Phosphatase Production Research Articles

Enhanced osteo-angiogenic coupling by a bioactive cell-free fat extract (CEFFE) delivered through electrospun fibers.

Regeneration of functional bone tissue relies heavily on achieving adequate vascularization in engineered bone constructs following implantation. This process requires the close integration of osteogenesis and angiogenesis. Cell-free fat extract (CEFFE or FE), a recently emerging acellular fat extract containing abundant growth factors, holds significant potential for regulating osteo-angiogenic coupling and promoting regeneration of vascularized bone tissue. However, its specific role in modulating the coupling between angiogenesis and osteogenesis remains unclear. Our previous research demonstrated that FE-decorated electrospun fibers of polycaprolactone/gelatin (named FE-PDA@PCL/GT) exhibited pro-vasculogenic capabilities both in vitro and in vivo (D. Li, Q. Li, T. Xu, X. Guo, H. Tang, W. Wang, W. Zhang and Y. Zhang, Pro-vasculogenic fibers by PDA-mediated surface functionalization using cell-free fat extract (CEFFE), Biomacromolecules 2024, 25, 1550-1562). Herein, we firstly demonstrated that the FE-PDA@PCL/GT fibers also significantly stimulated osteogenesis in a mouse calvaria osteoblast-like cell line MC3T3-E1 cells, as evidenced by the increased production of alkaline phosphatase (ALP), mineral deposits, and collagen I, as well as the upregulated expression of osteogenic marker genes in the osteoblasts. Using a transwell co-culture system, we further demonstrated that the release of FE from the FE-PDA@PCL/GT fibers not only promoted osteogenesis and angiogenesis but also markedly enhanced the paracrine functions and reciprocal communications between endothelial cells and osteoblasts. This dynamic interaction played a key role in the observed enhancement of osteo-angiogenic coupling. With the confirmed pro-osteogenic and pro-angiogenic properties of FE-PDA@PCL/GT, it is envisaged that these newly engineered bioactive fibers can be used to develop highly biomimicking bone constructs. These constructs are designed to promote native-like cell-scaffold and cell-cell interactions, which are essential for the effective regeneration of defected bone tissue with adequate vasculature.

Consortium of Lactobacillus crispatus 2029 and Ligilactobacillus salivarius 7247 Strains Shows In Vitro Bactericidal Effect on Campylobacter jejuni and, in Combination with Prebiotic, Protects Against Intestinal Barrier Dysfunction.

Background/Objectives:Campylobacter jejuni (CJ) is the etiological agent of the world's most common intestinal infectious food-borne disease, ranging from mild symptoms to fatal outcomes. The development of innovative synbiotics that inhibit the adhesion and reproduction of multidrug-resistant (MDR) CJ in animals and humans, thereby preserving intestinal homeostasis, is relevant. We have created a synbiotic based on the consortium of Lactobacillus crispatus 2029 (LC2029), Ligilactobacillus salivarius 7247 (LS7247), and a mannan-rich prebiotic (Actigen®). The purpose of this work was to study the in vitro anti-adhesive and antagonistic activities of the created synbiotic against MDR CJ strains, along with its role in preventing intestinal barrier dysfunction, which disrupts intestinal homeostasis. Methods: A complex of microbiological, immunological, and molecular biological methods was used. The ability of the LC2029 and LS7247 consortium to promote intestinal homeostasis in vitro was assessed by the effectiveness of controlling CJ-induced TLR4 activation, secretion of pro-inflammatory cytokines, development of intestinal barrier dysfunction, and production of intestinal alkaline phosphatase (IAP). Results: All MDR CJ strains showed marked adhesion to human Caco-2, pig IPEC-J2, chicken CPCE, and bovine BPCE enterocytes. For the first time, we found that the prebiotic and cell-free culture supernatant (CFS) from the consortium of LC2029 and LS7247 strains exhibit an additive effect in inhibiting the adhesion of MDR strains of CJ to human and animal enterocytes. CFS from the LC2029 and LS7247 consortium increased the permeability of the outer and inner membranes of CJ cells, which led to extracellular leakage of ATP and provided access to the peptidoglycan of the pathogen for the peptidoglycan-degrading bacteriocins nisin and enterolysin A produced by LS7247. The LC2029 and LS7247 consortium showed a bactericidal effect on CJ strains. Co-cultivation of the consortium with CJ strains resulted in a decrease in the viability of the pathogen by 6 log. CFS from the LC2029 and LS7247 consortium prevented the growth of CJ-induced TLR4 mRNA expression in enterocytes. The LC2029 and LS7247 consortium inhibited a CJ-induced increase in IL-8 and TNF-α production in enterocytes, prevented CJ-induced intestinal barrier dysfunction, maintained the transepithelial electrical resistance of the enterocyte monolayers, and prevented an increase in intestinal paracellular permeability and zonulin secretion. CFS from the consortium stimulated IAP mRNA expression in enterocytes. The LC2029 and LS7247 consortium and the prebiotic Actigen represent a new synergistic synbiotic with anti-CJ properties that prevents intestinal barrier dysfunction and preserves intestinal homeostasis. Conclusions: These data highlight the potential of using a synergistic synbiotic as a preventive strategy for creating feed additives and functional nutrition products based on it to combat the prevalence of campylobacteriosis caused by MDR strains in animals and humans.

