Measuring Phosphatase Activity Research Articles

Synthesis of N, N,O and O-carboxymethyl chitosan derivatives of controllable substitution degrees and their utilization as electrospun scaffolds for bone tissue engineering

Most chitosan (CS) carboxymethylation approaches are weighed down by insufficient description protocols regarding the reaction specificity and the degree of substitution (DS). Here, we provide three carboxymethylation protocols of enhanced specificity towards the amine (N-), amine/hydroxy (N,O-) and hydroxy (O-) groups of CS. The DS for all samples was found to be ∼70 %, as confirmed by NMR analysis. To illustrate the modified materials' potential in bone tissue regeneration, each derivative was blended with poly(vinyl alcohol) to prepare scaffolds via electrospinning. Both materials and electrospun membranes were characterized in terms of their physicochemical properties by Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry and water contact angle measurements. The electrospun membranes' swelling ratio, degradation, tensile strength, as well as morphology were also examined through scanning electron microscopy before and after heat treatment at 120 °C, which was used as a method of physical stabilization, leading to significantly enhanced degradation rate and mechanical strength. The three electrospun scaffold types were loaded with MC3T3-E1 pre-osteoblastic cells and their cell adhesion, viability and growth rate were assessed. Finally, osteogenic differentiation was examined by means of alkaline phosphatase activity measurement, calcium mineralization and formation of extracellular matrix markers, with all materials showing promising prospects.

Effect of microcapsules’ mechanical stability on the encapsulated cells’ behavior

Stem cell microencapsulation offers immunoprotection, utilizing natural polymers such as sodium alginate (SA), chitosan, and derivatives such as trimethyl chitosan (TMC). Additive minerals improve microcapsule properties and cell differentiation. Layering increases mechanical strength and shields cells from the host’s immune system. This study explores the impact of incorporating nano-hydroxyapatite (nHA) and graphene oxide (GO) into the three-layer alginate–TMC–alginate microcapsules housing human adipose mesenchymal stem cells (haMSCs). The experimental design focused on optimizing the mechanical stability, with the ideal GO and nHA contents identified as 0.81% and 34% w/w, respectively. Rheological analysis revealed enhanced mechanical consistency, evidenced by higher storage modulus and lower loss factor in the optimized sample. Viability of the microencapsulated haMSCs, assessed through acridine orange/propidium iodide staining and MTT assays, affirmed the nontoxic nature of the microcapsules. Osteogenic potential was evaluated via alkaline phosphatase (ALP) activity measurement and immunocytochemistry staining. Microcapsules containing nHA and GO exhibited significantly elevated ALP activity, underscoring the pivotal role of these additives in enhancing mesenchymal stem cell differentiation. The highest osteocalcin deposition occurred in the optimized and SA/nHA 40% w/w groups in the differentiation medium. This comprehensive investigation highlights the potential of three-layer microcapsules with nHA and GO for bone tissue engineering applications.

Efficient three-dimensional (3D) human bone differentiation on quercetin-functionalized isotropic nano-architecture chitinous patterns of cockroach wings

Developing cost-effective, biocompatible scaffolds with nano-structured surface that truthfully replicate the physico-(bio)chemical and structural properties of bone tissue's extracellular matrix (ECM) is still challenging. In this regard, surface functionalization of natural scaffolds to enhance capability of mimicking 3D niches of the bone tissue has been suggested as a solution. In the current study, we aimed to investigate the potential of chitin-based cockroach wings (CW) as a natural scaffold for bone tissue engineering. To raise the osteogenic differentiation capacity of such a scaffold, a quercetin coating was also applied (hereafter this scaffold is referred as QCW). Moreover, the QCW scaffold exhibited effective antibacterial properties against gram-positive S. aureus bacteria. With respect to bone regeneration, the QCW scaffold optimally induced the differentiation of adipose-derived human mesenchymal stem cells (AD-hMSCs) into osteoblasts, as validated by mineralization assays, alkaline phosphatase (ALP) activity measurements, expression of pre-osteocyte marker genes, and immunocytochemical staining. Confirmation of the potent biocompatibility and physicochemical characteristics of the QCW scaffold through a series of in vitro and in vivo analysis revealed that surface modification had significant effect on multi-purpose features of obtained scaffold. Altogether, surface modification of QCW made it as an affordable bioinspired scaffold for bone tissue engineering.

In vitro biodegradation, blood, and cytocompatibility studies of a bioactive lithium silicate glass-ceramic

