Association between serum alkaline phosphatase and clinical prognosis in patients with acute liver failure following cardiac arrest: a retrospective cohort study.

BACKGROUND: Acute liver failure (ALF) following cardiac arrest (CA) poses a significant healthcare challenge, characterized by high morbidity and mortality rates. This study aims to assess the correlation between serum alkaline phosphatase (ALP) levels and poor outcomes in patients with ALF following CA. METHODS: A retrospective analysis was conducted utilizing data from the Dryad digital repository. The primary outcomes examined were intensive care unit (ICU) mortality, hospital mortality, and unfavorable neurological outcome. Multivariable logistic regression analysis was employed to assess the relationship between serum ALP levels and clinical prognosis. The predictive value was evaluated using receiver operator characteristic (ROC) curve analysis. Two prediction models were developed, and model comparison was performed using the likelihood ratio test (LRT) and the Akaike Information Criterion (AIC). RESULTS: A total of 194 patients were included in the analysis (72.2% male). Multivariate logistic regression analysis revealed that a one-standard deviation increase of ln-transformed ALP were independently associated with poorer prognosis: ICU mortality (odds ratios (OR) = 2.49, 95% confidence interval (CI) 1.31-4.74, P = 0.005), hospital mortality (OR = 2.21, 95% CI 1.18-4.16, P = 0.014), and unfavorable neurological outcome (OR = 2.40, 95% CI 1.25-4.60, P = 0.009). The area under the ROC curve for clinical prognosis was 0.644, 0.642, and 0.639, respectively. Additionally, LRT analyses indicated that the ALP-combined model exhibited better predictive efficacy than the model without ALP. CONCLUSIONS: Elevated serum ALP levels upon admission were significantly associated with poorer prognosis of ALF following CA, suggesting its potential as a valuable marker for predicting prognosis in this patient population.

Osteogenic effect of poly(lactic-co-glycolic acid) microcapsules with different molecular weights encapsulating bone morphogenetic protein 2.

OBJECTIVES: This study aimed to explore the effects of bone morphogenetic protein 2 (BMP-2) encapsula-ted in poly(lactic-co-glycolic acid) (PLGA) microcapsules with different molecular weights on the osteogenic ability of osteoblasts. METHODS: PLGA microcapsules with different molecular weights (12 000, 30 000) encapsulating BMP-2, were prepared using a dual-channel microinjection pump. The morphology and structure of the microcapsules were characterized by optical microscopy and scanning electron microscopy. The sustained-release performance of the microcapsules was characterized by phosphate buffered saline immersion method. The cell compatibility of the microcapsules was detected by the Calcein-AM/PI staining and CCK-8 method. The chemotactic effect of BMP-2-encapsulated microcapsules on MC3T3-E1 cells after 48 h of treatment was detected by the Transwell assay. The alkaline phosphatase activity assay and Alizarin Red S staining were used to characterize the effect of microcapsules on the osteogenic ability of MC3T3-E1 cells. RESULTS: Both types of microcapsules with different molecular weights exhibited smooth surfaces, as well as uniform and good cell compatibility. The chemotactic effect of the 12 000 microcapsules was outstanding. The 30 000 microcapsules had a longer sustained-release time, and the initial burst release was reduced by approximately 25% compared with the 12 000 microcapsules. In addition, 30 000 microcapsules performed better in long-term osteogenesis induction than 12 000 microcapsules. CONCLUSIONS: In this study, the release of BMP-2 is regulated by adjusting the molecular weight of PLGA, and the results indicate that 30 000 microcapsules can better induce the long-term osteogenic ability of MC3T3-E1 cells.

Differences in Bone Metabolism between Children with Prader-Willi Syndrome during Growth Hormone Treatment and Healthy Subjects: A Pilot Study.