Enhanced Production of Acid Phosphatase in Bacillus subtilis: From Heterologous Expression to Optimized Fermentation Strategy

Enhanced Production of Acid Phosphatase in Bacillus subtilis: From Heterologous Expression to Optimized Fermentation Strategy

Innovative Biocompatible Blend Scaffold of Poly(hydroxybutyrate-co-hydroxyvalerate) and Poly(ε-caprolactone) for Bone Tissue Engineering: In Vitro and In Vivo Evaluation.

This study evaluated the biocompatibility of dense and porous forms of Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), Poly(ε-caprolactone) (PCL), and their 75/25 blend for bone tissue engineering applications. The biomaterials were characterized morphologically using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), and the thickness and porosity of the scaffolds were determined. Functional assessments of mesenchymal stem cells (MSCs) included the MTT assay, alkaline phosphatase (ALP) production, and morphological and cytochemical analyses. Moreover, these polymers were implanted into rats to evaluate their in vivo performance. The morphology and FTIR spectra of the scaffolds were consistent with the expected results. Porous polymers were thicker than dense polymers, and porosity was higher than 92% in all samples. The cells exhibited good viability, activity, and growth on the scaffolds. A higher number of cells was observed on dense polymers, likely due to their smaller surface area. ALP production occurred in all samples, but enzyme activity was more intense in PCL samples. The scaffolds did not interfere with the osteogenic capacity of MSCs, and mineralized nodules were present in all samples. Histological analysis revealed new bone formation in all samples, although pure PHBV exhibited lower results compared to the other blends. In vivo results indicated that dense PCL and the dense 75/25 blend were the best materials tested, with PCL tending to improve the performance of PHBV in vivo.

Considerations of growth factor and material use in bone tissue engineering using biodegradable scaffolds in vitro and in vivo

Bone tissue engineering aims to harness materials to develop functional bone tissue to heal ‘critical-sized’ bone defects. This study examined a robust, coated poly(caprolactone) trimethacrylate (PCL-TMA) 3D-printable scaffold designed to augment bone formation. Following optimisation of the coatings, three bioactive coatings were examined, i) elastin-like polypeptide (ELP), ii) poly(ethyl acrylate) (PEA), fibronectin (FN) and bone morphogenetic protein-2 (BMP-2) applied sequentially (PEA/FN/BMP-2) and iii) both ELP and PEA/FN/BMP-2 coatings applied concurrently. The scaffold material was robust and showed biodegradability. The coatings demonstrated a significant (p < 0.05) osteogenic response in vitro in alkaline phosphatase gene upregulation and alkaline phosphatase production. The PCL-TMA scaffold and coatings supported angiogenesis and displayed excellent biocompatibility following evaluation on the chorioallantoic membrane assay. No significant (p < 0.05) heterotopic bone formed on the scaffolds within a murine subcutaneous implantation model, compared to the positive control of BMP-2 loaded collagen sponge following examination by micro-computed tomography or histology. The current studies demonstrate a range of innovative coated scaffold constructs with in vitro efficacy and clearly illustrate the importance of an appropriate in vivo environment to validate in vitro functionality prior to scale up and preclinical application.

