Alkaline Phosphatase Activity Assay Research Articles

Peptide-Driven Assembly of Magnetic Beads for Potentiometric Sensing of Bacterial Enzyme at a Subcellular Level.

Bacterial enzymes with different subcellular localizations play a critical ecological role in biogeochemical processing. However, precisely quantifying enzymes localized at certain subcellular levels, such as extracellular enzymes, has not yet been fully realized due to the complexity and dynamism of the bacterial outer membrane. Here we present a magneto-controlled potentiometric sensing platform for the specific detection of extracellular enzymatic activity. Alkaline phosphatase (ALP), which is one of the crucial hydrolytic enzymes in the ocean, was selected as the target enzyme. Magnetic beads functionalized with an ALP-responsive self-assembled peptide (GGGGGFFFpYpYEEE, MBs-peptides) prevent negatively charged peptides from entering the bacterial outer membrane, thereby enabling direct potentiometric sensing of extracellular ALP both attached to the bacterial cell surface and released into the surrounding environment. The dephosphorylation-triggered assembly of peptide-coupled magnetic beads can be directly and sensitively measured by using a magneto-controlled sensor. In this study, extracellular ALP activity of Pseudomonas aeruginosa at concentrations ranging from 10 to 1.0 × 105 CFU mL-1 was specifically and sensitively monitored. Moreover, this magneto-controlled potentiometric method enabled a simple and accurate assay of ALP activity across different subcellular localizations.

Organic-inorganic nHA-Gelatin/Alginate high strength macroporous cryogel promotes bone regeneration

Macroporous cryogel has the advantages of nutrient exchange and cell growth, and is an ideal material for tissue regeneration. In order to strengthen the machenical properties of cryogel for the widely use, a high strength gelatin/sodium alginate/nano hydroxyapatite (nHA) porous cryogel (GA-HA cryogel) was prepared by a simple freeze-thaw process. The mechanical strength of GA-HA cryogel increased significantly with the increase of nHA content. In vitro studies showed that GA-HA cryogel had good biocompatibility and no obvious cytotoxicity to MC3T3-E1 cells. The results of alkaline phosphatase activity assay and osteocalcin immunofluorescence staining showed that GA-HA1 porous hydrogel system could significantly increase the expression of MC3T3-E1 alkaline phosphatase and osteocalcin when the content of nHA was 1 ​%. In addition, porous GA-HA cryogel showed good performance in promoting bone regeneration in rat skull defect model. Therefore, the high-strength double network cryogel prepared in this study can provide new applications in bone repair and tissue regeneration.

Indoxyl sulfate exacerbates alveolar bone loss in chronic kidney disease through ferroptosis.

The purpose of this study was to determine whether indoxyl sulfate (IS) is involved in alveolar bone deterioration and to elucidate the mechanism underlying alveolar bone loss in chronic kidney disease (CKD) patients. Mice were divided into the control group, CP group (ligature-induced periodontitis), CKD group (5/6 nephrectomy), and CKD + CP group. The concentration of IS in the gingival crevicular fluid (GCF) was determined by HPLC. The bone microarchitecture was evaluated by micro-CT. MC3T3-E1 cells were stimulated with IS, and changes in mitochondrial morphology and ferroptosis-related factors were detected. RT-PCR, western blotting, alkaline phosphatase activity assays, and alizarin red S staining were utilized to assess how IS affects osteogenic differentiation. Compared with that in the other groups, alveolar bone destruction in the CKD + CP group was more severe. IS accumulated in the GCF of mice with CKD. IS activated the aryl hydrocarbon receptor (AhR) invitro, inhibited MC3T3-E1 cell osteogenic differentiation, caused changes in mitochondrial morphology, and activated the SLC7A11/GPX4 signaling pathway. An AhR inhibitor attenuated the aforementioned changes induced by IS. IS activated the AhR/SLC7A11/GPX4 signaling pathway, inhibited osteogenesis in MC3T3-E1 cells, and participated in alveolar bone resorption in CKD model mice through ferroptosis.

