monitor direct bindings of TE and its main components to GSK3-3β. TE promoted vertebral and cranial bone formation in zebrafish larvae, elevating key osteogenic genes, such as sp7, runx2a, runx2b, and alpl. Among TE components, kaempferol and patuletin significantly enhanced vertebral formation, while isorhamnetin showed moderate effects. Patulitrin and quercetagetin did not increased vertebral formation. In MC3T3-E1 cells, TE increased ALP activity, mineralization, and the expression of SP7, RUNX2, and ALP. It also induced GSK-3β phosphorylation at serine 9 and promoted β-catenin nuclear translocation. Inhibition of β-catenin signaling reversed TE-induced osteogenic effects. Molecular docking suggested strong GSK-3β binding by TE components, with patuletin showing notable inhibition GSK-3β activity (half-maximal inhibitory concentration = 379.3 ng/mL) and enhancing vertebral formation. CETSA confirmed that TE and its main components, kaempferol and patuletin, degrades GSK-3β. Additionally, TE alleviated PDS-induced osteoporosis in both cellular and zebrafish models. By targeting GSK-3β and activating β-catenin-mediated pathways, TE shows promise as a novel anti-osteoporotic agent. This study highlights the potential of TE for therapeutic use in bone health, warranting further clinical trials to confirm its applicability.

Synthesis and Structure-Activity Relationships of Novel Benzofuran Derivatives with Osteoblast Differentiation-Promoting Activity.

Osteoporosis is caused by an imbalance between bone resorption and formation, which decreases bone mass and strength and increases the risk of fracture. Therefore, osteoporosis is treated with oral resorption inhibitors, such as bisphosphonates, and parenteral osteogenic drugs, including parathyroid hormone and antisclerostin antibodies. However, orally active osteogenic drugs have not yet been developed. In the present study, to find novel candidates for oral osteogenic drugs, various benzofuran derivatives were synthesized and their effects on osteoblast differentiation were examined in mouse mesenchymal stem cells (ST2 cells). Among the compounds tested, 3-{4-[2-(2-isopropoxyethoxy)ethoxy]phenyl}benzofuran-5-carboxamide (23d) exhibited potent osteoblast differentiation-promoting activity, estimated as EC200 for increasing alkaline phosphatase activity, and good oral absorption in female rats, resulting in high Cmax/EC200. Dual-energy X-ray absorptiometry scanning revealed that 23d at 10 mg/kg/d for 8 weeks increased femoral bone mineral density in ovariectomized rats with an elevation in plasma bone-type alkaline phosphatase activity, and micro-computed tomography showed that it increased bone volume, mineral contents, and strength in femoral diaphysis cortical, but not trabecular bone during the experiment period. 23d potently inhibited cyclin-dependent kinase 8 (CDK8) activity, suggesting that its osteoblastogenic activity is mediated by the suppression of CDK8, as previously reported for diphenylether derivatives. In conclusion, the structure-activity relationships of novel benzofuran derivatives were clarified and 3,5-disubstituted benzofuran was identified as a useful scaffold for orally active osteogenic compounds. Compound 23d exhibited potent osteoblastogenic activity through CDK8 inhibition and osteogenic effects in ovariectomized rats, indicating its potential as an orally active anti-osteoporotic drug.

MiR-181a/MSC-Loaded Nano-Hydroxyapatite/Collagen Accelerated Bone Defect Repair in Rats by Targeting Ferroptosis Pathway.

Background: The reparative regeneration of jawbone defects poses a significant challenge within the field of dentistry. Despite being the gold standard, autogenous bone materials are not without drawbacks, including a heightened risk of postoperative infections. Consequently, the development of innovative materials that can surpass the osteogenic capabilities of autologous bone has emerged as a pivotal area of research. Methods: Mesenchymal stem cells (MSCs), known for their multilineage differentiation potential, were isolated from human umbilical cords and transfected with miR-181a. The osteogenic differentiation of miR-181a/MSC was investigated. Then, physicochemical properties of miR-181a/MSC-loaded nano-hydroxyapatite (nHAC) scaffolds were characterized, and their efficacy and underlying mechanism in rat calvarial defect repair were explored. Results: miR-181a overexpression in MSCs significantly promoted osteogenic differentiation, as evidenced by increased alkaline phosphatase activity and expression of osteogenic markers. The miR-181a/MSC-loaded nHAC scaffolds exhibited favorable bioactivity and accelerated bone tissue repair and collagen secretion in vivo. Mechanistic studies reveal that miR-181a directly targeted the TP53/SLC7A11 pathway, inhibiting ferroptosis and enhancing the osteogenic capacity of MSCs. Conclusions: The study demonstrates that miR-181a/MSC-loaded nHAC scaffolds significantly enhance the repair of bone defects by promoting osteogenic differentiation and inhibiting ferroptosis. These findings provide novel insights into the molecular mechanisms regulating MSC osteogenesis and offer a promising therapeutic strategy for bone defect repair.

