Enhanced Alkaline Phosphatase Activity Research Articles

High molecular weight hyaluronic acid alleviates ovariectomy-induced bone loss in mice

BackgroundThe rapid decline in ovarian function associated with menopause promotes osteoclast differentiation and increases bone resorption, disrupting of bone homeostasis and increasing the risk of osteoporosis. Hyaluronic acid (HA) is a polysaccharide ubiquitously present in the connective tissues. Recent reports indicate that high-molecular-weight HA (HMW-HA) promotes osteoblast proliferation, enhances alkaline phosphatase activity and mineral deposition, and promotes the expression of bone differentiation markers, such as Runx2 and osteocalcin. HMW-HA also inhibits the expression of the receptor activator of nuclear factor kappa-B ligand (RANKL) in osteoblasts. These results suggest that HMW-HA may be an effective therapeutic agent against postmenopausal osteoporosis. Therefore, this study aimed to examine whether HMW-HA alleviates ovariectomy (OVX)-induced bone loss in mice.MethodsEight-week-old female C57BL/6 J mice were randomly divided into the following five groups: Group 1: Sham/saline, Group 2: OVX/saline, Group 3: OVX/HMW-HA [15 mg/kg]; Group 4: OVX/HMW-HA [30 mg/kg]; and Group 5: OVX/HMW-HA [60 mg/kg]. Mice were administered HMW-HA or saline subcutaneously starting from 1 week after OVX and changes in bone mass were analyzed at 5 weeks using three-dimensional micro-computed tomography (3D-μCT). In addition, changes in osteoclast parameters were analyzed histologically.ResultsThe reduction in trabecular bone volume and trabecular number was significantly ameliorated in the OVX/HMW-HA group compared with that observed in the OVX/saline group, along with a significant inhibition of the increase in trabecular spacing. In addition, the OVX/HMW-HA group exhibited a significant reduction in osteoclast surface area and number compared with the OVX/saline group, with no significant differences compared with the sham group. In vitro experiments revealed that depletion of HMW-HA from the culture medium by hyaluronidase treatment increased RANKL expression in the bone marrow stromal cell line ST2. These data suggest that HMW-HA alleviates OVX-induced bone loss by downregulating osteoclast formation and/or activity in mice.ConclusionHMW-HA is a potential novel therapeutic agent for osteoporosis.

Biochar promotes the dissolution of inorganic inactive phosphorus by mediating the bacterial community during corn stover and cattle manure composting.

Phosphorus (P) is a macroelement primarily found in insoluble forms in nature. Enhancing the effectiveness of P is crucial for sustainable agricultural development and ecosystems. The research employed a combination of sequential extraction methods, high-throughput sequencing techniques, microbial culturing, and ecological network analysis of bacterial communities, along with module comparison, to explore the dynamics of different P fractions in calcareous soils. The objective of incorporating biochar into the composting of maize stover and cattle dung was to uncover potential microbial processes that could facilitate the activation of inorganic non-labile P. Findings revealed that during the composting process with biochar, bacterial populations played three distinct roles in the transformation of inorganic non-labile P compounds (such as occluded P and Ca10-P). Primarily, the introduction of biochar significantly increased both the diversity and abundance of bacterial communities. Additionally, it enhanced the ability of phosphate-solubilizing bacteria to maintain the structure of bacterial ecological networks by boosting their complexity, interconnectedness, and stability. Moreover, the incorporation of biochar stimulated the P-related metabolic activities within the bacterial community, significantly enriching key metabolic pathways such as the citrate (TCA) cycle, glycolysis/gluconeogenesis, the pentose phosphate pathway, galactose metabolism, starch, and sucrose metabolism, as well as the metabolism of amino and nucleotide sugars. Moreover, biochar addition intensified the connections between key operational taxonomic units (OTUs) and non-labile P while simultaneously increasing the total organic carbon concentration and enhancing alkaline phosphatase activity. This study provides valuable insights for enhancing P effectiveness in calcareous soils.

The impact of graphene quantum dots on osteogenesis potential of Wharton's jelly mesenchymal stem cells in fibrin hydrogel scaffolds.