Effects of 10-MDP calcium salt on osteoblasts and fibroblasts

Objective10-methacryloyloxidecyl dihydrogen phosphate monomer (10-MDP) is commonly used as a bonding monomer in universal adhesives. Adhesives that contain this monomer can directly contact the surrounding periodontium due to the chemical binding of 10-MDP with hydroxyapatite in hard tissue to form calcium salts. However, the effect of these calcium salts on the periodontium in the case of subgingival fillings remains poorly understood. The objective of this study was to investigate effects of 10-MDP calcium salts on osteoblasts and fibroblasts in the periodontal tissues. MethodsThis study investigated the effects of different concentrations of 10-MDP calcium salts on the migration, proliferation, and differentiation of osteoblasts (MC3T3-E1) and fibroblasts (L929); additionally, the effect on apoptosis and matrix metalloproteinases (MMPs) expression in these cells was evaluated. Cell proliferation assay, alkaline phosphatase (ALP) activity assay, Western blotting, and quantitative real-time polymerase chain reaction were performed to determine the effects. ResultsThe 10-MDP calcium salts (within a concentration of 0.5 mg/mL) showed no cytotoxicity and did not seem to influence the apoptosis, mitochondrial membrane potential, and reactive oxygen species (ROS) levels in the cells. However, they had an inhibitory effect on the secretion of MMP2 and MMP9 in the osteoblasts and fibroblasts. The ALP activity assay and Alizarin Red staining did not reveal any significant effects of the 10-MDP calcium salts on osteoblast differentiation. SignificanceThese results suggest that applying 10-MDP-containing adhesives to subgingival fillings may be safe and beneficial for the periodontal tissues.

SPA inhibits hBMSC osteogenic differentiation and M1 macrophage polarization by suppressing SETD2 in acute suppurative osteomyelitis

To clarify the impact of SETD2 on macrophage function in pediatric patients with acute suppurative osteomyelitis and to elucidate the precise underlying mechanism. To gain insights into the potential functions of SETD2, a comprehensive study was conducted utilizing a co-culture model of human bone mesenchymal stem cells (hBMSCs) and bone marrow-derived macrophages (THP-1). A range of techniques were employed, including quantitative polymerase chain reaction, western blotting, ELISA, alkaline phosphatase activity assays, alizarin red S staining, luciferase reporter gene assays, and chromatin immunoprecipitation, to unravel the intricate interactions and molecular mechanisms involving SETD2 in this system. It was observed that SETD2 expression was reduced in THP-1 cells stimulated by staphylococcal protein A (SPA). Furthermore, the downregulation of SETD2 resulted in elevated M1 macrophage polarization and glycolysis, effects that were mitigated by SPA stimulation. Notably, SPA-stimulated THP-1 cells exhibited an increase in HIF-1α expression, which exhibited an inverse correlation with SETD2 levels. Moreover, it was discovered that SETD2 functioned as a catalyst for H3K36me3 and bound to the HIF-1α gene, which, in turn, regulated HIF-1α expression. Furthermore, the suppression of HIF-1α abrogated the consequences of SETD2 downregulation on glycolysis and M1 macrophage polarization. Lastly, the study demonstrated that M1 macrophage polarization serves as a mediator for BMP4’s inhibitory effect on osteogenic differentiation of hBMSCs. This research has uncovered a previously unknown role of SETD2 in macrophages during osteomyelitis, revealing its significance in the pathogenesis of this condition. These findings suggest SETD2 as a novel target for the treatment of osteomyelitis.