Halophilic Phosphate-Solubilizing Microbes (Priestia megaterium and Bacillus velezensis) Isolated from Arabian Sea Seamount Sediments for Plant Growth Promotion.

Arabian Sea is a highly productive Ocean owing to deep upwelling with reports on phosphorus cycling in ocean sediments. In this study, microbes from sea mounts of the Arabian Sea at varying depths (400m, 900m) were screened to isolate and characterize phosphate-solubilizing bacteria (PSB) with plant growth-promoting properties. Out of the seven morphologically different PSBs, two bacterial strains with maximum phosphate solubilization index were identified as Priestia megaterium (H1) and Bacillus velezensis (H2) based on biochemical and molecular characteristics. Different factors influencing phosphatase production were optimized, which showed maximum solubilization at temperature of 30°C (97.5μg/mL), glucose as best carbon source (70µg/mL), 1-M NaCl (114.1µg/mL), and pH 8 (134.3µg/mL) indicating their halophilic and alkaliphilic characteristics. Alkaline phosphatase enzyme was extracted and partially purified from both PSBs wherein H2 strains showed greater specific activity (24.83 U/mg). Metabolomics studies through HPLC revealed maximum production of gluconic acid (483.75mg/L) in addition to lactic, oxalic, acetic, and succinic acid during solubilization. Biopriming effect of PSBs on tomato seed germination showed high germination index (80%) in consortia of both isolates which was also validated through root colonization by SEM analysis. Further studies using pot assay experiments also showed comparable results in marine PSB consortia with positive control (Phosphobacteria) for plant growth attributes including root height and weight. These findings suggest that the halophilic PSB strains from marine sediments could be used as potential bio-inoculants to enhance plant growth and combat saline stress for sustainable Agriculture.

Bone Tissue Engineering with Adipose-Derived Stem Cells in Polycaprolactone/Graphene Oxide/Dexamethasone 3D-Printed Scaffolds.

We fabricated three-dimensional (3D)-printed polycaprolactone (PCL) and PCL/graphene oxide (GO) (PGO) scaffolds for bone tissue engineering. An anti-inflammatory and pro-osteogenesis drug dexamethasone (DEX) was adsorbed onto GO and a 3D-printed PGO/DEX (PGOD) scaffold successfully improved drug delivery with a sustained release of DEX from the scaffold up to 1 month. The physicochemical properties of the PCL, PGO, and PGOD scaffolds were characterized by various analytical techniques. The biological response of these scaffolds was studied for adherence, proliferation, and osteogenic differentiation of seeded rabbit adipose-derived stem cells (ASCs) from DNA assays, alkaline phosphatase (ALP) production, calcium quantification, osteogenic gene expression, and immunofluorescence staining of osteogenic marker proteins. The PGOD scaffold was demonstrated to be the best scaffold for maintaining cell viability, cell proliferation, and osteogenic differentiation of ASCs in vitro. In vivo biocompatibility of PGOD was confirmed from subcutaneous implantation in nude mice where ASC-seeded PGOD can form ectopic bones, demonstrated by microcomputed tomography (micro-CT) analysis and immunofluorescence staining. Furthermore, implantation of PGOD/ASCs constructs into critical-sized cranial bone defects in rabbits form tissue-engineered bones at the defect site, observed using micro-CT and histological analysis.