Bone tissue engineering is a promising approach to overcome the limitations of traditional autograft bone transplantation. Graphene quantum dots (GQDs) have been suggested as an enhancement for osteogenic differentiation. This study aimed to investigate the ability of the fibrin hydrogel scaffold in the presence of graphene quantum dots to promote osteogenic differentiation of human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs). The hWJ-MSCs were isolated from the Wharton's jelly of the human umbilical cord using a mechanical method. Fibrin hydrogel scaffolds were prepared by mixing 15 µl of thrombin solution with fibrinogen solution. GQDs were incorporated into the scaffolds at concentrations of 0, 5, and 10 µg/ml. Cell viability was determined through DAPI staining and the MTT assay. Osteogenic differentiation was assessed by measuring alkaline phosphatase (ALP) activity, quantifying calcium deposition using Alizarin Red S staining, and analyzing the gene expression of BGLAP, COL1A1, Runx-2 and ALP via qPCR. Scanning electron microscopy (SEM) was employed to analyze the scaffold architecture. SEM analysis revealed that the fibrin hydrogel exhibited a suitable architecture for tissue engineering, and DAPI staining confirmed cell viability. The MTT results indicated that the GQDs and fibrin hydrogel scaffold exhibited no cytotoxic effects. Furthermore, the incorporation of GQDs at a concentration of 10 µg/ml significantly enhanced ALP activity, calcium deposition, and the expression of osteogenesis-related genes compared to the control. The findings suggest that the combination of fibrin hydrogel and GQDs can effectively promote the osteogenic differentiation of hWJ-MSCs, contributing to the advancement of bone tissue engineering.

Integrated MicroRNA-mRNA Analyses of the Osteogenic Differentiation of Human Dental Pulp Stem Cells by a Helioxanthin Derivative.

Dental pulp stem cells (DPSCs) demonstrate high proliferative and multilineage differentiation potential. As previously reported, the helioxanthin derivative 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH) has been demonstrated to induce the osteogenic differentiation of DPSCs. However, the mechanism of osteogenesis induced by TH in DPSCs remains unknown. The objective of this study was to identify functional extracellular vesicle (EV) microRNAs (miRNAs), and the principal genes involved in the TH-induced osteogenesis of DPSCs. DPSCs were derived from dental pulp extracted from the third molars of three healthy subjects, and were cultured with or without TH. miRNAs were extracted from DPSC-derived EVs. The gene expression patterns of mRNA and miRNA were compared using RNA-Seq and miRNA-Seq. To investigate miRNA/mRNA interacting networks, functional analyses were performed by Ingenuity Pathway Analysis. Alkaline phosphatase (ALP) staining demonstrated that treatment with TH resulted in enhanced ALP activity in DPSCs after 7 days. The expression levels of ALP and type 1 collagen alpha 1 were significantly higher in TH-induced DPSCs on day 7. RNA-Seq and miRNA-Seq analyses identified 869 differentially expressed genes (DEGs) and 18 miRNA-DEGs. Gene Ontology analysis of the mRNA-Seq results showed that TH induced several biological activities associated with signal transduction, cell adhesion, and cell differentiation. Integrated miRNA-mRNA analyses showed that these miRNAs contain the targeting information of 277 mRNAs of the DEGs. Among them, 17 target genes known to be involved in the differentiation of osteoblasts, and 24 target genes known to be involved in the differentiation of bone cells were identified. Quantitative real-time PCR showed that WNT5a expression in DPSCs was upregulated by 48 h of TH treatment. Upstream regulator analysis indicated that WNT3a, FOS, and RAC1 may be responsible for gene expression changes in DPSCs after TH treatment. EV miRNA regulatory networks might play crucial roles in TH-induced osteogenic differentiation of DPSCs. Our results presented herein offer valuable insights that will facilitate further research into the mechanism of osteogenesis of DPSCs, which is expected to lead to the clinical application of TH-induced DPSCs for bone regeneration. Furthermore, EVs derived from TH-induced DPSCs might be useful as therapeutic tools for bone defects.

Fabrication of 3D microfibrous composite polycaprolactone/hydroxyapatite scaffolds loaded with piezoelectric poly (lactic acid) nanofibers by sequential near-field and conventional electrospinning for bone tissue engineering