LL-37 regulates odontogenic differentiation of dental pulp stem cells in an inflammatory microenvironment

BackgroundInflammation often causes irreversible damage to dental pulp tissue. Dental pulp stem cells (DPSCs), which have multidirectional differentiation ability, play critical roles in the repair and regeneration of pulp tissue. However, the presence of proinflammatory factors can affect DPSCs proliferation, differentiation, migration, and other functions. LL-37 is a natural cationic polypeptide that inhibits lipopolysaccharide (LPS) activity, enhances cytokine production, and promotes the migration of stem cells. However, the potential of LL-37 in regenerative endodontics remains unknown. This study aimed to investigate the regulatory role of LL-37 in promoting the migration and odontogenic differentiation of DPSCs within an inflammatory microenvironment. These findings establish an experimental foundation for the regenerative treatment of pulpitis and provide a scientific basis for its clinical application.Materials and methodsDPSCs were isolated via enzyme digestion combined with the tissue block adhesion method and identified via flow cytometry. The impact of LL-37 on the proliferation of DPSCs was evaluated via a CCK-8 assay. The recruitment of DPSCs was assessed through a transwell assay. The mRNA expression levels of inflammatory and aging-related genes were assessed via reverse transcription‒polymerase chain reaction (RT‒PCR), western blotting, and enzyme‒linked immunosorbent assay (ELISA). The odontogenic differentiation of DPSCs was assessed through alkaline phosphatase (ALP) staining, alizarin red staining, and RT‒PCR analysis.ResultsLL-37 has the potential to enhance the migration of DPSCs. In an inflammatory microenvironment, LL-37 can suppress the expression of genes associated with inflammation and aging, such as TNF-α, IL-1β, IL-6, P21, P38 and P53. Moreover, it promotes odontogenic differentiation in DPSCs by increasing ALP activity, increasing calcium nodule formation, and increasing the expression of dentin-related genes such as DMP1, DSPP and BSP.ConclusionThese findings suggest that the polypeptide LL-37 facilitates the migration of DPSCs and plays a crucial role in resolving inflammation and promoting cell differentiation within an inflammatory microenvironment. Consequently, LL-37 has promising potential as an innovative therapeutic approach for managing inflammatory dental pulp conditions.

Extract of Curculigo capitulata Ameliorates Postmenopausal Osteoporosis by Promoting Osteoblast Proliferation and Differentiation.

Postmenopausal osteoporosis (PMOP) is a bone disease characterized by bone thinning and an increased risk of fractures due to estrogen deficiency. Current PMOP therapies often result in adverse side effects. The traditional medicinal plant Curculigo capitulata is commonly used to strengthen bones and support kidney function, but its role in treating PMOP is not well understood. This study aims to investigate the therapeutic effects of the total extract of Curculigo capitulata (Eocc) on PMOP and to explore the underlying mechanisms. The major components of the extract were identified using HPLC. Transcriptomics was employed to predict potential targets. An osteogenic differentiation model of MC3T3-E1 cells was used in vitro. The osteogenic potential of the Eocc was assessed through CCK-8 cell viability assays, alkaline phosphatase (ALP) staining, Alizarin Red staining, Western blotting, and qPCR. MCF-7 and HEK-293 cells were utilized to evaluate the estrogen-like activity of Eocc. Apoptosis rates were detected by flow cytometry. In vivo, a bilateral ovariectomized mouse model of PMOP was used to further validate the in vitro findings through histopathological analysis and WB results. The results demonstrated that the Eocc promoted the proliferation of MC3T3-E1 cells, increased ALP activity, and stimulated the formation of osteogenic mineralized nodules. It also upregulated the expression of osteogenic markers (Runx2, OCN, OPN, and BSP) at both the protein and mRNA levels. The Eocc induced the activation of ERα both in vitro and in vivo, initiating the Src/PI3K/AKT signaling pathway, leading to the phosphorylation of GSK3β and subsequent osteogenesis. The activation of this pathway also stimulated the phosphorylation of mTOR and p70S6K while downregulating cleaved caspase-3 and caspase-9. Additionally, the Eocc reduced apoptosis during osteogenic differentiation and promoted cell proliferation. These findings suggest that the Eocc facilitates osteoblast proliferation and differentiation, improving bone integrity in PMOP mice, and may represent a promising therapeutic candidate for managing PMOP.