Despite therapy with growth hormone (GH) in children with Prader-Willi syndrome (PWS), low bone mineral density and various orthopedic deformities have been observed often. Therefore, this study aimed to analyze bone markers, with an emphasis on vitamin K-dependent proteins (VKDPs), in normal-weight children with PWS undergoing GH therapy and a low-energy dietary intervention. Twenty-four children with PWS and 30 healthy children of the same age were included. Serum concentrations of bone alkaline phosphatase (BALP), osteocalcin (OC), carboxylated-OC (Gla-OC), undercarboxylated-OC (Glu-OC), periostin, osteopontin, osteoprotegerin (OPG), sclerostin, C-terminal telopeptide of type I collagen (CTX-I), and insulin-like growth factor-I (IGF-I) were determined using immunoenzymatic methods. OC levels and the OC/CTX-I ratios were lower in children with PWS than in healthy children (p = 0.011, p = 0.006, respectively). Glu-OC concentrations were lower (p = 0.002), but Gla-OC and periostin concentrations were higher in patients with PWS compared with the controls (p = 0.005, p < 0.001, respectively). The relationships between IGF-I and OC (p = 0.013), Gla-OC (p = 0.042), and the OC/CTX-I ratio (p = 0.017) were significant after adjusting for age in children with PWS. Bone turnover disorders in children with PWS may result from impaired bone formation due to the lower concentrations of OC and the OC/CTX-I ratio. The altered profile of OC forms with elevated periostin concentrations may indicate more intensive carboxylation processes of VKDPs in these patients. The detailed relationships between the GH/IGF-I axis and bone metabolism markers, particularly VKDPs, in children with PWS requires further research.

Photobiomodulation Dose-Response on Adipose-Derived Stem Cell Osteogenesis in 3D Cultures.

Osteoporosis and other degenerative bone diseases pose significant challenges to global healthcare systems due to their prevalence and impact on quality of life. Current treatments often alleviate symptoms without fully restoring damaged bone tissue, highlighting the need for innovative approaches like stem cell therapy. Adipose-derived mesenchymal stem cells (ADMSCs) are particularly promising due to their accessibility, abundant supply, and strong differentiation potential. However, ADMSCs tend to favor adipogenic pathways, necessitating the use of differentiation inducers (DIs), three-dimensional (3D) hydrogel environments, and photobiomodulation (PBM) to achieve targeted osteogenic differentiation. This study investigated the combined effects of osteogenic DIs, a fast-dextran hydrogel matrix, and PBM at specific wavelengths and fluences on the proliferation and differentiation of immortalized ADMSCs into osteoblasts. Near-infrared (NIR) and green (G) light, as well as their combination, were used with fluences of 3 J/cm2, 5 J/cm2, and 7 J/cm2. The results showed statistically significant increases in alkaline phosphatase levels, a marker of osteogenic differentiation, with G light at 7 J/cm2 demonstrating the most substantial impact on ADMSC differentiation. Calcium deposits, visualized by Alizarin red S staining, appeared as early as 24 h post-treatment in PBM groups, suggesting accelerated osteogenic differentiation. ATP luminescence assays indicated increased proliferation in all experimental groups, particularly with NIR and NIR-G light at 3 J/cm2 and 5 J/cm2. MTT viability and LDH membrane permeability assays confirmed enhanced cell viability and stable cell health, respectively. In conclusion, PBM significantly influences the differentiation and proliferation of hydrogel-embedded immortalized ADMSCs into osteoblast-like cells, with G light at 7 J/cm2 being particularly effective. These findings support the combined use of 3D hydrogel matrices and PBM as a promising approach in regenerative medicine, potentially leading to innovative treatments for degenerative bone diseases.

Levels of B-ALP and TRAP-5b in a patient with short root anomaly: a case report.

The purpose of this study was to observe the changes in bone-specific alkaline phosphatase (B-ALP) and tartrate-resistant acid phosphatase-5b (TRAP-5b) in a patient diagnosed with short root anomaly (SRA). The detailed clinical data and history of related clinical symptoms of the SRA patient were retrieved. Oral examination showed that the shape and color of the tooth crown were normal. Tooth 11 and 12 were missing, and the mobility degree of other teeth was II-III. Panoramic radiograph examination showed that the root length only reached the neck of the tooth. Laboratory results showed that blood spectrum, chromosome and trace elements were normal. Endocrinological evaluation indicated that hormone levels were within normal limits; however, both B-ALP and TRAP-5b were higher than the normal range. The present case shows that SRA may be related to an imbalance in osteoblast/osteoclast metabolism, which provides a new direction for the etiological research of this disease.

Novel Role of the ALPI Gene Associated with Constipation Caused by Complement Component 3 Deficiency.