Near-field electrospinning (NFES) has recently gained considerable interest in fabricating tissue engineering scaffolds. This technique combines the advantages of both 3D printing and electrospinning. It allows for the production of fibers with smaller resolution and the ability to make regular structures with suitable pores. In this study, a microfibrous composite scaffold of polycaprolactone (PCL)/hydroxyapatite (HA) was prepared by NFES in the first step. The microfibrous scaffold had a fiber spacing of 414.674 ± 24.9 μm with an average fiber diameter of 94.695 ± 16.149 μm. However, due to the large fiber spacing, the surface area was insufficient for cell adhesion. Therefore, the hybrid scaffold was prepared by adding aligned and random electrospun poly (L-lactic acid) (PLLA) nanofibers to the microfibrous scaffold. Cellular studies showed that cell adhesion to the hybrid scaffold increased by 334 % compared to the microfibrous scaffold. These nanofibers also exhibited piezoelectric properties, which helped stimulate bone regeneration. Aligned nanofibers in the hybrid scaffold enhanced alkaline phosphatase activity and the intensity of alizarin red staining 1.5 and 1.6 times, respectively, compared to the microfibrous scaffold. Furthermore, the elastic modulus and ultimate tensile strength increased by 268 % and 130 %, respectively, by adding aligned nanofibers to the microfibrous scaffold. Therefore, the hybrid microfibrous composite scaffold of PCL/HA containing aligned electrospun PLLA nanofibers with improved properties showed the potential for bone regeneration.

NQO1 promotes osteogenesis and suppresses angiogenesis in DPSCs via MAPK pathway modulation

BackgroundInfluence on stem cells’ angiogenesis and osteogenesis of NAD(P)H Quinone Dehydrogenase 1(NQO1) has been established, but its impact on dental pulp stem cells (DPSCs) is unexplored. An important strategy for the treatment of arteriosclerosis is to inhibit calcium deposition and to promote vascular repair and angiogenesis. This study investigated the function and mechanism of NQO1 on angiogenesis and osteogenesis of DPSCs, so as to provide a new ideal for the treatment of arteriosclerosis.MethodsCo-culture of human DPSCs and human umbilical vein endothelial cells (HUVECs) was used to detect the angiogenesis ability. Alkaline phosphatase (ALP) activity, alizarin red staining (ARS), and transplantation of HA/tricalcium phosphate with DPSCs were used to detect osteogenesis.ResultsNQO1 suppressed in vitro tubule formation, migration, chemotaxis, and in vivo angiogenesis, as evidenced by reduced CD31 expression. It also enhanced ALP activity, ARS, DSPP expression and osteogenesis and boosted mitochondrial function in DPSCs. CoQ10, an electron transport chain activator, counteracted the effects of NQO1 knockdown on these processes. Additionally, NQO1 downregulated MAPK signaling, which was reversed by CoQ10 supplementation in DPSCs-NQO1sh.ConclusionsNQO1 inhibited angiogenesis and promoted the osteogenesis of DPSCs by suppressing MAPK signaling pathways and enhancing mitochondrial respiration.

Heraclenin promotes the osteogenic differentiation of bone marrow stromal cells by activating the RhoA/ROCK pathway.

Osteoporosis is a devastating skeletal disease, the pathogenesis of which is related to abnormal bone metabolism, featured by the imbalance between osteoblastic bone formation and osteoclastic bone resorption. Stem cell-based therapies have been demonstrated to improve osteoporosis treatment. Previously, the linear furanocoumarin heraclenin was reported to enhance osteoblast differentiation and mineralization in mouse mesenchymal stem cells (MSCs), suggesting its potential for osteogenic differentiation and bone regeneration. Our study was designed to confirm the promotive role of heraclenin on osteogenic differentiation of human bone MSCs (BMSCs) and explore the underlying mechanisms. Human BMSCs were treated for 24, 48, and 72h with heraclenin (5, 10, 20, 40, and 80 μM), and cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. To further evaluate the cytotoxicity of heraclenin, cell suspension obtained from BMSCs treated with heraclenin (5, 10, and 20 μM) for 72h was subjected to a MUSE™ cell analyzer for cell viability and count assay. BMSCs were incubated in osteogenic induction medium for 7 days. Then, osteogenic differentiation and mineralization of BMSCs were assessed through alkaline phosphatase (ALP) and Alizarin Red S staining. The expression of osteogenesis markers including ALP, osteocalcin (OCN), osterix (OSX), and runt-related transcription factor 2 (RUNX2) was detected via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The effects of heraclenin on the RhoA/ROCK pathway were estimated through western blotting. Y-27632, the ROCK inhibitor, was used to confirm the role of the RhoA/ROCK pathway in heraclenin-mediated osteogenic differentiation of BMSCs. Heraclenin (5-80 μM) was non-toxic on human BMSCs. Heraclenin treatment (5-20 μM) dose-dependently enhanced ALP activity and calcium deposition. Furthermore, heraclenin promoted ALP, OCN, OSX, and RUNX2 mRNA and protein expression. Mechanically, heraclenin treatment increased RhoA and ROCK1 mRNA expression, stimulated the translocation of ROCK from the cytosolic to the membrane fraction, and elevated the protein levels of phosphorylated cofilin (p-cofilin) and active RhoA. Additionally, treatment with Y-27632 overturned the promotion of heraclenin on ALP activity, calcium deposition, the expression of osteogenesis markers, and the RhoA/ROCK signaling pathway. Heraclenin facilitates the osteogenic differentiation of human BMSCs through the activation of the RhoA/ROCK pathway.