Biosolubilization Potential of Rock Phosphate and Phosphatase Production by some Phosphate Solubilizing Fungi

Biosolubilization Potential of Rock Phosphate and Phosphatase Production by some Phosphate Solubilizing Fungi

Geographic bioprospection of maize rhizoplane-associated bacteria for consortia construction and impact on plant growth and nutrient uptake under low P availability

Application of beneficial microbial consortia for improving plant growth and productivity is considered a major approach to attain sustainable crop production. The construction of plant growth promoting (PGP) bacterial consortia (BC) is reliant on the design of microbial systems based on tuned inter-species interactions with known ecological functions. In this study, maize rhizoplane-associated bacteria were isolated from seven distinct agricultural regions in Morocco. Taxonomic and functional (related to phosphorus “P” use) diversity of 107 rhizoplane bacterial isolates were explored to construct BC while preserving the diversity of the niche they were isolated from. Thirty-six BC were generated, including 28 intra-zone consortia, seven intra-region consortia and one global BC. Quantification of three functional genes: glucose dehydrogenase encoding gene (gcd), pyrroloquinoline quinone (pqqC), and alkaline phosphatase encoding gene (phoD), involved in P cycling, confirmed the presence of gcd in nineteen BC, pqqC in eight BC and phoD in only one BC. In vitro functional characterization revealed that all BC were able to solubilize/mineralize P (50–88 ppm) through the production of organic acids and acid phosphatase (25 – 280 nmol.h−1). Assessment of above- and below-ground parameters of 45-day old maize plants identified five potential niche-constructed “intra-zone” BC (BC-6, -11, -12, -14, and -18) notably in terms of plant biomass, shoot nutrient (N, P, K) uptake and induced root morphological and physiological traits. These BC were associated with increased rhizosphere available P (80 ppm) and decreased microbial biomass P (17 ppm) while the remaining BC significantly increased microbial biomass P (30 ppm) at the expense of a decreased rhizosphere available P (35 ppm) with no significant effect on plant nutrient uptake and biomass. These findings demonstrate that intra-zone BC constructed from the same niche outperformed the intra- and inter-region consortia, supporting the niche-conservatism approach to construct P-efficient BC. This study lays a technical foundation for the construction of synthetic microbial consortia for plant growth and nutrient acquisition, through the optimization of inter-species bacterial interactions.

Multifunctional Sr,Mg-Doped Mesoporous Bioactive Glass Nanoparticles for Simultaneous Bone Regeneration and Drug Delivery.

Mesoporous bioactive glass nanoparticles (MBGNs) doped with therapeutical ions present multifunctional systems that enable a synergistic outcome through the dual delivery of drugs and ions. The aim of this study was to evaluate influence of co-doping with strontium and magnesium ions (SrMg-MBGNs) on the properties of MBGNs. A modified microemulsion-assisted sol-gel synthesis was used to obtain particles, and their physicochemical properties, bioactivity, and drug-loading/release ability were evaluated. Indirect biological assays using 2D and 3D cell culture models on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and endothelial EA.hy926 cells, respectively, were used to determine biocompatibility of MBGNs, their influence on alkaline phosphatase (ALP) production, calcium deposition, and cytoskeletal organization. Results showed that Sr,Mg-doping increased pore volume and solubility, and changed the mesoporous structure from worm-like to radial-dendritic, which led to a slightly accelerated drug release compared to pristine MBGNs. Biological assays confirmed that particles are biocompatible, and have ability to slightly induce ALP production and calcium deposition of hBM-MSCs, as well as to significantly improve the proliferation of EA.hy926 compared to biochemical stimulation via vascular endothelial growth factor (VEGF) administration or regular media. Fluorescence staining revealed that SrMg-MBGNs had a similar effect on EA.hy926 cytoskeletal organization to the VEGF group. In conclusion, Sr,Mg-MBGNs might be considered promising biomaterial for biomedical applications.