Serum metabolomics and 16S rRNA amplicon sequencing reveal the role of puerarin in alleviating bone loss aggravated by antidiabetic agent pioglitazone in type 2 diabetic mice

Ethnopharmacological relevancePioglitazone (PIO) was an anti type 2 diabetes (T2D) agent but caused bone loss and bone marrow fat accumulation. Puerarin (PUE) was a natural component of herbal medicine extracted from Pueraria lobata (Willd.) Ohwi and reduced glycemia and improved bone mass as a supplementary drug. A combination of PIO and PUE might be good for maintaining bone mass and blood glucose. Aim of the studyWe aimed to elucidate the potential correlation and underlying mechanisms of dietary supplement PUE in reducing side effects caused by PIO. Materials and methodsIn vitro, alkaline phosphatase (ALP) staining, alizarin S (ARS) staining and qRT-PCR were performed to detect the osteogenesis activity in MC3T3-E1 cells. In vivo, we established the T2D model by treating C57BL6/J mice with high-fat diets and streptozotocin (STZ). Micro-CT, hematoxylin and eosin (H&E) staining and tartrate-resistant acid phosphatase (TRAcP) staining were performed to observe the difference in skeletal phenotype. Serum metabolomics and 16S rRNA amplicon sequencing were applied to analyze the potential effect of the combination of PIO and PUE. ResultsWe showed that the PUE could increase ALP activity and mineralization nodes of MC3T3-E1 with PIO. PIO could aggravate bone loss but PUE alleviated the effect caused by PIO in T2D mice. PUE promoted alpha-linolenic acid metabolism and glycerophospholipid metabolism, and affected the alpha diversity of the gut microbiome by regulating the genera of Alloprevotella, Fusobacterium, Rodentibacter, etc. Correlation analysis indicated that sphingosine-1-phosphate, nonadecylic acid, and margaric acid were associated with the effect of PUE. ConclusionsTaken together, we demonstrated that PIO combined with PUE was able to lower blood sugar levels without causing bone loss. The effect of PUE mainly correlated with the genua of Alloprevotella, Fusobacterium, Rodentibacter, and Alistipes. Also, alpha-linolenic acid metabolism and glycerophospholipid metabolism were major targets of PUE.

Improvement of osteogenic differentiation potential of placenta-derived mesenchymal stem cells by metformin via AMPK pathway activation

BackgroundPlacenta-derived human mesenchymal stem cells (PL-MSCs) have gained a lot of attention in the field of regenerative medicine due to their availability and bone-forming capacity. However, the osteogenic differentiation capacity of these cells remains inconsistent and could be improved to achieve greater efficiency. Although metformin, a widely used oral hypoglycemic agent, has been shown to increase bone formation in various cell types, its effect on osteogenic differentiation of PL-MSCs has not yet been investigated. Therefore, the objective of this study was to examine the effect of metformin on the osteogenic differentiation capacity of PL-MSCs and the underlying mechanisms.MethodsThe PL-MSCs were treated with 0.5 to 640 µM metformin and their osteogenic differentiation capacity was examined by an alkaline phosphatase (ALP) activity assay, Alizarin red S staining and expression levels of osteogenic genes. The role of adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling in mediating the effect of metformin on the osteogenic differentiation capacity of PL-MSCs was also investigated by determining levels of phosphorylated AMPK (pAMPK)/AMPK ratio and by using compound C, an AMPK inhibitor.ResultsThe results showed that 10–160 µM metformin significantly increased the viability of PL-MSCs in a dose- and time-dependent manner. Furthermore, 80–320 µM metformin also increased ALP activity, matrix mineralization, and expression levels of osteogenic genes, runt-related transcription factor 2 (RUNX2), osterix (OSX), osteocalcin (OCN) and collagen I (COL1), in PL-MSCs. Metformin increases osteogenic differentiation of PL-MSCs, at least in part, through the AMPK signaling pathway, since the administration of compound C inhibited its enhancing effects on ALP activity, matrix mineralization, and osteogenic gene expression of PL-MSCs.ConclusionsThis study demonstrated that metformin at concentrations of 80–320 μM significantly enhanced osteogenic differentiation of PL-MSCs in a dose- and time-dependent manner, primarily through activation of the AMPK signaling pathway. This finding suggests that metformin could be used with other conventional drugs to induce bone regeneration in various bone diseases. Additionally, this study provides valuable insights for future osteoporosis treatment by highlighting the potential of modulating the AMPK pathway to improve bone regeneration.