Complement component 3 (C3) deficiency has recently been reported as one of the novel causes of constipation. To identify a unique gene specific to constipation caused by C3 deficiency, the total RNA extracted from the mid colon of C3 knockout (C3 KO) mice was hybridized to oligonucleotide microarrays, and the function of the candidate gene was verified in in vitro and in vivo models. C3 KO mice used for microarrays showed definite phenotypes of constipation. Overall, compared to the wild type (WT), 1237 genes were upregulated, and 1292 genes were downregulated in the C3 KO mice. Of these, the major genes included were lysine (K)-specific demethylase 5D (KDM5D), olfactory receptor 870 (Olfr870), pancreatic lipase (PNLIP), and alkaline phosphatase intestinal (ALPI). Specifically, the ALPI gene was selected as a novel gene candidate based on alterations during loperamide (Lop)-induced constipation and intestinal bowel disease (IBD). The upregulation of ALPI expression treated with acetate recovered the expression level of mucin-related genes in primary epithelial cells of C3 KO mice as well as most phenotypes of constipation in C3 KO mice. These results indicate that ALPI plays an important role as the novel gene associated with C3 deficiency-induced constipation.

Nanocrystal hydroxyapatite carrying traditional Chinese medicine for osteogenic differentiation.

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

Combined DeRitis ratio and alkaline phosphatase on the prediction of portal vein tumor thrombosis in patients with hepatocellular carcinoma.

Portal vein tumor thrombosis (PVTT) is one of the common complications of HCC and represents a sign of poor prognosis. PVTT signifies advanced liver cancer, deteriorating liver function, and heightened susceptibility to intrahepatic dissemination, systemic metastasis, and complications related to portal hypertension. It is important to seek novel strategies for PVTT arising from HCC. Portal vein tumor thrombus (PVTT) in hepatocellular carcinoma (HCC) represents a worse liver function, less treatment tolerance, and poor prognosis. This study aimed to investigate the diagnostic value of the combination of the DeRitis ratio (AST/ALT) and alkaline phosphatase (ALP) index (briefly named DALP) in predicting the occurrence risk of PVTT in patients with HCC. We performed a retrospective study enrolling consecutive patients with HCC from January 2017 to December 2020 in Hebei Medical University Third Hospital. ROC analysis was performed to estimate the predictive effectiveness and optimal cut-off value of DALP for PVTT occurrence in patients with HCC. Kaplan-Meier analysis revealed the survival probabilities in each subgroup according to the risk classification of DALP value. Univariate and multivariate Logistics regression analyses were applied to determine the independent risk for poor prognosis. ROC analysis revealed that the optimal cut-off value for DALP was 1.045, with an area under the curve (AUC) of 0.793 (95% CI 0.697-0.888). Based on the DALP classification (three scores: 0-2) with distinguishable prognoses, patients in the score 0 group had the best prognosis with a 1-year overall survival (OS) of 100%, whereas score 2 patients had the worst prognosis with 1-year OS of 72.4%. Similarly, there was a statistically different recurrence-free survival among the three groups. Besides, this risk classification was also associated with PVTT progression in HCC patients (odds ratio [OR] 5.822, P < 0.0001). Pathologically, patients in the score 2 group had more advanced tumors considering PVTT, extrahepatic metastasis, and ascites than those in score 0, 1 groups. Moreover, patients with a score of 2 had more severe hepatic inflammation than other groups. Combination of DeRitis ratio and ALP index presented a better predictive value for PVTT occurrence in patients with HCC, contributing to the tertiary prevention.

Whole genome sequencing in adults with clinical hallmarks of hypophosphatasia negative for ALPL variants.

BACKGROUND: Hypophosphatasia (HPP) is a rare disease caused by deficient activity of tissue-nonspecific alkaline phosphatase (ALP), encoded by the ALPL gene. The primary objective was to explore novel ALPL variants by whole genome sequencing (WGS) in patients with HPP who previously tested negative by standard methods for ALPL variants. The secondary objective was to search for genes beyond ALPL that may reduce ALP activity or contribute to HPP symptoms. METHODS AND RESULTS: WGS was performed in 16 patients clinically diagnosed with HPP who had ALP activity below the normal range and tested negative for ALPL variants. Genetic variants in ALPL and genes possibly associated with low ALP activity or phenotypic overlap with HPP were assessed. All 16 patients had ALP activity below the normal range. WGS did not identify any novel disease-causing ALPL variants. Positive findings for other gene variants were identified in 4 patients: 1 patient presented with variants in COL1A1, NLRP12, and SCN9A, coding for collagen, type, I alpha-1 chain, nod-like receptor pyrin domain containing 12, and sodium voltage-gated channel alpha subunit 9, respectively; 1 presented with a heterozygous, likely pathogenic variant in P3H1 coding for prolyl 3 hydroxylase 1; 1 presented with a heterozygous pathogenic variant in SGCE, coding for sarcoglycan epsilon; and 1 presented with a heterozygous variant of uncertain significance in VDR, encoding vitamin D receptor. CONCLUSION: Genomic analysis did not identify novel ALPL variants or a pattern of disease-causing variants in genes other than ALPL to explain the HPP phenotype in these patients. REGISTRATION: Clinicaltrials.gov identifier: NCT04925804.

Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation.

Periodontal regeneration is a challenge, and tissue engineering based on periodontal ligament stem cells (PDLSCs) has been shown to be a promising alternative to this process. However, the need for scaffolds has limited the therapeutic use of PDLSCs. In this context, scaffold-free tissue engineering using the cell sheet (CS) technique has been developed as an alternative approach to improve tissue regeneration. Previously, we showed that Protease-activated receptor-1 (PAR1) can regulate PDLSCs. Herein, we evaluate whether PAR1 influences osteogenesis in CSs produced from PDLSCs, without the use of scaffolds. PDLSCs were isolated and immunophenotyped. Then, CSs were obtained by supplementing the culture medium with ascorbic acid (50 µg/mL), and PAR1 was activated through its agonist peptide (100 nM). Scaffold-free 3D CSs were successfully produced from PDLSCs, and they showed higher proliferation potential than isolated PDLSCs. Also, PAR1 activation decreased senescence and improved osteogenic differentiation of CSs by increasing mineralized nodule deposition and alkaline phosphatase concentration; PAR1 also modulated osteogenic markers at the gene and protein levels. We further demonstrated that this effect was regulated by Wnt, TGF-ßI, MEK, p38 MAPK, and FGF/VEGF signaling pathways in PDLSCs (p < 0.05%). Overall, PAR1 activation increased osteogenic activity in CSs, emerging as a promising scaffold-free therapeutic approach for periodontal regeneration.

Energy level as a theranostic factor for successful therapy of tissue injuries with polyphosphate: the triad metabolic energy - mechanical energy - heat.

Rationale: Tissue regeneration of skin and bone is an energy-intensive, ATP-consuming process that, if impaired, can lead to the development of chronic clinical pictures. ATP levels in the extracellular space including the exudate of wounds, especially chronic wounds, are low. This deficiency can be compensated by inorganic polyphosphate (polyP) supplied via the blood platelets to the regenerating site. Methods: The contribution of the different forms of energy derived from polyP (metabolic energy, mechanical energy and heat) to regeneration processes was dissected and studied both in vitro and in patients. ATP is generated metabolically during the enzymatic cleavage of the energy-rich anhydride bonds between the phosphate units of polyP, involving the two enzymes alkaline phosphatase (ALP) and adenylate kinase (ADK). Exogenous polyP was administered after incorporation into compressed collagen or hydrogel wound coverages to evaluate its regenerative activity for chronic wound healing. Results: In a proof-of-concept study, fast healing of chronic wounds was achieved with the embedded polyP, supporting the crucial regeneration-promoting activity of ATP. In the presence of Ca2+ in the wound exudate, polyP undergoes a coacervation process leading to a conversion of fibroblasts into myofibroblasts, a crucial step supporting cell migration during regenerative tissue repair. During coacervation, a switch from an endothermic to an exothermic, heat-generating process occurs, reflecting a shift from an entropically- to an enthalpically-driven thermodynamic reaction. In addition, mechanical forces cause the appearance of turbulent flows and vortices during liquid-liquid phase separation. These mechanical forces orient the cellular and mineralic (hydroxyapatite crystallite) components, as shown using mineralizing SaOS-2 cells as a model. Conclusion: Here we introduce the energetic triad: metabolic energy (ATP), thermal energy and mechanical energy as a novel theranostic biomarker, which contributes essentially to a successful application of polyP for regeneration processes.

Copper (II)-catalyzed polydopamine mediated photothermal sensors for visual quantitative point-of-care testing.