A comprehensive investigation of strontium doped hydroxyapatite-merwinite composite: In-vitro biomineralization, mechanical testing, and cytotoxicity analysis

The advancement of biomaterials for reconstruction and replacement of damaged tissues is essential, which would possess biocompatibility, better mechanical, biochemical, and physical properties. Merwinite (Ca3MgSi2O8) has emerged as a promising material for hard tissue engineering, particularly in composite with hydroxyapatite (HAp). Strontium-doped hydroxyapatite (SrHAp) was prepared with varying dopant concentrations (1%, 3%, and 5% Sr) on the calcium site of HAp. The materials were analyzed by XRD, FTIR and SEM/EDX techniques. XRD analysis revealed average crystallite sizes ranging from 46 to 50 nm for SrHAp. It was found that the Merwinite-SrHAp composite doped with 5% strontium had a higher rate of apatite nucleation compared to pure composite, indicating that strontium doping enhances the bioactivity of the composite. The composite scaffolds, prepared in an 80:20 ratio of SrHAp to merwinite, exhibited compressive strengths ranging from 63.43 MPa (1% Sr doping) to 80.68 MPa (5% Sr doping). Cytotoxicity assessment against MG63 cell line via MTT assay demonstrated cell inhibition rates ranging from 11.71% (at 6.5 μg/mL) to 68.35% (at 100 μg/mL) for the 5% Sr-doped composite. Alkaline phosphatase (ALP) investigation revealed enhanced ALP activity for cells cultured on the 5% Sr-doped composite compared to control samples, indicating increased cell differentiation and mineralization potential.

Alendronate releasing silk fibroin 3D bioprinted scaffolds for application in bone tissue engineering: Effects of alginate concentration on printability, mechanical properties and stability

3D bioprinting uses biomaterials combined with cells to develop living constructs. This study explores the optimization of natural polymers, including silk fibroin, gelatin, and alginate, as bioink for 3D bioprinting in bone tissue engineering. The physicochemical properties of the bioink were thoroughly examined, revealing high water uptake and reduced degradation rate due to the addition of silk fibroin. The compressive modulus increased with higher alginate concentration. Rheological analysis confirmed shear-thinning properties and viscoelasticity of the ink. Through meticulous parameter optimization, the ink achieved the highest print accuracy with 4 % w/v alginate content. The printed scaffolds exhibited both macro and micro porosity, making them suitable for bone tissue regeneration. Furthermore, the scaffolds remained stable in culture medium for 36 days. The optimal composition for the hydrogel was determined to be a blend of 5 % fibroin, 7 % gelatin, and 4 % alginate in equal ratios. The bioink demonstrated excellent biocompatibility and, when supplemented with alendronate, enhanced alkaline phosphatase activity in MG-63 osteoblast-like cells. This finding indicates the commitment of cells toward the osteoblastic phenotype. Overall, this study successfully optimized the bioink and bioprinting process for bone tissue engineering applications, highlighting its promising potential for future advancements in the field.

The core phoD-harboring bacteria promote wheat phosphorus uptake by enhancing alkaline phosphatase activity under long-term fertilization

The core phoD-harboring bacteria promote wheat phosphorus uptake by enhancing alkaline phosphatase activity under long-term fertilization

Enhancing immune modulation and bone regeneration on titanium implants by alleviating the hypoxic microenvironment and releasing bioactive ions

Bone implantation inevitably causes damage to surrounding vasculature, resulting in a hypoxic microenvironment that hinders bone regeneration. Although titanium (Ti)-based devices are widely used as bone implants, their inherent bioinert surface leads to poor osteointegration. Herein, a strontium peroxide (SrO2)-decorated Ti implant, Ti_P@SrO2, was constructed through coating with poly-L-lactic acid (PLLA) to alleviate the hypoxic microenvironment and transform the bioinert surface of the implant into a bioactive surface. PLLA degradation resulted in an acidic microenvironment and the release of SrO2 nanoparticles. The acidic microenvironment then accelerated the decomposition of SrO2, resulting in the release of O2 and Sr ions. O2 released from Ti_P@SrO2 can alleviate the hypoxic microenvironment, thus enhancing cell proliferation in an O2-insufficient microenvironment. Furthermore, under hypoxic and normal microenvironments, Ti_P@SrO2 enhanced alkaline phosphatase activity and bone-related gene expression in C3H10T1/2 cells with the continuous release of Sr ions. Meanwhile, Ti_P@SrO2 suppressed M1 polarization and promoted M2 polarization of bone marrow-derived monocytes under hypoxic and normal conditions. Furthermore, in a rat implantation model, the implant enhanced new bone formation and improved osteointegration after modification with SrO2. In summary, the newly designed O2- and Sr ion-releasing Ti implants are promising for applications in bone defects.