A functional analysis of a resorbable citrate-based composite tendon anchor

Rapid and efficient tendon fixation to a bone following trauma or in response to degenerative processes can be facilitated using a tendon anchoring device. Osteomimetic biomaterials, and in particular, bio-resorbable polymer composites designed to match the mineral phase content of native bone, have been shown to exhibit osteoinductive and osteoconductive properties in vivo and have been used in bone fixation for the past 2 decades. In this study, a resorbable, bioactive, and mechanically robust citrate-based composite formulated from poly(octamethylene citrate) (POC) and hydroxyapatite (HA) (POC-HA) was investigated as a potential tendon-fixation biomaterial. In vitro analysis with human Mesenchymal Stem Cells (hMSCs) indicated that POC-HA composite materials supported cell adhesion, growth, and proliferation and increased calcium deposition, alkaline phosphatase production, the expression of osteogenic specific genes, and activation of canonical pathways leading to osteoinduction and osteoconduction. Further, in vivo evaluation of a POC-HA tendon fixation device in a sheep metaphyseal model indicates the regenerative and remodeling potential of this citrate-based composite material. Together, this study presents a comprehensive in vitro and in vivo analysis of the functional response to a citrate-derived composite tendon anchor and indicates that citrate-based HA composites offer improved mechanical and osteogenic properties relative to commonly used resorbable tendon anchor devices formulated from poly(L-co-D, l-lactic acid) and tricalcium phosphate PLDLA-TCP.

Evaluation of Three Different Selective Media for Enumeration of Clostridium perfringens in Untreated and Treated Wastewater.

Current and emerging legislation in North Carolina and other regions calls for the enumeration of Clostridium perfringens as a surrogate indicator for protozoan parasites in various types of waters. Past studies that have evaluated selective media for the detection of this bacterium have provided limited, conflicting, and inconclusive results. In this study, membrane filtration was used to enumerate C. perfringens as culturable spores or total culturable cells in 19 samples of untreated and 25 samples of partially treated wastewaters on 3 candidate media, Tryptose Sulfite Cycloserine Agar (TSC), CP ChromoSelect Agar (CPCS), and membrane Clostridium perfringens Agar (m-CP) in parallel, and the results were compared. Presumptive isolates from each agar were further subjected to phenotypic confirmation tests for acid phosphatase production and stormy fermentation to further determine the performance of each agar. The CPCS agar was determined to have the highest enumerative capacity of total C. perfringens cells when compared to both TSC agar and m-CP agar (p-value < 0.05), but there was no significant difference in its ability to detect spores when compared to TSC agar (p-value > 0.05). The overall specificity of CPCS agar as determined by agreement of results from both confirmation tests was 0.81, while the specificity of TSC agar was only 0.28. Based on its performance, ease of preparation and use and consistency of colony characteristics, CPCS agar is recommended as the preferred medium for C. perfringens enumeration in wastewater.

Exosome-loaded hyaluronic acid hydrogel composite with oxygen-producing 3D printed polylactic acid scaffolds for bone tissue repair and regeneration

Bone defects can interfere with bone healing by disrupting the local environment, resulting in vascular damage and hypoxia. Under these conditions, insufficient oxygen availability is a significant factor that exacerbates disease by blocking angiogenesis or osteogenesis. Exosomes play a crucial role in intercellular communication and modulation of inflammation to aid bone regeneration. However, the distance between exosomes and areas of damage can hinder efficient bone generation and cell survival. To overcome this limitation, we fabricated a continuous oxygen-supplying composite scaffold, with the encapsulation of calcium peroxide in a polylactic acid three-dimensional (3D) printing construct (CPS), as both an oxygen source and hydroxyapatite (HAP) precursor. Furthermore, bone marrow mesenchymal stem cell (BMSC)-derived exosomes were incorporated into hyaluronic acid (HA) hydrogels to stimulate cell growth and modulate inflammation. The release of exosomes into cells leads to an increase in alkaline phosphatase production. In vivo results demonstrated that the composite scaffold regulated the inflammatory microenvironment, relieved tissue hypoxia, and promoted new bone formation. These results indicate that the synergistic effect of exosomes and oxygen promoted the proliferation of BMSCs, alleviated inflammation and exhibited excellent osteogenic properties. In conclusion, this osteogenic functional composite scaffold material offers a highly effective approach for bone repair.