Novel artificial tricalcium phosphate and magnesium composite graft facilitates angiogenesis in bone healing

BackgroundBone grafting is the standard treatment for critical bone defects, but autologous grafts have limitations like donor site morbidity and limited availability, while commercial artificial grafts may have poor integration with surrounding bone tissue, leading to delayed healing. Magnesium deficiency negatively impacts angiogenesis and bone repair. Therefore, incorporating magnesium into a synthetic biomaterial could provide an excellent bone substitute. This study aims to evaluate the morphological, mechanical, and biological properties of a calcium phosphate cement (CPC) sponge composed of tetracalcium phosphate (TTCP) and monocalcium phosphate monohydrate (MCPM), which could serve as an excellent bone substitute by incorporating magnesium. MethodsThis study aims to develop biomedical materials composed mainly of TTCP and MCPM powder, magnesium powder, and collagen. The materials were prepared using a wet-stirred mill and freeze-dryer methods. The particle size, composition, and microstructure of the materials were investigated. Finally, the biological properties of these materials, including 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay for biocompatibility, effects on bone cell differentiation by alkaline phosphatase (ALP) activity assay and tartrate-resistant acid phosphatase (TRAP) activity assay, and endothelial cell tube formation assay for angiogenesis, were evaluated as well. ResultsThe data showed that the sub-micron CPC powder, composed of TTCP/MCPM in a 3.5:1 ratio, had a setting time shorter than 15 minutes and a compressive strength of 4.39±0.96 MPa. This reveals that the sub-micron CPC powder had an adequate setting time and mechanical strength. We found that the sub-micron CPC sponge containing magnesium had better biocompatibility, including increased proliferation and osteogenic induction effects without cytotoxicity. The CPC sponge containing magnesium also promoted angiogenesis. ConclusionIn summary, we introduced a novel CPC sponge, which had a similar property to human bone promoted the biological functions of bone cells, and could serve as a promising material used in bone regeneration for critical bone defects.

Comparing the Soluble Form of Recombinant Human Insulin-like Growth Factor-1 (rhIGF-1) in Escherichia coli Using Thioredoxin as Fused and Co-expressed Protein.

Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. Escherichia coli is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1. In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli. The rhIGF-1 was expressed in the E. coli Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose-derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process. Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity. It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in E. coli cells.

PSO/SDF-1 composite hydrogel promotes osteogenic differentiation of PDLSCs and bone regeneration in periodontitis rats

Periodontitis is an inflammatory disease characterized by the destruction of periodontal tissues, and the promotion of bone tissue regeneration is the key to curing periodontitis. Psoralen is the main component of Psoralea corylifolia Linn, and has multiple biological effects, including anti-osteoporosis and osteogenesis. We constructed a novel hydrogel loaded with psoralen (PSO) and stromal cell-derived factor-1 (SDF-1) for direct endogenous cell homing. This study aimed to evaluate the synergistic effects of PSO/SDF-1 on periodontal bone regeneration in patients with periodontitis. The results of CCK8, alkaline phosphatase (ALP) activity assay, and Alizarin Red staining showed that PSO/SDF-1 combination treatment promoted cell proliferation, chemotaxis ability, and ALP activity of PDLSCs. qRT-PCR and western blotting showed that the expression levels of alkaline phosphatase (ALP), dwarf-associated transcription factor 2 (RUNX2), and osteocalcin (OCN) gene were upregulated. Rat periodontal models were established to observe the effect of local application of the composite hydrogel on bone regeneration. These results proved that the PSO/SDF-1 combination treatment significantly promoted new bone formation. The immunohistochemical (IHC) results confirmed the elevated expression of ALP, RUNX2, and OCN osteogenic genes. PSO/SDF-1 composite hydrogel can synergistically regulate the biological function and promote periodontal bone formation. Thus, this study provides a novel strategy for periodontal bone regeneration.

Heat stress promotes osteogenic and odontogenic differentiation of stem cells from apical papilla via glucose-regulated protein 78-mediated autophagy