Dental Pulp Stem Cell Conditioned Medium Enhance Osteoblastic Differentiation and Bone Regeneration.

Cell-free approaches, utilizing mesenchymal stem cell secretome, have promising prospects in various fields of regenerative medicine. In this study, we examined in vitro and in vivo the potential of dental pulp stem cell-conditioned medium (DPSC-CM) for bone regeneration. The secretome of undifferentiated stem cells from dental pulp were collected, and the effects of this DPSC-CM were assessed for osteodifferentiation of osteoblast-like cells (MG-63) and osteoblasts deriving from DPSC. Cell proliferation, alkaline phosphatase (ALP) activity, gene expression of Runt-related transcription factor 2 (Runx2), Bone Sialoprotein (BSP), Osteocalcin (OCN), and extracellular matrix mineralization were evaluated. The rat caudal vertebrae critical size defect model was to investigate the effect of DPSC-CM in vivo. Results showed that DPSC-CM induced cell growth, and increased ALP activity and the expression of key marker genes at an early stage of osteoblastic differentiation compared to control. A rat bone defect model was used to illustrate the effect of DPSC-CM in vivo. The bone density within the defects were improved using conditioned medium, even though there was no significant difference between the control and DPSC-CM groups. The analysis of DPSC-CM by human growth factor antibody array revealed the presence of several factors involved in osteogenesis. Taken together, these findings indicate that DPSC-CM is a promising therapeutic candidate for bone regenerative therapy, accelerating the maturation of osteoblastic cells. And even though safety and efficiency of DPSC-CM have to be confirmed in preclinical studies, these results represent a first step toward clinical application.

OPTIMIZED SOLID LIPID NANOPARTICLES FOR ENHANCED ORAL BIOAVAILABILITY AND OSTEOGENIC EFFECT OF IPRIFLAVONE: FORMULATION, CHARACTERIZATION, AND IN VITRO EVALUATION

Objective: This study aimed to enhance the oral bioavailability of Ipriflavone (IP) and evaluate its osteogenic effect on human osteosarcoma cells (MG-63) by developing Ipriflavone-loaded Solid Lipid Nanoparticles (IP-SLN). Methods: IP-SLNs were prepared using a modified solvent evaporation method with probe sonication. Formulation optimization employed Central Composite Design (CCD) with independent variables, including lipid amount, surfactant concentration, and sonication time. Characterization was performed using Transmission Electron Microscopy (TEM). In vitro drug release and ex vivo permeation studies were conducted to assess drug release kinetics and bioavailability. Cytotoxicity, Alkaline Phosphatase (ALP) activity, and calcium deposition studies on MG-63 cells evaluated osteogenic effects. Results: TEM images showed round particles with an average diameter of 43.24±3 nm, a zeta potential of-9.53 mV, and a drug entrapment efficiency of 76.53±1.84%. In vitro drug release from IP-SLN was 79.02% compared to 14.21% from IP after 48 h, following the Korsmeyer-Peppas model and first-order kinetics. Ex vivo permeation of IP-SLN was approximately 2-fold higher than IP dispersion. Cytotoxicity studies revealed no toxicity on MG-63 cells. ALP activity and calcium deposition studies indicated that IP-SLN stimulated osteoblast differentiation, increasing alkaline phosphatase activity and mineralization. Pharmacokinetic studies demonstrated that IP-SLN increased the relative bioavailability by 515% compared to ipriflavone. Conclusion: IP-SLN formulations significantly improved the oral bioavailability and osteogenic effects of ipriflavone on MG-63 cells, suggesting potential for novel therapeutic applications in osteoporosis treatment.