Determination of Antagonistic Activities of Endophytic Bacteria Isolated from Different Wheat Genotypes Against Fusarium culmorum

This study aimed to evaluate the physiological and biochemical properties and enzyme activities of endophytic bacteria obtained from different wheat genotypes, as well as their effectiveness against Fusarium culmorum, which causes root and crown rot in wheat. The results obtained from double culture tests of isolates against F. culmorum showed that the inhibition rate varied between 80.56% and 13.90%. The inhibition rate against F. culmorum was 80.59% for Bacillus subtilis (MM11), 69.41% for Stenotrophomonas maltophilia (EY5), and 61.10% for Enterobacter sp. (MY3) under in vitro conditions, the most effective isolates. Pseudomonas putida (EM9) and Pseudomonas orientalis (MM21) isolates gave positive results in all tests in the production of amylase, cellulase, phosphatase, ACC deaminase, and siderophore. To identify six promising isolates, 16S rRNA gene-based sequence analysis was utilized. The efficacy of bacterial strains against F. culmorum, pot experiments were conducted in a growth room (in vivo). The results demonstrated that the combination of S. maltophilia, Enterobacter sp., and B. subtilis (MY3+EY5+MM11) yielded the most favorable outcomes in terms of disease severity, plant height, wet weight, dry weight, root wet weight, and root dry weight. The combination of Stenotrophomonas rhizophila, P. putida, and P. orientalis (EY1+EM9+MM21) exhibited promising results. Utilizing effective bacterial strains is anticipated to reduce the dependence on and costs associated with chemical fertilizers and pesticides while minimizing their environmental impact. Furthermore, these strains show potential for commercial applications pending further validation procedures. The findings from this study significantly contribute to the field of biological control strategies against F. culmorum by leveraging the diverse capabilities of endophytic bacteria.

Eggshell waste bioprocessing for sustainable acid phosphatase production and minimizing environmental hazards

BackgroundThe Environmental Protection Agency has listed eggshell waste as the 15th most significant food industry pollution hazard. Using eggshell waste as a renewable energy source has been a hot topic recently. Therefore, finding a sustainable solution for the recycling and valorization of eggshell waste by investigating its potential to produce acid phosphatase (ACP) and organic acids by the newly-discovered B. sonorensis was the target of the current investigation.ResultsDrawing on both molecular and morphological characterizations, the most potent ACP-producing B. sonorensis strain ACP2, was identified as a local bacterial strain obtained from the effluent of the paper and pulp industries. The use of consecutive statistical experimental approaches of Plackett–Burman Design (PBD) and Orthogonal Central Composite Design (OCCD), followed by pH-uncontrolled cultivation conditions in a 7 L bench-top bioreactor, revealed an innovative medium formulation that substantially improved ACP production, reaching 216 U L−1 with an ACP yield coefficient Yp/x of 18.2 and a specific growth rate (µ) of 0.1 h−1. The metals Ag+, Sn+, and Cr+ were the most efficiently released from eggshells during the solubilization process by B. sonorensis. The uncontrolled pH culture condition is the most suitable and favoured setting for improving ACP and organic acids production. Quantitative and qualitative analyses of the produced organic acids were carried out using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Lactic acid, citric acid, and hydroxybenzoic acid isomer were the most common organic acids produced throughout the cultivation process. The findings of TGA, DSC, SEM, EDS, FTIR, and XRD analysis emphasize the significant influence of organic acids and ACP activity on the solubilization of eggshell particles.ConclusionsThis study emphasized robust microbial engineering approaches for the large-scale production of a newly discovered acid phosphatase, accompanied by organic acids production from B. sonorensis. The biovalorization of the eggshell waste and the production of cost-effective ACP and organic acids were integrated into the current study, and this was done through the implementation of a unique and innovative medium formulation design for eggshell waste management, as well as scaling up ACP production on a bench-top scale.

Soil phosphorus transformation and plant uptake driven by phosphate-solubilizing microorganisms.