Background/purposeHeat stress is essential for improving the efficacy of mesenchymal stem cell (MSC)-based regeneration medicine. However, it is still unclear whether and how heat stress influences the differentiation of stem cells from apical papilla (SCAPs). This research aimed to explore the potential mechanism of glucose-regulated protein 78 (GRP78) in regulating differentiation under heat stress in SCAPs. Materials and methodsThe proliferation ability was assessed using the 5-Ethynyl-2’- deoxyuridine (EdU) assay, cell counting kit assay (CCK-8), and flow cytometry (FCM). The osteogenic and odontogenic differentiation capacities were investigated through Western blot, quantitative reverse transcription polymerase chain reaction (qRT-PCR), alkaline phosphatase (ALP) staining and activity assay, alizarin red S (ARS) staining, as well as immunofluorescence staining. Western blot and transmission electron microscopy (TEM) were used to detect autophagy. ResultsHeat stress enhanced the osteogenic and odontogenic differentiation of SCAPs, but it did not significantly affect proliferation. Besides, GRP78 has been confirmed to modulate the differentiation induced by heat stress. Autophagy triggered by GRP78 enhanced osteogenic and odontogenic differentiation of SCAPs, while the knockdown of GRP78 or the inhibitor of autophagy suppressed the differentiation. ConclusionHeat stress induces osteogenic and odontogenic differentiation of SCAPs through GRP78-mediated autophagy.

Potential probiotic Lactobacillus delbrueckii subsp. lactis KUMS-Y33 suppresses adipogenesis and promotes osteogenesis in human adipose-derived mesenchymal stem cell

Today, probiotics are considered to be living microorganisms whose consumption has a certain number of beneficial effects on the consumer. The present study aimed to investigate the effect of a new probiotic extract (Lactobacillus delbrueckii subsp. lactis KUMS Y33) on the differentiation process of human adipose-derived stem cells (hADSCs) into adipocytes and osteocytes and, as a result, clarify its role in the prevention and treatment of bone age disease. Several bacteria were isolated from traditional yogurt. They were evaluated to characterize the probiotic’s activity. Then, the isolated hADSCs were treated with the probiotic extract, and then osteogenesis and adipogenesis were induced. To evaluate the differentiation process, oil red O and alizarin red staining, a triglyceride content assay, an alkaline phosphatase (ALP) activity assay, as well as real-time PCR and western blot analysis of osteocyte- and adipocyte-specific genes, were performed. Ultimately, the new strain was sequenced and registered on NBCI. In the probiotic-treated group, the triglyceride content and the gene expression and protein levels of C/EBP-α and PPAR-γ2 (adipocyte-specific markers) were significantly decreased compared to the control group (P < 0.05), indicating an inhibited adipogenesis process. Furthermore, the probiotic extract caused a significant increase in the ALP activity, the expression levels of RUNX2 and osteocalcin, and the protein levels of collagen I and FGF-23 (osteocyte-specific markers) in comparison to the control group (P < 0.05), indicating an enhanced osteogenesis process. According to the results of the present study, the probiotic extract inhibits adipogenesis and significantly increases osteogenesis, suggesting a positive role in the prevention and treatment of osteoporosis and opening a new aspect for future in-vivo study.

FBLN5 was Regulated by PRDM9, and Promoted Senescence and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells.

Periodontal ligament stem cells (PDLSCs) are ideal seed cells for periodontal tissue regeneration. Our previous studies have indicated that the histone methyltransferase PRDM9 plays an important role in human periodontal ligament stem cells (hPDLSCs). Whether FBLN5, which is a downstream gene of PRDM9, also has a potential impact on hPDLSCs is still unclear. Senescence was assessed using β-galactosidase and Enzyme-linked immunosorbent assay (ELISA). Osteogenic differentiation potential of hPDLSCs was measured through Alkaline phosphatase (ALP) activity assay and Alizarin red detection, while gene expression levels were evaluated using western blot and RT-qPCR analysis. FBLN5 overexpression promoted the osteogenic differentiation and senescence of hPDLSCs. FBLN5 knockdown inhibited the osteogenic differentiation and senescence of hPDLSCs. Knockdown of PRDM9 decreased the expression of FBLN5 in hPDLSCs and inhibited senescence of hPDLSCs. Additionally, both FBLN5 and PRDM9 promoted the expression of phosphorylated p38 MAPK, Erk1/2 and JNK. The p38 MAPK pathway inhibitor SB203580 and the Erk1/2 pathway inhibitor PD98059 have the same effects on inhibiting the osteogenic differentiation and senescence of hPDLSCs. The JNK pathway inhibitor SP600125 reduced the senescence of hPDLSCs. FBLN5 promoted senescence and osteogenic differentiation of hPDLSCs via activation of the MAPK signaling pathway. FBLN5 was positively targeted by PRDM9, which also activated the MAPK signaling pathway.