BACKGROUND: Temperature sensing is commonly used in point-of-care (POC) detection technologies, yet the portability and convenience of use are frequently compromised by the complexity of thermosensitive processes and signal transduction. Especially, multi-step target recognition reactions and temperature measurement in the reaction vessel present challenges in terms of stability and integration of detection devices. To further combine photothermal reaction and signal readout in one assay, these two processes enable to be integrated into miniaturized microfluidic chips, thereby facilitating photothermal sensing and achieving a simple visual temperature sensing as POC detection. RESULTS: A copper ion (Cu2+)-catalyzed photothermal sensing system integrated onto a microfluidic distance-based analytical device (µDAD), enabling the visual, portable, and sensitive quantitative detection of multiple targets, including ascorbic acid, glutathione, and alkaline phosphatase (ALP). The polydopamine nanoparticles (PDA NPs) were synthesized by the regulation of free Cu2+ through redox or coordination reactions, facilitating the transduction of distinct photothermal response signals and providing the versatile Cu2+-responsive sensing systems. Promoted by integration with a photothermal µDAD, the system combines PDA's photothermal responsiveness and thermosensitive gas production of ammonium bicarbonate for improved sensitivity of ALP detection, reaching the detection limit of 9.1 mU/L. The system has successfully achieved on-chip detection of ALP with superior anti-interference capability and recoveries ranging from 96.8 % to 104.7 %, alongside relative standard deviations below 8.0 %. SIGNIFICANCE AND NOVELTY: The µDAD design accommodated both the photothermal reaction of PDA NPs and thermosensitive gas production reaction, achieving the rapid sensing of visual distance signals. The µDAD-based Cu2+-catalyzed photothermal sensing system holds substantial potential for applications in biochemical analysis and clinical diagnostics, underscored by the versatile Cu2+ regulation mechanism for a broad spectrum of biomarkers.

Assessment of hemodynamic and blood parameters that may reflect macroscopic quality of porcine kidneys during normothermic machine perfusion using whole blood.

PURPOSE: Using ex vivo normothermic machine perfusion (NMP) with whole blood we assessed marginal porcine kidneys under reperfusion. The aim was to link measureable machine and clinical blood parameters with the currently used visual assessment. This could serve as a baseline for a standardized evaluation score to identify potentially transplantable kidneys in the future. METHODS: Kidneys and autologous whole blood were procured from slaughterhouse pigs (n = 33) and were perfused for 4 h using NMP. The hemodynamic parameters arterial pressure (AP), renal blood flow (RBF) and intrarenal resistance (IRR) were measured. Activity of aspartate transaminase (AST), gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and lactate were assessed in blood at 0/1/2/4 h. Kidneys were grouped into "potentially transplantable" (PT) or "not transplantable" (NT) based on their overall macroscopic appearance after NMP by an experienced physician. RESULTS: PT-kidneys (n = 20) had a significantly lower IRR and higher RBF than NT-kidneys (n = 13). GGT, ALP and LDH did not differ significantly, but at 4 h, AST was significantly higher in PT-kidneys compared to NT-kidneys. Lactate levels kept increasing during NMP in NT-kidneys and were significantly higher at 1/2/4 h than in PT-kidneys. CONCLUSION: The immediately assessed macroscopic aspects of examined kidneys correlated with hemodynamic parameters, increased lactate and lower AST in this study. In the future, NMP with whole blood could be a useful tool to extend the donor pool by allowing the assessment of otherwise unknown characteristics of marginal kidneys before transplantation.

Alkaline phosphatase-activated prodrug system based on a bifunctional CuO NP tandem nanoenzyme for on-demand bacterial inactivation and wound disinfection.

Nanozymes are promising antimicrobials, as they produce reactive oxygen species (ROS). However, the intrinsic lack of selectivity of ROS in distinguishing normal flora from pathogenic bacteria deprives nanozymes of the necessary selectivities of ideal antimicrobials. Herein, we exploit the physiological conditions of bacteria (high alkaline phosphatase (ALP) expression) using a novel CuO nanoparticle (NP) nanoenzyme system to initiate an ALP-activated ROS prodrug system for use in the on-demand precision killing of bacteria. The prodrug strategy involves using 2-phospho-L-ascorbic acid trisodium salt (AAP) that catalyzes the ALP in pathogenic bacteria to generate ascorbic acid (AA), which is converted by the CuO NPs, with intrinsic ascorbate oxidase- and peroxidase-like activities, to produce ROS. Notably, the prodrug system selectively kills Escherichia coli (pathogenic bacteria), with minimal influence on Staphylococcus hominis (non-pathogenic bacteria) due to their different levels of ALP expression. Compared to the CuO NPs/AA system, which generally depletes ROS during storage, CuO NPs/AAP exhibits a significantly higher stability without affecting its antibacterial activity. Furthermore, a rat model is used to indicate the applicability of the CuO NPs/AAP fibrin gel in wound disinfection in vivo with negligible side effects. This study reveals the therapeutic precision of this bifunctional tandem nanozyme platform against pathogenic bacteria in ALP-activated conditions.