Phosphorus (P) is an important nutrient for plants, and a lack of available P greatly limits plant growth and development. Phosphate-solubilizing microorganisms (PSMs) significantly enhance the ability of plants to absorb and utilize P, which is important for improving plant nutrient turnover and yield. This article summarizes and analyzes how PSMs promote the absorption and utilization of P nutrients by plants from four perspectives: the types and functions of PSMs, phosphate-solubilizing mechanisms, main functional genes, and the impact of complex inoculation of PSMs on plant P acquisition. This article reviews the physiological and molecular mechanisms of phosphorus solubilization and growth promotion by PSMs, with a focus on analyzing the impact of PSMs on soil microbial communities and its interaction with root exudates. In order to better understand the ability of PSMs and their role in soil P transformation and to provide prospects for research on PSMs promoting plant P absorption. PSMs mainly activate insoluble P through the secretion of organic acids, phosphatase production, and mycorrhizal symbiosis, mycorrhizal symbiosis indirectly activates P via carbon exchange. PSMs can secrete organic acids and produce phosphatase, which plays a crucial role in soil P cycling, and related genes are involved in regulating the P-solubilization ability. This article reviews the mechanisms by which microorganisms promote plant uptake of soil P, which is of great significance for a deeper understanding of PSM-mediated soil P cycling, plant P uptake and utilization, and for improving the efficiency of P utilization in agriculture.

Cardamonin inhibits osteogenic differentiation by downregulating Wnt/beta-catenin signaling and alleviates subchondral osteosclerosis in osteoarthritic mice.

Osteoarthritis (OA) is a common degenerative joint disease, and subchondral osteosclerosis is an important pathological change that occurs in its late stages. Cardamonin (CD) is a natural flavonoid isolated from Alpinia katsumadai that has anti-inflammatory activity. The objectives of this study were to investigate the therapeutic effects and potential mechanism of CD in regulating OA subchondral osteosclerosis at in vivo and in vitro settings. Eight-week-old male C57BL/6J mice were randomly divided into four groups: sham operation, anterior cruciate ligament transection (ACLT)-induced OA model, low-dose and high-dose CD treated ACLT-OA model groups. Histological assessment and immunohistochemical examinations for chondrocyte metabolism-related markers metalloproteinase-13, ADAMTS-4, Col II, and Sox-9 were performed. Microcomputed tomography was used to assess the sclerosis indicators in subchondral bone. Further, MC3T3-E1 (a mouse calvarial preosteoblast cell line) cells were treated with various concentrations of CD to reveal the influence and potential molecular pathways of CD in osteogenic differentiations. Animal studies suggested that CD alleviated the pathological changes in OA mice such as maintaining integrity and increasing the thickness of hyaline cartilage, decreasing the thickness of calcified cartilage, decreasing the Osteoarthritis Research Society International score, regulating articular cartilage metabolism, and inhibiting subchondral osteosclerosis. In vitro investigation indicated that CD inhibited alkaline phosphatase expression and production of calcium nodules during osteogenic differentiation of MC3T3-E1 cells. In addition, CD inhibited the expression of osteogenic differentiation-related indicators and Wnt/β-catenin pathway-related proteins. In conclusion, CD inhibits osteogenic differentiation by downregulating Wnt/β-catenin signaling and alleviating subchondral osteosclerosis in a mouse model of OA.

Investigating the Role of Amino Acids in Short Peptides for Hydroxyapatite Binding and Osteogenic Differentiation of Mesenchymal Stem Cells to Aid Bone Regeneration.