Translating proteome and transcriptome dynamics of periodontal ligament stem cell-derived secretome/conditioned medium in an in vitro model of periodontitis

BackgroundPeriodontal ligament stem cells (PDLSCs) have been proposed as therapeutic candidates in periodontal diseases and periodontium defects. Paracrine factors of PDLSCs, namely, secretome, can contribute to tissue regeneration comparable to direct stem cell application. This study explored restoration effects of PDLSC-derived secretome/conditioned medium (PDLSC-CM) on PDLSCs themselves in an inflammatory microenvironment and identified its action mechanisms using proteomics and transcriptomic profiling.MethodsPDLSC-CM was prepared from cells under healthy culture conditions. Mass spectrometry and liquid chromatography–tandem mass spectrometry (LC–MS/MS) were then performed to analyze the PDLSC-CM proteome. Osteogenic differentiation of PDLSCs under inflammatory conditions or in the presence of PDLSC-CM was then characterized in assays of alkaline phosphatase activity, intracellular calcium levels, protein expression of osteogenic markers, and matrix mineralization. Furthermore, the transcriptomic profile was assessed to identify significantly enriched signaling pathways and associated molecular networks by RNA sequencing.ResultsLC–MS/MS proteomics identified a total of 203 proteins and distinguished 187 significant protein changes in PDLSC-CM compared to control-CM. LPS-treated PDLSCs significantly attenuated osteogenic differentiation. When PDLSCs were treated with PDLSC-CM alone, their osteogenic activity was significantly upregulated compared to the control group. Moreover, the LPS-impaired osteogenesis of PDLSCs was reconstituted by PDLSC-CM treatment. RNA sequencing revealed 252, 1,326, and 776 differentially expressed genes in the control vs. LPS, control vs. PDLSC-CM, and LPS vs. LPS + PDLSC-CM groups, respectively.ConclusionThis study suggest that PDLSC-CM restores the osteogenic potential of PDLSCs in an inflammatory environment through secretory functions representing potential repair and regenerative mechanisms.

Nanofibrous polyelectrolyte complex incorporated BSA-alginate composite bioink for 3D bioprinting of bone mimicking constructs

Although several bioinks have been developed for 3D bioprinting applications, the lack of optimal printability, mechanical properties, and adequate cell response has limited their practical applicability. Therefore, this work reports the development of a composite bioink consisting of bovine serum albumin (BSA), alginate, and self-assembled nanofibrous polyelectrolyte complex aggregates of gelatin and chitosan (PEC-GC). The nanofibrous PEC-GC aggregates were prepared and incorporated into the bioink in varying concentrations (0 % to 3 %). The bioink samples were bioprinted and crosslinked post-printing by calcium chloride. The average nanofiber diameter of PEC-GC was 62 ± 15 nm. It was demonstrated that PEC-GC improves the printability and cellular adhesion of the developed bioink and modulates the swelling ratio, degradation rate, and mechanical properties of the fabricated scaffold. The in vitro results revealed that the bioink with 2 % PEC-GC had the best post-printing cell viability of the encapsulated MG63 osteosarcoma cells and well oragnized stress fibers, indicating enhanced cell adhesion. The cell viability was >90 %, as observed from the MTT assay. The composite bioink also showed osteogenic potential, as confirmed by the estimation of alkaline phosphatase activity and collagen synthesis assay. This study successfully fabricated a high-shape fidelity bioink with potential in bone tissue engineering.