Rice residue management alternatives in rice–wheat cropping system: impact on wheat productivity, soil organic carbon, water and microbial dynamics

In the Indo-Gangetic Plains (IGP), rice–wheat cropping system (RWCS) predominates, producing large quantity of crop residue and its management is major concern. Farmers usually burn the residue to clear the field for succeding crop, and burning damages soil microbes, resulted in loss of soil organic matter. Hence, current study was conducted to assess the impact of different Happy seeder based residue management options on changes in microbial dynamics, enzyme activities and soil organic matter content and also to know that alternative method for attaining sustainable wheat productivity in sandy loam soils of Haryana, India. Results revealed that Zero tillage wheat (ZTW) with partial and full residue retention treatments sown with Happy seeder (after using chopper and spreader), and ZTW with anchored stubbles significantly enhanced soil microbial count by 47.9–60.4%, diazotropic count by 59.0–73.1% and actinomycetes count by 47.3–55.2%, grain yield by 9.8–11.3% and biomass yield by 7.4–9.6% over conventional tilled (CT) residue burning and residue removal plots. ZTW sown with surface retention of rice crop residue increased the organic carbon by 0.36–0.42% and the soil moisture content by 13.4–23.6% over CTW without residue load. Similarly, ZTW sown with Happy seeder with full residue enhanced alkaline phosphatase activity from 95.3 µg TPF g−1 soil 24 h−1 in 2018–2019 to 98.6 µg TPF g−1 soil 24 h−1 in 2019–2020 over control plots. Likely, microbial population and enzymatic activity showed strong positive correlation under variable residue retention practices. However, increased microbial population reduced the soil pH from 7.49 to 7.27 under ZTW with residue retention plots. The wheat yield enhanced by 9.8–11.3% during 2018–2019 and 2019–2020 under ZTW with Happy seeder with full residue load over residue burning and residue removal plots. ZTW sown with Happy seeder under full residue retention, achieved maximum net return 43.16–57.08 × 103 ₹ ha−1) and B-C ratio (1.52 to 1.70) over CTW without residue. Therefore, rice residue needs to be managed by planting wheat using appropriate machinery under ZT for sustaining higher productivity in RWCS and improve soil health and environment under IGP regions.

In situ co-deposition synthesis for collagen-Astragalus polysaccharide composite with intrafibrillar mineralization as potential biomimetic-bone repair materials.

A hybrid material possessing both componential and structural imitation of bone tissue is the preferable composites for bone defect repair. Inspired by the microarchitecture of native bone, this work synthesized in vitro a functional mineralized collagen fibril (MCF) material by utilizing the method of in situ co-precipitation, which was designed to proceed in the presence of Astragalus polysaccharide (APS), thus achieving APS load within the biomineralized collagen-Astragalus polysaccharide (MCAPS) fibrils. Transmission electron microscope (TEM), selected area electron diffraction (SAED) and scanning electronic microscopy (SEM) identified the details of the intrafibrillar mineralization of the MCAPS fibrils, almost mimicking the secondary level of bone tissue microstructure. A relatively uniform and continuous mineral layer formed on and within all collagen fibrils and the mineral phase was identified as typical weak-crystalline hydroxyapatite (HA) with a Ca/P ratio of about 1.53. The proliferation of bone marrow-derived mesenchymal stem cells (BMSC) and mouse embryo osteoblast precursor cells (MC3T3-E1) obtained a significant promotion by MCAPS. As for the osteogenic properties of MCAPS, a distinct increase in the alkaline phosphatase (ALP) activity and the number of calcium nodules (CN) in BMSC and MC3T3-E1 was detected. The up-regulation of three osteogenic-related genes of RUNX-2, BMP-2 and OCN were confirmed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to further verify the osteogenic performance promotion of MCAPS. A period of 14 days of culture demonstrated that MCAPS-L exhibited a preferable efficacy in enhancing ALP activity and CN quantity, as well as in promoting the expression of osteogenic-related genes over MCAPS-M and MCAPS-H, indicating that a lower dose of APS within the material of MCAPS is more appropriate for its osteogenesis promotion properties.