Calcium Phosphate Loaded with Curcumin Prodrug and Selenium Is Bifunctional in Osteosarcoma Treatments.

Although SeO32- ions have been loaded onto calcium phosphate to treat a wide range of cancers, the quest to promote bone tissue regeneration is still ongoing. Curcumin (cur), an herbal extraction, can selectively inhibit tumor cells and promote osteogenesis. In this study, SeO32- ions were co-precipitated in biomimetic calcium phosphate (Se@BioCaP), and modified curcumin prodrug (mcur) was adsorbed on diverse Se@BioCaP surfaces (mcur-Se@BioCaP-Ads). Co-precipitation yielded Se@BioCaP with a significantly higher Se content and exhibited a tailorable micro-/nanostructure. The favorable pH-responsive release of Se and mcur from mcur-Se@BioCaP-Ads showed a synergistic anticancer efficiency in OS cells, enhancing OS cell inhibition more than a single dose of them, which might be associated with ROS production in OS cells. In addition, increased alkaline phosphatase activity and calcium nodule formation in MC3T3-E1 pre-osteoblasts were also verified. These results suggest this novel mcur-Se@BioCaP-Ads has promising and widespread potential in OS treatments.

A Pt@ZIF-8/ALN-ac/GelMA composite hydrogel with antibacterial, antioxidant, and osteogenesis for periodontitis

Periodontitis is a chronic inflammatory disease caused by oral pathogens, and the osteogenic potential of human periodontal ligament stem cells (hPDLSCs) is severely impaired under the inflammatory environment. Current clinical periodontitis treatment strategies such as surgical interventions and antibiotic delivery still suffer from poor antibacterial efficacy, difficulty in ameliorating excessive inflammatory responses and slow periodontal tissue regeneration. Here, we have innovatively developed a non-surgical treatment strategy based on a functional composite hydrogel. A composite hydrogel system (Pt@ZIF-8/ALN-ac/Gel) containing bioactive zeolite imidazolate framework-8 (ZIF-8) integrated with platinum nanoparticles (Pt@ZIF-8) and alendronate acrylamide (ALN-ac) was constructed on the basis of gelatin methacryloyl (GelMA) to achieve enhanced antibacterial effect and reactive oxygen species (ROS) scavenging ability while promoting the osteogenic potential of hPDLSCs. We confirmed that Pt@ZIF-8/ALN-ac/Gel was able to continuously release Zn2+ and exerted an obvious antibacterial effect against Porphyromonas gingivalis. In vitro experiments proved that Pt@ZIF-8/ALN-ac/Gel had good biocompatibility, while efficiently featuring excellent reactive oxygen species (ROS) scavenging capacity, increasing alkaline phosphatase activity, and promoting extracellular matrix mineralization by hPDLSCs. In vivo, Pt@ZIF-8/ALN-ac/Gel significantly inhibited the alveolar bone deterioration and reduced osteoclast activation and inflammation, thereby promoting the regeneration of damaged tissues. These findings demonstrated superior therapeutic efficacy in the reported clinical periodontitis treatment, exhibiting great potential for application.

The effect of the composition of hepatoprotective action on biochemical and morphostructural changes in the body of laying hens under thermal stress

To reduce the negative effects of hyperthermia on the body of farm animals and poultry, various drugs and feed additives are currently used. Which do not have sufficiently adaptogenic and antitoxic properties. We studied the effect of a hepatoprotective composition consisting of dried live yeast, amorphous silicon dioxide, propylene glycol, calcium propionate, ascorbic acid, manganese, copper and zinc chelates, methionine and choline chloride on the variability of biochemical and morphological parameters of the body of laying hens under temperature stress. It was simulated by increasing the air temperature in a building where laying hens were kept from 18.0 ± 1.0 °C to 28.0 ± 1.0 °C for 48 hours. Due to hyperthermia, changes in biochemical and morphofunctional parameters were observed in the tissues and organs of birds. The obtained values of the biochemical parameters of blood serum in the birds of the control group indicated the intensity of the adaptive capabilities of their body. A complex of morphological changes confirmed a violation of protein metabolism and the regenerative-compensatory process. Pathological changes in the structure of the duodenum, characteristic of catarrhal-necrotic duodenitis, were identified. The stress reaction was also reflected in the condition of the heart muscle, in which an inflammatory process developed against the background of granular dystrophy of cardiomyocytes. The results of biochemical studies of blood serum in birds of the experimental group indicated an increase in the anti-stress response to a temperature stimulus under the influence of the studied composition (tendency to increase glucose and calcium, increase alkaline phosphatase activity by 47.4 %). The introduction of a hepatoprotective composition into the diet of laying hens during periods of temperature stress did not lead to disruption of the structure of tissues and organs, preserving cellular metabolic mechanisms. The