Label-free and sensitive assay for ICP-MS determination of alkaline phosphatase activity via manganese target

As an essential and universal enzyme, alkaline phosphatase (ALP) has been identified as an important biomarker of various diseases, thus highly sensitive assay of ALP activity is of great significance. Herein, a label-free and sensitive ICP-MS based assay was developed for ALP activity detection using MnO2 nanosheets (MnO2 NS) as the element target source. ALP triggered the hydrolysis of ascorbic acid-2-phosphate (AAP) to produce reductive ascorbic acid (AA), followed with the decomposition of MnO2 NS to Mn2+ ions, which were quantified directly by ICP-MS after selective separation of MnO2 NS and Mn2+ ions simply by filtration, and no further elemental labelling was needed. Under the selected conditions, the limit of detection (LOD) was 0.007 U L-1 for ALP. Additionally, this assay was also extended to investigate the inhibition effect of ALP inhibitor (Na3VO4) with reasonable results. To our knowledge, this is the first endeavor for ALP activity detection using ICP-MS, which holds great promise for the early clinical diagnosis and biomedical research on ALP.

Morinda officinalis polysaccharide promotes the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells via microRNA-210-3p/scavenger receptor class A member 3.

Morinda officinalis polysaccharide (MOP) is the bioactive ingredient extracted from the root of Morinda officinalis, and Morinda officinalis is applied to treat osteoporosis (OP). The purpose of this study was to determine the role of MOP on human bone marrow mesenchymal stem cells (hBMSCs) and the underlying mechanism. HBMSCs were isolated from bone marrow samples of patients with OP and treated with MOP. Quantitative real-time polymerase chain reaction was adopted to quantify the expression of microRNA-210-3p (miR-210-3p) and scavenger receptor class A member 3 (SCARA3) mRNA. Cell Counting Kit-8 assay was employed to detect cell viability; Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling assay and flow cytometry were adopted to detect apoptosis; Alkaline Phosphatase (ALP) activity assay kit was applied to detect ALP activity; Western blot was executed to quantify the expression levels of SCARA3, osteogenic and adipogenic differentiation markers. Ovariectomized rats were treated with MOP. Bone mineral density (BMD), serum tartrate-resistant acid phosphatase 5b (TRACP 5b), and N-telopeptide of type I collagen (NTx) levels were assessed by BMD detector and Enzyme-linked immunosorbent assay kits. It was revealed that MOP could promote hBMSCs' viability and osteogenic differentiation and inhibit apoptosis and adipogenic differentiation. MOP could also upregulate SCARA3 expression through repressing miR-210-3p expression. Treatment with MOP increased the BMD and decreased the TRACP 5b and NTx levels in ovariectomized rats. MOP may boost the osteogenic differentiation and inhibit adipogenic differentiation of hBMSCs by miR-210-3p/SCARA3 axis.

Abstract TMP3: Overexpression of Alk1 in Brain Endothelial Cells Alleviates the Severity of Brain Arteriovenous Malformations in Alk1 Mutant Mice