Acemannan coated, cobalt-doped biphasic calcium phosphate nanoparticles for immunomodulation regulated bone regeneration.

Biomaterials are used as scaffolds in bone regeneration to facilitate the restoration of bone tissues. The local immune microenvironment affects bone repair but the role of immune response in biomaterial-facilitated osteogenesis has been largely overlooked and it presents a major knowledge gap in the field. Nanomaterials that can modulate M1 to M2 macrophage polarization and, thus, promote bone repair are known. This study investigates a novel approach to accelerate bone healing by using acemannan coated, cobalt-doped biphasic calcium phosphate nanoparticles to promote osteogenesis and modulate macrophage polarization to provide a prohealing microenvironment for bone regeneration. Different concentrations of cobalt were doped in biphasic calcium phosphate nanoparticles, which were further coated with acemannan polymer and characterized. The nanoparticles showed >90% cell viability and enhanced cell proliferation along with osteogenic differentiation as demonstrated by the enhanced alkaline phosphatase activity and osteogenic calcium deposition. The morphology of MC3T3-E1 cells remained unchanged even after treatment with nanoparticles. Acemannan coated nanoparticles were also able to decrease the expression of M1 markers, iNOS, and CD68 and enhance the expression of M2 markers, CD206, CD163, and Arg-1 as indicated by RT-qPCR, flow cytometry, and ICC studies. The findings show that acemannan coated nanoparticles can create a supportive immune milieu by inducing and promoting the release of osteogenic markers, and by causing a reduction in inflammatory markers, thus helping in efficient bone regeneration. As per our knowledge, this is the first study showing the combined effect of acemannan and cobalt for bone regeneration using immunomodulation. The work presents a novel approach for enhancing osteogenesis and macrophage polarization, thus, offering a potent strategy for effective bone regeneration.

The Enrichment of Whey Protein Isolate Hydrogels with Poly-γ-Glutamic Acid Promotes the Proliferation and Osteogenic Differentiation of Preosteoblasts.

Osseous disease accounts for over half of chronic pathologies, but there is a limited supply of autografts, the gold standard; hence, there is a demand for new synthetic biomaterials. Herein, we present the use of a promising, new dairy-derived biomaterial: whey protein isolate (WPI) in the form of hydrogels, modified with the addition of different concentrations of the biotechnologically produced protein-like polymeric substance poly-γ-glutamic acid (γ-PGA) as a potential scaffold for tissue regeneration. Raman spectroscopic analysis demonstrated the successful creation of WPI-γ-PGA hydrogels. A cytotoxicity assessment using preosteoblastic cells demonstrated that the hydrogels were noncytotoxic and supported cell proliferation from day 3 to 14. All γ-PGA-containing scaffold compositions strongly promoted cell attachment and the formation of dense interconnected cell layers. Cell viability was significantly increased on γ-PGA-containing scaffolds on day 14 compared to WPI control scaffolds. Significantly, the cells showed markers of osteogenic differentiation; they synthesised increasing amounts of collagen over time, and cells showed significantly enhanced alkaline phosphatase activity at day 7 and higher levels of calcium for matrix mineralization at days 14 and 21 on the γ-PGA-containing scaffolds. These results demonstrated the potential of WPI-γ-PGA hydrogels as scaffolds for bone regeneration.

A Biomimetic Nanogenerator to Enhance Bone Regeneration by Restoring Electric Microenvironments.

Piezoelectric materials have received increasing attention in bone regeneration due to their prominent role in bioelectricity in bone homeostasis. This study aimed to develop bioactive barium titanate-chitosan-graphene oxide piezoelectric nanoparticles (BCG-NPs) to improve biocompatibility and stimulate bone repair. Butterfly loops, hysteresis loops, and in vitro microcurrent studies on BCG-NPs confirmed their good piezoelectric properties. BCG-NPs exhibited enhanced alkaline phosphatase activity, mineralized nodule formation, and expression of osteogenic-associated proteins and genes in human umbilical cord Wharton's jelly-derived mesenchymal stem cells by creating microelectric environments in response to noninvasive ultrasound stimulation. Further, BCG-NPs upregulated intracellular calcium ions via electrical stimulation. They acted synergistically with piezo-type mechanosensitive ion channel component 1 and calcium-permeable cation channel transient receptor potential vanilloid 4 to activate osteogenic differentiation. In conclusion, ultrasound-assisted BCG-NPs created a microelectric environment that putatively promoted bone repair in a noninvasive manner.