Label-Free Imaging of Mesenchymal Stem Cell Spheroid Differentiation with Flexible-Probe SECM and a Microfluidic Device.

Mesenchymal stem cells (MSCs) have emerged as an indispensable source for stem cell research and preclinical studies due to their capacity for in vitro proliferation and their potential to differentiate into mesodermal lineages, particularly into osteoblasts. This capability has propelled their application in the fields of bone regeneration and osteochondral repair. Traditional methodologies for assessing the differentiation status of MSCs necessitate invasive procedures such as cell lysis or fixation. In this study, we introduce a nondestructive technique that utilizes an integrated label-free approach to evaluate the osteogenic maturation of MSC spheroid aggregates. This method employs scanning electrochemical microscopy (SECM) with a flexible probe in conjunction with a top-removable microfluidic device designed for easy SECM access. By tracking the production rate of p-aminophenol (PAP) in the generation/collection mode and assessing morphological changes via the negative feedback mode using [Ru(NH3)6]Cl3 (Ruhex), we can discern variations in the alkaline phosphatase (ALP) activity indicative of osteogenic differentiation. This innovative strategy enables the direct evaluation of osteogenic differentiation in MSC spheroids cultured within microwell arrays without necessitating any labeling procedures. The utilization of a flexible microelectrode as the probe that scans in contact mode (with probe-substrate distances potentially as minimal as 0 µm) affords enhanced resolution compared to the traditional stiff-probe technique. Furthermore, this method is compatible with subsequent molecular biology assays, including gene expression analysis and immunofluorescence, thereby confirming the electrochemical findings and establishing the validity of this integrative approach.

Tissue scaffolds mimicking hierarchical bone morphology as biomaterials for oral maxillofacial surgery with augmentation: structure, properties, and performance evaluation forin vitrotesting.

In this study, tissue scaffolds mimicking hierarchical morphology are constructed and proposed for bone augmentation. The scaffolds are fabricated using lyophilization, before coating them with collagen (Col). Subsequently, the Col-coated scaffolds undergo a second lyophilization, followed by silk fibroin (SF) coating, and a third lyophilization. Thereafter, the scaffolds are divided into six groups with varying ratios of Col to SF: Col/SF = 7:3, 5:5, 3:7, 10:0, and 0:10, with an SF scaffold serving as the control group. The scaffold morphology is examined using a scanning electron microscope, while molecular and structural formations are characterized by Fourier transform infrared spectrometer and differential scanning calorimeter, respectively. Physical and mechanical properties including swelling and compression are tested. Biological functions are assessed throughin vitroosteoblast cell culturing. Biomarkers indicative of bone formation-cell viability and proliferation, alkaline phosphatase activity, and calcium content-are analyzed. Results demonstrate that scaffolds coated with Col and SF exhibit sub-porous formations within the main pore. The molecular formation reveals interactions between the hydrophilic groups of Col and SF. The scaffold structure contains bound water and SF formation gets disrupted by Col. Physical and mechanical properties are influenced by the Col/SF ratio and morphology due to coating. The biological functions of scaffolds with Col and SF coating show enhanced potential for promoting bone tissue formation, particularly the Col/SF (7:3) ratio, which is most suitable for bone augmentation in small defect areas.

A high-calcium environment induced ectopic calcification of renal interstitial fibroblasts via TFPI-2-DCHS1-ALP/ENPP1 axis to participate in Randall's plaque formation.