Background and purpose: Brain arteriovenous malformation (bAVM) can cause life threatening intra-cranial hemorrhage. Currently, there is no effective medical therapy available to treat bAVM patients. Mutation of activin receptor-like kinase 1 ( Alk1 also called ACVRL1 ) causes a familiar form of bAVM. Genetically overexpression of Alk1 in endothelial cells (ECs) recurs the phenotype of Alk1 deficient mice. We hypothesize that transduction of Alk1 gene into brain ECs using an adeno associated viral vector (AAV) may lead to cure of bAVM in Alk1 mutant mice. Methods: To identify a AAV capsid that can transduce brain ECs after intravenous (i.v.) delivery, several engineered AAV capsids were screened. A single-stranded CBA promoter driven tdTomato transgene was packaged in these capsids and delivered i.v. to wild-type mice. Tissues were collected 4 weeks later. After identifying a capsid, cc84, that can transduce brain ECs effectively after i.v. deliver, we package CMV-Alk1 transgene into AAV.cc84. Brain AVMs were induced on PdgfbiCre; Alk1 f/f mice through intra-brain injection of AAV1-VEGF followed by intraperitoneal injection of 1.25 mg/25g tamoxifen two weeks later. AAV.cc84-Alk1 was delivered to PdgfbiCre; Alk1 f/f mice through i.v. injection at the time of tamoxifen treatment Histological analyses were used to analyze viral transduction in the brain and liver, vessel density, and abnormal vessels in the brain and liver inflammation. Liver and kidney function were analyzed by colorimetric assays for Alkaline Phosphatase Activity (ALP), Alanine Transaminase Activity ALT), and Creatinine levels (Cr). Results: After i.v. injection, AAV-cc84 transduced a high percentage of brain ECs and a few hepatocytes. AAV.cc84-Alk1 treatment reduced vessel density (p=0.008), abnormal vessels (p=0.034), mouse mortality rates (20% vs 60% respectively) compared to the AAV.cc84-RFP (control vector) treated PdgfbiCre; Alk1 f/f mice. The levels of ALP, ALT and Cr were similar between AAV injected and uninjected mice. Conclusion: AAV.cc84 mediated EC overexpression of Alk1 reduced bAVM severity in Alk1 deficient mice indicating that AAV.cc84 is a promising gene therapy vector to treat bAVMs.

Bioactivated lubricant-infused surfaces: A dual-action strategy for enhancing osseointegration and preventing implant-associated infections

Implant-associated infections (IAIs), caused by bacterial adhesions and biofilm formations on the implant surfaces, have been the most critical threat for orthopedic surgeries. A promising strategy to prevent IAIs involves modifying the implant surface to inhibit undesirable adhesions. In particular, lubricant-infused surfaces demonstrate exceptional anti-fouling and antibacterial properties. However, their extreme anti-fouling nature also hinders the adhesion of essential biomolecules and cells, including osteogenic cells vital for bone-implant integration. Successful implantation of orthopedic implants demands strong osseointegration as well as antibacterial properties. In this study, we designed a bone morphogenetic protein 2 (BMP-2) immobilized lubricant-infused surface (BILS) that promotes selective osteoblast adhesion while effectively preventing undesired biosubstance adhesions. BILS exhibits excellent repellency against various liquids (sliding angle less than 5°), proteins (coverage less than 1%), and bacteria (colony-forming units 10-fold less than bare substrate). Notably, BILS enhances osteoblast adhesion, reaching 30% coverage after one day and proliferating up to 70% after three weeks. We also confirmed osteoblast differentiation on BILS through alkaline phosphatase activity and calcium assays as early and late markers, respectively. BILS-applied intramedullary nails were implanted into a rabbit femoral fracture model to investigate the antibacterial and osteogenic effects of BILS in vivo. Through histological, radiographic, and biomechanical tests, BILS was confirmed to exhibit both antibacterial as well as osseointegration properties, suggesting that BILS is a rational approach to orthopedic implants without side effects.

Biomimetic mineralization of platelet lysate/oxidized dextran cryogel as a macroporous 3D composite scaffold for bone repair

Scaffold development approaches using autologous sources for tissue repair are of great importance in obtaining bio-active/-compatible constructs. Platelet-rich plasma (PRP) containing various growth factors and platelet lysate (PL) derived from PRP are autologous products that have the potential to accelerate the tissue repair response by inducing a transient inflammatory event. Considering the regenerative capacity of PRP and PL, PRP/PL-based scaffolds are thought to hold great promise for tissue engineering as a natural source of autologous growth factors and a provider of mechanical support for cells. Here, a bio-mineralized PRP-based scaffold was developed using oxidized dextran (OD) and evaluated for future application in bone tissue engineering. Prepared PL/OD scaffolds were incubated in simulated body fluid (SBF) for 7, 14 and 21 d periods. Mineralized PL/OD scaffolds were characterized using Fourier transform infrared spectroscopy, x-ray diffraction spectroscopy, scanning electron microscopy (SEM), thermogravimetric analysis, porosity and compression tests. SEM and energy-dispersive x-ray spectroscopy analyses revealed mineral accumulation on the PL/OD scaffold as a result of SBF incubation. In vitro cytotoxicity and in vitro hemolysis tests revealed that the scaffolds were non-toxic and hemocompatible. Additionally, human osteoblasts (hOBs) exhibited good attachment and spreading behavior on the scaffolds and maintained their viability throughout the culture period. The alkaline phosphatase activity assay and calcium release results revealed that PL/OD scaffolds preserved the osteogenic properties of hOBs. Overall, findings suggest that mineralized PL/OD scaffold may be a promising scaffold for bone tissue engineering.