Incorporating pH/NIR responsive nanocontainers into a smart self-healing coating for a magnesium alloy with controlled drug release, bacteria killing and osteogenesis properties

Magnesium (Mg)-based orthopedic implant materials can potentially be protected from deterioration using a protective polymer coating. However, this coating is susceptible to excessive corrosion and accidental scratches. Moreover, the inadequate bone integration and infections associated with bone implants present additional challenges that hinder their effective use. In this work, a spin-spray layer-by-layer (SSLbL) assembly technique was employed to develop a smart self-healing coating for Mg alloy WE43. This coating was based on paeonol-encapsulated nanocontainers (PMP) that were modified with a stimuli-responsive polydopamine (PDA). The leached paeonol could form a compact chelating layer when complexed with Mg2+ ions. Dynamic reversible hydrogen bonds were formed between assembly units, which ensured that the hybrid coating possessed rapid and cyclic self-healing properties. Under 808 nm near-infrared (NIR) laser irradiation, the self-healing coating exhibited antibacterial properties due to the synergistic effects of hyperthermia, reactive oxygen species (ROS), and paeonol. In addition, the incorporation of nanoparticles into the hybrid coating led to improvements in the cytocompatibility and osteogenic properties of the implant material. The smart coating enhanced alkaline phosphatase activity, extracellular matrix (ECM) mineralization, and the expression of osteogenic genes. This study presents a promising opportunity to explore the application of a smart self-healing coating for a Mg alloy. Statement of significanceHerein, we report a self-healing coating comprised of polyethyleneimine and nanocontainer-crosslinked hyaluronic acid to achieve drug-controlled release, antimicrobial activity, and osteogenesis performance. The formation of hydrogen bonds between HA and PEI facilitated the self-assembly process, thereby improving the coating's corrosion resistance and adhesion strength. The hybrid coating exhibited a rapid and cyclic self-healing activity due to paeonol and dynamic reversible bonds. The release of paeonol was controlled by pH and NIR stimuli owing to polydopamine modification. In vitro testing revealed that the hybrid coating achieved effective bacteria eradication through synergistic effects of hyperthermia, reactive oxygen species, and paeonol. Moreover, the smart coating was found to enhance alkaline phosphatase activity, extracellular matrix mineralization, and the expression of osteogenic genes.

Icariin promotes osteogenic differentiation through the mmu_circ_0000349/mmu-miR-138-5p/Jumonji domain-containing protein-3 axis

Circular RNAs (circRNAs) regulate Jumonji domain-containing protein-3 (JMJD3) by sponging with microRNAs (miRNAs). This study aimed to investigate the role of icariin on specific circRNA/miRNA/JMJD3 axis in osteogenic differentiation of MC3T3-E1 cells. CircRNA sequencing was performed on the MC3T3-E1 cells induced by osteogenic differentiation medium for 1 d (negative control (NC) group) and 14 d (osteogenesis group). And mmu_circ_0000349 was verified using Sanger sequencing, ribonuclease R degradation, and actinomycin D assay. The function of mmu_circ_0000349 was validated by detecting the expressions of osteogenic differentiation markers, alkaline phosphatase (ALP), and runt-related transcription (RUNX2), via real-time quantitative PCR (qPCR) and Western blotting or ALP and alizarin red staining assay. Dual luciferase reporter gene assay confirmed the relationship between mmu_circ_0000349 and mmu-miR-138-5p (or mmu-miR-138-5p and JMJD3). Meanwhile, the JMJD3 binding to mmu_circ_0000349 was screened using an RNA pull-down assay. qPCR and Western blotting confirmed the effect of icariin on the mmu_circ_0000349/mmu-miR-138-5p/JMJD3 axis and osteogenic differentiation. As MC3T3-E1 osteogenic differentiation progressed, the JMJD3 expression level increased. A total of 361 circRNAs exhibited differences between the NC and osteogenesis groups. After validation, mmu_circ_0000349 was further analyzed as it exhibited the largest expression. And mmu_circ_0000349 was identified as a stable circular structure. Overexpression of mmu_circ_0000349 increased the expression levels of ALP and RUNX2, enhanced ALP activity, and increased the number of mineralized nodules; contrarily, inhibition of mmu_circ_0000349 exerted opposite effects. The data also confirmed that mmu_circ_0000349 regulated JMJD3 by sponging with mmu-miR-138-5p. With the increase in icariin concentration and time for treatment, the expression levels of mmu_circ_0000349, JMJD3, ALP, and RUNX2 also increased, whereas that of mmu-miR-138-5p decreased. In conclusion, Icariin promoted osteogenic differentiation by regulating the mmu_circ_0000349/mmu-miR-138-5p/JMJD3 pathway. Therefore, this provides a theoretical basis for the treatment of diseases related to osteogenic differentiation.