Effects of Biochar and Cladophora glomerata on Wheat (Triticum aestivum L.) Growth and Rhizosphere Enzyme Activities

The positive effects of biochar on both soil quality and plant growth and also on plant growth of macroalgae have been reported in studies. Studies on biochar and macroalgae interaction are quite limited. This study was carried out according to randomized plot design in greenhouse conditions to determine the effects of biochar and Cladophora glomerata applications and interaction on the growth of wheat (Triticum aestivum L.) and some enzyme activities in the rhizosphere. Biochar and C. glomerata interaction increased wheat root (90%) and shoot dry weight (84.2%), root length (43.1%) and plant height (84.2%) compared to control. Biochar application increased alkaline phosphatase activity by 66.3%, while C. glomerata increased β-glucosidase activity by 49%. The interaction of both applications increased catalase activity by 62.1% compared to control. These findings confirm the potential of biochar and C. glomerata to improve wheat production by inducing growth.

Orphan nuclear receptor NR4A1 regulates both osteoblastogenesis and adipogenesis in human mesenchymal stem cells.

The nuclear receptor subfamily 4 group A member 1 (NR4A1) gene plays a crucial role in both osteoporosis and adipogenesis. The present study investigated the mechanisms by which NR4A1 influences osteoblastogenesis and adipogenesis in human bone marrow‑derived mesenchymal stem cells (BMD‑MSCs). NR4A1 was overexpressed or knocked down in mouse MC3T3‑E1 osteoblast cells and 3T3‑L1 adipocyte cells, as well as in PCS‑500‑012, a BMD‑MSC line. The alkaline phosphatase (ALP) assay and Alizarin Red S staining were performed using MC3T3‑E1 and BMD‑MSCs to assess ALP activity and mineralization, while Oil Red O staining was used to assess the lipid content in 3T3‑L1 cells and BMD‑MSCs. Total RNA was isolated from control, NR4A1‑overexpressing and NR4A1 small interfering RNA (siRNA; siNR4A1)‑treated BMD‑MSCs. RNA sequencing (RNA‑seq) was performed to identify differentially expressed genes, followed by ingenuity pathway analysis (IPA) to determine the role of NR4A1 in osteoblastogenesis and adipogenesis. NR4A1 or Nr4a1 knockdown tended to increase ALP activity and significantly increased calcification in BMD‑MSCs (P<0.005) and MC3T3‑E1 cells (P<0.005), respectively. By contrast, NR4A1 or Nr4a1 overexpression significantly decreased ALP activity and calcification. NR4A1 or Nr4a1 knockdown and overexpression significantly decreased and increased adipogenesis, respectively, in BMD‑MSCs (P<0.005 and <0.05, respectively) and 3T3‑L1 cells (P<0.005 in both). Treatments of BMD‑MSCs with an NR4A1 antagonist, 1,1‑bis(3'‑indolyl)‑1‑(p‑hydroxyphenyl) methane and siNR4A1 showed similar results. RNA‑seq and IPA in control, NR4A1 knockdown and NR4A1 overexpressing cells indicated that Notch signaling mediated the effects of NR4A1 in osteoblastogenesis and adipogenesis. Expression of mastermind‑like transcriptional coactivator 3 was reduced in the Notch signaling pathway in cells treated with siNR4A1. In conclusion, NR4A1 suppressed osteoblastogenesis and promotes adipogenesis in human BMD‑MSCs. The present study also suggested that NR4A1 plays a role in the progression of osteoporosis and adipogenesis by modulating the Notch signaling cascade.