Cellulose acetate microwell plates for high-throughput colorimetric assays.

Single use plasticware (SUP) in scientific, diagnostic, and academic laboratories makes a significant contribution to plastic waste generation worldwide. Polystyrene (PS) microwell plates form a part of this waste. These plates are the backbone of high throughput colorimetric measurements in academic, research, and healthcare settings for detection/quantification of wide-ranging analytes including proteins, carbohydrates, nucleic acids, and enzyme activity. Polystyrene (PS) microwell plates serve as a platform for holding samples and reagents, where mixing initiates chemical reaction(s), and the ensuing color changes are quantified using a microplate reader. However, these plates are rarely reused or recycled, contributing to the staggering amounts of plastic waste generated in scientific laboratories. Here, we are reporting the fabrication of cellulose acetate (CA) microwell plates as a greener alternative to non-biodegradable PS plates and we demonstrate their application in colorimetric assays. These easy to fabricate, lighter weight, customizable, and environmentally friendly plates were fabricated in 96- and 384-well formats and made water impermeable through chemical treatment. The plates were tested in three different colorimetric analyses: (i) bicinchoninic acid assay (BCA) for protein quantification; (ii) chymotrypsin (CT) activity assay; and (iii) alkaline phosphatase (AP) activity assay. Color intensities were quantified using a freely available smartphone application, Spotxel® Reader (Sicasys Software GmbH). To benchmark the performance of this platform, the same assays were performed in commercial PS plates too and quantified using a UV/Vis microplate reader. The two systems yielded comparable linear correlation coefficients, LOD and LOQ values, thereby validating the CA plate-cell phone based analytical method. The CA microwell plates, coupled with smart phone optical data capture, provide greener, accessible, and scalable tools for all laboratory settings and are particularly well-suited for resource- and infrastructure-limited environments.

Ratio-fluorescent and naked-eye visualized dual-channel sensing strategy for Cu2+ and alkaline phosphatase activity assay.

The levels of Cu2+ and alkaline phosphatase (ALP) are the important indicators of the developed stage of the relative diseases. Herein, a binary ratio-fluorescent and smartphone-assisted visual strategy basing on 4'-aminomethyl-4, 5', 8-trimethylpsoralen (AMT) and the oxidation of o-phenylenediamine was developed. Under the action of Cu2+, the fluorescent molecule, 3-diaminophenazine (DAP) formed which can act as a fluorescent acceptor of the ratio-fluorescent sensor. The emission spectrum of AMT overlapped with the excitation spectrum of DAP and, thus, it can act as the fluorescent donor of the ratio-fluorescent sensor. With the increasing concentration of Cu2+ and ALP, the fluorescent intensity of AMT decreased and the fluorescent intensity of DAP increased. The dual-emission reverse change ratio-fluorescent sensor realized the sensitive detection Cu2+ and ALP with the detection limits of 2nM and 0.03 U/mL, respectively. In addition, the acceptable recoveries were obtained when the Cu2+ and ALP in spiked samples were detected. Furthermore, the relative activity of ALP was assessed by increasing the concentrations of the inhibitor Na3VO4 and IC50 of 25μM was obtained. Importantly, the target concentration-dependent color change of DAP allowed us to utilize R/B ratio values to design the smartphone-assisting visual detection model of Cu2+ and ALP activity with the detection limits of 0.1μM and 0.18 U/mL. This simple, flexible, dual-mode sensor strategy has a potential for disease diagnosis and drug screening.