Effect of icariin surface treatment on the resorption of denuded roots after replantation in rat.

Limiting the incidence of resorption associated with delayed replantation of avulsed teeth is critical for long-term tooth survival. In this study, we assessed whether icariin, a natural product with anti-osteoclastic properties, could reduce root resorption in a rat model of tooth replantation. Cytocompatibility of icariin (10, 20, 40 and 80 μM) was evaluated by CCK-8 proliferation assay in vitro, and an osteoclastogenesis assay was performed to evaluate the effect of icariin on the differentiation of rat bone marrow macrophages and human peripheral blood monocytes into tartrate-resistant acid phosphatase-stained (TRAP+ ) multinucleated giant cells (MNGCs). Differentiation of human periodontal ligament stem cells (hPDLSCs) treated with icariin (10 μM) was also evaluated at 5, 10 and 21 days of osteogenic induction. The first maxillary molars of five-week-old male Sprague-Dawley rats were extracted, denuded of PDL, then treated either with neutralized collagen solution (Carrier control) or icariin in collagen (3 μg/μL) before replantation into their sockets. The animals were euthanized 2 weeks post-surgery for micro-computed tomography (micro-CT) imaging and histological analyses. Icariin was cytocompatible and significantly reduced the differentiation of TRAP+ MNGCs in a dose-dependent manner compared to the control. Moreover, icariin enhanced alkaline phosphatase activity, expression of osteogenic marker genes and proteins, and calcium deposition in hPDLSCs. Micro-CT imaging of the replanted samples demonstrated a significantly higher volume of remaining roots in the icariin-treated group than in the control group. Histological analysis revealed a marked number of resorptive lacunae with TRAP activity in the control group, whereas icariin-treated samples showed signs of functional healing and reduced osteoclastic activity. Icariin was biocompatible and demonstrated potent anti-osteoclastic and pro-osteogenic properties that reduced resorption and promoted functional healing of denuded roots in a rat maxillary first molar model of replantation. These findings indicate that root surface treatment with icariin may be a clinically relevant and practical method for improving the retention and survival of teeth with compromised PDL after delayed replantation following traumatic avulsion.

Understanding the mechanisms and implications of acacetin in mitigating diabetic osteoporosis: Insights from a zebrafish model

Osteoporosis, a condition characterised by reduced bone mineral density is more prevalent in individuals with diabetes. Acacetin, a natural flavonoid, possesses anti-inflammatory and antioxidant properties. However, its protective role in diabetic osteoporosis remains unexplored. Hence, the present study aimed to investigate the antihyperglycemic, antioxidant, and osteoprotective effects of acacetin using streptozotocin (STZ)-induced diabetic osteoporosis models in larval and adult zebrafish. The larvae were subjected to assays such as embryotoxicity, detection of oxidative stress markers (reactive oxygen species (ROS), superoxide levels, lipid peroxidation), apoptosis, and gene expression related to diabetes and osteoporosis. In adult zebrafish, body weight, blood glucose level, alkaline phosphatase, and tartrate resistant acid phosphatase (TRAP) activity were estimated, along with assays like alizarin red staining, histopathology, and gene expression. Interaction of acacetin and RANK/RANKL/OPG pathway was detected using molecular docking analysis. Acacetin exhibited antihyperglycemic effects in the zebrafish model, as shown by reduced blood glucose levels and increased body weight. Acacetin also demonstrated antioxidant properties by decreasing ROS, superoxide radicals, and lipid peroxidation levels. Additionally, acacetin treatment resulted in reduction of apoptosis and tartrate-resistant acid phosphatase levels and improved bone histology with increased bone density and enhanced alkaline phosphatase activity. In gene expression analysis, acacetin treatment has upregulated osteoprotegerin gene and downregulated RANKL gene. In conclusion, acacetin holds potential as a therapeutic agent for diabetic osteoporosis by targeting hyperglycemia, oxidative stress, apoptosis, and the RANK/RANKL/OPG signalling pathway.