Effects of icariin on dental pulp stem cells and its potential applications in dentin repair

ObjectivesAs dental pulp therapy evolves towards regenerative approaches, biomolecules such as icariin, derived from Epimedium flowers, are being evaluated for their therapeutic potential. This study investigates icariin's effectiveness in promoting odontogenic differentiation in human dental pulp stem cells (hDPSCs) in vitro and as a pulp-capping agent in vivo. DesignThe study explored the effects of icariin on hDPSCs at concentrations of 10, 20, and 40 µM. Cell viability and migration assays were conducted to evaluate cytotoxicity and chemotaxis. Odontogenic differentiation was assessed using alkaline phosphatase staining and alizarin red S (ARS) staining, complemented by real-time PCR and Western blot analyses of key markers such as RUNX family transcription factor 2 (RUNX2), collagen type I alpha 1 chain (COL1A1), alkaline phosphatase (ALPL), and dentin sialophosphoprotein (DSPP). Additionally, the in vivo effects of icariin were tested in a rat maxillary molar model, where icariin-treated collagen sponges were used for direct pulp capping to evaluate its potential to induce reparative dentin formation. ResultsIcariin showed no cytotoxic effects on hDPSCs at any tested concentration, enhanced migratory activity in a dose-dependent manner, and significantly increased alkaline phosphatase activity and calcium deposition. Gene and protein expression analyses revealed a dose-dependent increase in odontogenic differentiation markers in icariin-treated hDPSCs. In vivo, icariin effectively promoted reparative dentin formation in exposed rat pulp. ConclusionsIcariin enhances odontogenic differentiation of hDPSCs and has promising potential as a pulp-capping agent for vital pulp therapy.

Osteogenic potential of human bone marrow stromal cell line HS-27A.

Human cell line HS-27A represents an immortalized subpopulation of human stromal cells derived from bone marrow. HS-27A cells meet the criteria set by the International Society of Cell Therapy (ISCT) for the classification as mesenchymal stem cells (MSCs) by expression of surface molecules CD73, CD90, CD105 and HLA-ABC with no expression of CD14, CD31, CD34, CD45 and HLA-DR. We hypothesized that these cells may undergo osteogenesis similar to human bone marrow-derived stromal cells (BMSCs) and serve as a model of osteogenic cell responses under normal and inflammatory conditions. HS-27A cells were treated with: standard osteogenic factors, i.e., ascorbic acid (Asc), dexamethasone (Dex) and β-glycerophosphate (BGP); recombinant human bone morphogenetic protein 2 (rhBMP-2), MEK1/2 kinase inhibitor (PD98059) and/or lipopolysaccharide (LPS). Cells were examined for alkaline phosphate (ALP) activity, mRNA expression of osteoblastic markers (qRT-PCR) and mineralization of extracellular matrix. Besides, we analyzed mRNA expression of proinflammatory cytokines IL-1β and TNF-α in LPS-treated cells and evaluated transfection efficiency of these cells with Lipofectamine 3000 for potential future genetic manipulations. We determined that HS-27A cell line increase alkaline phosphatase activity (P<0.05), while mineralization of extracellular matrix remains low. Cells treatment with rhBMP-2 and PD98059 resulted in increased mRNA levels for osteogenesis-related transcription factor MSX-2, bone sialoprotein and osteocalcin (all P<0.05). In osteogenic cultures with and without rhBMP-2, addition of LPS led to increased ALP activity, mRNA levels for collagen type I and osteocalcin as well for IL-1β and TNF-α (all P<0.05). We also show that HS-27A cells can be transfected with phMGFP plasmid using Lipofectamine 3000 with low efficiency that may be sufficient for some genetic manipulations. Thus, HS-27A cell line appear as a useful in vitro cell culture model to study short-term osteogenic and inflammatory-related responses of human bone marrow stromal cells.

Nanocrystal hydroxyapatite carrying traditional Chinese medicine for osteogenic differentiation

Developing biomaterials with high osteogenic properties is crucial for achieving rapid bone repair and regeneration. This study focuses on the application of nanocrystal hydroxyapatite (nHAp) as a drug carrier to load Fu Yuan Huo Xue Decoction (FYHXD), a traditional Chinese medicine derived from Angelica sinensis, aiming to achieve improved efficacy in treating bone diseases such as osteoporosis. Through a facile physical adsorption approach, the FTIR result emerges new characteristic absorption peaks in the range of 1200–950 cm−1, proving the successful absorption of FYHXD onto the nHAp with a loading efficiency of 39.76 %. The modified nHAp exhibits a similar shape to the bone-derived hydroxyapatite nanocrystals, and their diameter increases slightly after modification. The drug release assay implies the rapid release of FYHXD in the first 10 h, followed by a continuously slow release within 70 h. The developed nHAp effectively enhances the adhesion, spreading, and proliferation of MC3T3-E1 cells in vitro, and significantly promotes their osteogenic differentiation, as indicated by increased alkaline phosphatase activity. Overall, the biocomposites hold great promise as active ingredients for integration into bone-associated biomaterials, offering the potential to stimulate spontaneous osteogenesis without requiring exogenous osteogenic factors.