Bone defects show a slow rate of osteoconduction and imperfect reconstruction, and the current treatment strategies to treat bone defects suffer from limitations like immunogenicity, lack of cell adhesion, and the absence of osteogenic activity. In this context, bioactive supramolecular peptides and peptide gels offer unique opportunities to develop biomaterials that can play a dominant role in the biomineralization of bone tissues and promote bone formation. In this article, we have demonstrated the potential of six tetrapeptides for specific binding to hydroxyapatite (HAp), a major inorganic component of the bone, and their effect on the growth and osteogenic differentiation of mesenchymal stem cells (MSCs). We adopted a simplistic approach of rationally designing amphiphilic peptides by incorporating amino acids, Ser, pSer, Pro, Hyp, Asp, and Glu, which are present in either collagenous or noncollagenous proteins and render properties like antioxidant, calcification, and mineralization. A total of six tetrapeptides, Trp-Trp-His-Ser (WWHS), Trp-Trp-His-pSer (WWHJ), Trp-Trp-His-Pro (WWHP), Trp-Trp-His-Hyp (WWHO), Trp-Trp-His-Asp (WWHD), and Trp-Trp-His-Glu (WWHE), were synthesized. Four peptides were found to self-assemble into nanofibrillar gels resembling the extracellular matrix (ECM), and the remaining two peptides (WWHJ, WWHP) self-assembled into nanorods. The peptides showed excellent cell adhesion, encapsulation, proliferation, and migration and induced the differentiation of mesenchymal stem cells (MSCs), as evident from the enhanced mineralization, resulting from the upregulation of osteogenic markers, RUNX 2, COL I, OPN, and OCN, alkaline phosphatase (ALP) production, and calcium deposition. The peptides also induced the downregulation of inflammatory markers, TNF-α and iNOS, and the upregulation of the anti-inflammatory marker, IL-10, resulting in M2 macrophage polarization. RANKL and TRAP genes were downregulated in a coculture system of MC3T3-E1 and RAW 264.7 cells, implying that peptides promote osteogenesis and inhibit osteoclastogenesis. The peptide-based biomaterials developed in this work can enhance bone regeneration capacity and show strong potential as scaffolds for bone tissue engineering.

Effect of polydatin on the viability and odontogenic differentiation of human dental pulp stem cells: An in-vitro study

Background/purposeVarious materials have been used to promote human dental pulp stem cells (hDPSCs) differentiation to produce dentin bridge formation with less-than-optimal results. Polydatin (PD), a naturally present material with osteogenic properties can be a promising material in the pulp regeneration/repair process. The aim of this study was to evaluate the effect of (PD) on the viability and differentiation of human dental pulp stem cells. Materials and methodsPD effect on hDPSCs in terms of cellular viability, alkaline phosphatase (ALP) production, and messenger RNAs (mRNA) of odontogenic markers production using quantitative reverse transcription polymerase chain reaction (RT-qPCR) were evaluated. In addition, mineral deposits were detected with Alizarin red stain. ResultsThe viable hDPSCs in the presence of 0.01 μM and 0.1 μM PD were significantly higher than the control on days 3 and 7, respectively. In addition, ALP activity of hDPSCs was significantly increased with 0.01, 0.1, and 1 μM of PD. In addition, increased expression mRNAs of ALP, osteocalcin (OC), osteonectin (ON), osteopontin (OP), Runt-related transcription factor-2 (RUNX-2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) was observed after PD treatment, however, the difference was not statistically significant. Furthermore, increased size of mineral deposits was observed with PD. ConclusionPD promoted the expression of markers associated with odontogenic differentiation and mineralized tissue deposition in hDPSCs.

Measurement of the Activity of Wildtype and Disease-Causing ALPK1 Mutants in Transfected Cells With a 96-Well Format NF-κB/AP-1 Reporter Assay.

Alpha-protein kinase 1 (ALPK1) is normally activated by bacterial ADP-heptose as part of the innate immune response, leading to the initiation of downstream signalling events that culminate in the activation of transcription factors such as NF-κB and AP-1. In contrast, disease-causing mutations in ALPK1 that cause ROSAH syndrome or spiradenoma allow ALPK1 to be activated in cells in the absence of bacterial infection (i.e., without ADP-heptose). This protocol describes a semi-quantitative reporter assay based on ALPK1 knockout HEK-Blue cells that measures the activity of transfected wildtype and disease-causing forms of ALPK1 by virtue of their ability to activate the transcription factors NF-κB and AP-1. These cells express a synthetic gene encoding alkaline phosphatase under the control of an NF-κB/AP-1-dependent promoter, and consequently, the activation of ALPK1 leads to the production of alkaline phosphatase, which is secreted into the culture media and can be measured colorimetrically at 645 nm after the addition of a detection reagent. Key features • Highly sensitive reporter assay allowing detection of low-level activity arising from ALPK1 mutants • Optimised in 96-well plate format, requiring only 60,000 transfected ALPK1 KO HEK-Blue cells per well • Rapid experimental design, taking only four days from start to finish • Suitable for screening ALPK1 variants of unknown significance in an arrayed 96-well format.