Randall's plaques (RP) serve as anchoring sites for calcium oxalate (CaOx) stones, but the underlying mechanism remains unclear. Renal interstitium with a high-calcium environment is identified as pathogenesis of RP formation where the role of human renal interstitial fibroblasts (hRIFs) was highlighted. Our study aims to elucidate the potential mechanism by which a high-calcium environment drives ectopic calcification of hRIFs to participate in RP formation. Alizarin Red staining demonstrated calcium nodules in hRIFs treated with high-calcium medium. Utilizing transcriptome sequencing, tissue factor pathway inhibitor-2 (TFPI-2) was found to be upregulated in high-calcium-induced hRIFs and RP tissues, and TFPI-2 promoted high-calcium-induced calcification of hRIFs. Subsequently, the downstream regulator of TFPI2 was screened by transcriptome sequencing analysis of hRIFs with TFPI-2 knockdown or overexpressed. Dachsous Cadherin Related 1 (DCHS1) knockdown was identified to suppress the calcification of hRIFs enhanced by TFPI-2. Further investigation revealed that TFPI-2/DCHS1 axis promoted high-calcium-induced calcification of hRIFs via disturbing the balance of ENPP1/ALP activities, but without effect on the canonical osteogenic markers, such as osteopontin (OPN), osteogenic factors runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2). In summary, our study mimicked the high-calcium environment observed in CaOx stone patients with hypercalciuria, and discovered that the high-calcium drove ectopic calcification of hRIFs via a novel TFPI-2-DCHS1-ALP/ENPP1 pathway rather than adaption of osteogenic phenotypes to participate in RP formation.

Rapid and sensitive detection of SARS-CoV-2 based on a phage-displayed scFv antibody fusion with alkaline phosphatase and NanoLuc luciferase.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent pandemic have led to devastating public health and economic losses. The development of highly sensitive, rapid and inexpensive methods to detect and monitor coronaviruses is essential for family diagnosis, preventing infections, choosing treatments and programs and laying the technical groundwork for viral diagnosis. This study established one-step immunoassays for rapid and sensitive detection of SARS-CoV-2 by using a single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) or NanoLuc (NLuc) luciferase. First, a high-affinity scFv antibody specific to the SARS-CoV-2 nucleocapsid (N) protein was screened from hybridoma cells-derived and phage-displayed library. Next, prokaryotic expression of the scFv-AP and scFv-NLuc fusion proteins were induced, leading to excellent antibody binding properties and enzyme catalytic activities. The scFv-AP fusion had a detection limit of 3 pmol per assay and was used to produce eye-readable biosensor readouts. Moreover, the scFv-NLuc protein was applied in a highly sensitive luminescence immunoassay, achieving a detection limit lower than 0.1 pmol per assay. Therefore, the scFv-AP and scFv-NLuc fusion proteins can be applied for the rapid and simple diagnosis of SARS-CoV-2 to safeguard human health and provide guidance for the detection of other pathogenic viruses.

Photobiomodulation Effect of Diode Laser on Differentiation of Osteoprogenitor Cells in Rat Bone Marrow.

BACKGROUND/AIM: Bone marrow cells contain nonhematopoietic cells with the ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Mechanical stress influences osteoblast differentiation of bone marrow cells into osteogenic, chondrogenic, and adipogenic lineages, measurable as the abundance of alkaline phosphatase-positive (ALP+) colony-forming unit-fibroblasts (CFU-F); however, the effect of diode laser irradiation on osteoblast differentiation is unknown. The aim of this study was to analyze the effects of photobiomodulation on the osteogenic differentiation of mesenchymal stem cells in the bone marrow, using the CFU-F assay. MATERIALS AND METHODS: Bone marrow cells isolated from rat tibiae were cultured and irradiated with a diode laser (wavelength 808 nm) at a total energy of 0 J (control), 50 J, and 150 J. RESULTS: On day 7 after irradiation, ALP+ CFU-F were most abundant in the 50 J group and the least abundant in the 150 J group. Mineralized nodule formation was observed after long-term culture (21 days). Compared with the control group, there were significantly more nodules in the 50 J group and significantly fewer nodules in the 150 J group. Osteocalcin mRNA expression was highest in the 50 J group, and there was no difference between the control and 150 J groups. CONCLUSION: Irradiation with 50 J was effective in stimulating osteogenesis in bone marrow stem cells. These findings suggest that diode laser irradiation can induce osteogenesis in rat bone marrow cells in an energy-dependent manner, and appears suitable for application in bone regeneration therapy.

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

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