Development of Biocompatible Coatings with PVA/Gelatin Hydrogel Films on Vancomycin-Loaded Titania Nanotubes for Controllable Drug Release.

This study investigates the utilization of poly(vinyl alcohol) (PVA)/gelatin hydrogel films cross-linked with glutaraldehyde as a novel material to coat the surface of vancomycin-loaded titania nanotubes (TNTs), with a focus on enhancing biocompatibility and achieving controlled vancomycin release. Hydrogel films have emerged as promising candidates in tissue engineering and drug-delivery systems due to their versatile properties. The development of these hydrogel films involved varying the proportions of PVA, gelatin, and glutaraldehyde to achieve the desired properties, including the gel fraction, swelling behavior, biocompatibility, and biodegradation. Among the formulations tested, the hydrogel with a PVA-to-gelatin ratio of 25:75 and 0.2% glutaraldehyde was selected to coat vancomycin-loaded TNTs. The coated TNTs demonstrated slower release of vancomycin compared with the uncoated TNTs. In addition, the coated TNTs demonstrated the ability to promote osteogenesis, as evidenced by increased alkaline phosphatase activity and calcium accumulation. The vancomycin-loaded TNTs coated with hydrogel film demonstrated effectiveness against both E. coli and S. aureus. These findings highlight the potential benefits and therapeutic applications of using hydrogel films to coat implant materials, offering efficient drug delivery and controlled release. This study contributes valuable insights into the development of alternative materials for medical applications, thereby advancing the field of biomaterials and drug delivery systems.

Effect of Bidens tripartita leaf supplementation on the organism of rats fed a hypercaloric diet high in fat and fructose

Herbs play an important role in folk medicine, and scientific research has confirmed the properties of their use as an alternative treatment, including the treatment and mild correction of metabolic disorders during disease. Trifid bur-marigold (Bidens tripartita) is a pharmacopoeial herbal raw material that is widely used in clinical practice as an external remedy for skin lesions and as an internal remedy for digestive and respiratory disorders. In this work, the general effect of dried leaves of B. tripartita on physiological activity and metabolic processes in model animals on a high-calorie diet was determined. For the experiment, three groups of 18 male white laboratory rats were formed and fed a hypercaloric diet (increased fat content and 20% fructose solution instead of water) for 27 days, in addition to 0.4% and 4.0% dried leaves of B. tripartita. The consumption of 0.4% and 4.0% of the medicinal plant resulted in a significant delay in the body weight gain and the average daily weight gain of the rats compared to the control group. Dried leaves of B. tripartita in the diet of rats decreased the relative weight of the thymus and increased the relative weight of the brain, and at a dose of 4.0%, increased the relative weight of the lungs and individual large intestines (cecum and colon). Dietary supplementation with B. tripartita caused an increase in globulin concentration and changes in protein coefficient. Blood parameters such as: urea, urea nitrogen, inorganic phosphorus, glucose and bilirubin levels changed depending on the dose. In the general blood test, consumption of dried leaves of B. tripartita caused a decrease in hematocrit, hemoglobin concentration and platelet count, but increased the number of eosinophils. Bidens tripartita at both concentrations significantly increased ALT activity with a corresponding change in the blood De Ritis ratio. The addition of 20 g of B. tripartita leaves to the diet increased alkaline phosphatase activity and decreased alpha-amylase activity, while 200 g increased blood gamma-glutamyltransferase activity. At the end of the experiment, the rats' orientation activity, determined in the open field, changed according to the herb dose consumed: 0.4% leaves caused an increase and 4.0% a decrease. Physical activity was reduced and emotional state increased, regardless of the dose of dried B. tripartita leaves, compared to the control group of animals. The results obtained show that the addition of B. tripartita dried leaves as a dietary supplement to a high-calorie diet is safe, does not cause pathological changes or side effects, and has a significant effect on metabolic processes. This provides theoretical support for the use of B. tripartita dried leaves in the manufacture of nutraceutical and pharmacological products for the correction of metabolic disorders in humans and animals. The doses and duration of their application require further studies.