This study investigates a novel non-stoichiometric lithia-silica glass-ceramic (Bioactive LD) designed for bone tissue engineering applications. Incorporating SiO2–Li2O–CaO–P2O5–K2O–SrO–ZnO, the glass-ceramic underwent optimized thermal treatments resulting in a composition predominantly of lithium disilicate (Li2Si2O5 - 87 %) with a minor lithium phosphate phase (Li3PO4 - 13 %). Structural analysis using X-ray diffraction (XRD) and scanning electron microscopy (SEM) revealed a refined microstructure with Li2Si2O5 crystals in rod form with a high aspect ratio of around 4:1. Expanding on previous work, this study investigates the in vitro osteoblastic response of Bioactive LD compared to a commercial glass-ceramic (E-Max CAD®). The evaluation, employing live/dead assays, resazurin assays, and alkaline phosphatase activity measurements at different time points, demonstrated comparable cell viability and proliferation on both glass-ceramics. The chemical stability of the samples was tested, and the Bioactive LD showed a higher amount of leached ions in the solution, without significant mass loss, consistent with the standardized values for a class 4 material (ISO 6872), with no significant change in its microstructure. The chemical solubility tests were investigated employing SEM and energy dispersive X-ray analysis (EDS), giving insights into the role of leached ions in the microstructure and bioactivity of the materials. Notably, Bioactive LD exhibits higher alkaline phosphatase activity, indicating enhanced stimulation of osteoblastic cells. Additionally, the Bioactive LD promotes mineralization matrix production, as evidenced by alizarin red S staining. The study also employs SEM-EDS to evaluate the formation of apatite mineralized matrix. Bioactive LD exhibits a homogeneous mineralized matrix with increased Calcium and Sulfur signals, suggesting early hydroxyapatite formation after Alizarin Red S assay. In contrast, Commercial LD shows limited reactivity and bioactivity, with no substantial calcium deposition. An indirect cytotoxicity test indicates minimal cytotoxicity for Bioactive LD, while Commercial LD exhibits slight cytotoxicity, consistent with findings from the literature. The results highlight Bioactive LD's potential for bone regeneration, emphasizing its enhanced bioactivity and the formation of a complex mineralized matrix that could contribute to improved bone integration properties.

Enhanced properties of nickel–silver codoped hydroxyapatite for bone tissue engineering: Synthesis, characterization, and biocompatibility evaluation

Hydroxyapatite (HAp) is the most well-known bioceramic and widely utilized in bone tissue regeneration. Hydroxyapatite is biocompatible and bioactive however, it lacks osteogenesis, angiogenesis, and antibacterial properties. In the current study, we synthesized and evaluated a novel nickel (Ni) and silver (Ag) codoped hydroxyapatite (HAp) in comparison to undoped HAp and individually doped HAp samples. Extensive physicochemical characterizations like XRD, TEM, FE-SEM/EDS, FTIR, Raman spectroscopy, and TGA were performed, confirming the crystal structure and morphology of the synthesized HAp samples. All HAp samples exhibited elongated spherical-like nanoparticle morphologies with lengths between 34 and 44 nm and widths between 21 and 26 nm. The presence of dopant atoms, Ag and Ni, were observed in the doped/codoped HAp samples by EDS elemental mapping. Biocompatibility assessments using pre-osteoblast cells indicated high cell viability for all the doped and undoped HAp samples. Osteoinduction potential through alkaline phosphatase (ALP) activity measurements and alizarin red S (ARS) staining revealed enhanced calcium deposition in the presence of Ni–Ag codoped HAp compared to other HAp samples and control groups. This highlights the importance of Ni–Ag co-doping in promoting osteogenesis, surpassing the effects of silver doped HAp and nickel doped HAp. The potential of this novel Ni–Ag codoped HAp to induce osteogenesis in pre-osteoblast cells makes it a promising material for various applications in bone tissue engineering.

ENHANCED OSTEOGENIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS BY FLEXIBLE β-TCP/PLA BONE GRAFTS WITH SILICATE ADDITIVE

In recent years, ceramics, polymers, and composites have been used to develop biologically and mechanically suitable bone scaffolds. β-tricalcium phosphate（β-TCP) is a widely used bioceramic in bone tissue engineering. It shows excellent osteoconductivity, osteoinductivity, and good biocompatibility properties, as its chemical composition is similar to the original chemical structure of bone. Herein, we designed β-TCP-PLA composite scaffolds containing two different concentrations of silicate additives. We aimed to investigate the effect of silicate-additive with varying concentrations (0.8% and 1%) on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) seeded flexible bone grafts. The morphological structure of β-TCP-PLA-based bone grafts was assessed by scanning electron microscopy (SEM) and the tensile strength of grafts was evaluated. The results showed that scaffolds had porous and flexible structures. hMSCs osteogenic differentiation was evaluated with the alkaline phosphatase (ALP) activity and DNA content measurements. Compared with β-TCP-PLA grafts, these designed synthetic flexible bone grafts with 0.8% and 1% silicate-additive significantly promoted hMSCs proliferation and osteogenic differentiation. Moreover, 0.8% silicate-additive β-TCP-PLA grafts showed increased ALP activity. The outcomes of the present study suggest that synthetic flexible bone grafts with silicate-additive might be useful for encouraging the regeneration of bone.

Low Magnesium Concentration Enforces Bone Calcium Deposition Irrespective of 1,25-Dihydroxyvitamin D3 Concentration.

Efficient coordination between Mg2+ and vitamin D maintains adequate Ca2+ levels during lactation. This study explored the possible interaction between Mg2+ (0.3, 0.8, and 3 mM) and 1,25-dihydroxyvitamin D3 (1,25D; 0.05 and 5 nM) during osteogenesis using bovine mesenchymal stem cells. After 21 days, differentiated osteocytes were subjected to OsteoImage analysis, alkaline phosphatase (ALP) activity measurements, and immunocytochemistry of NT5E, ENG (endoglin), SP7 (osterix), SPP1 (osteopontin), and the BGLAP gene product osteocalcin. The mRNA expression of NT5E, THY1, ENG, SP7, BGLAP, CYP24A1, VDR, SLC41A1, SLC41A2, SLC41A3, TRPM6, TRPM7, and NIPA1 was also assessed. Reducing the Mg2+ concentration in the medium increased the accumulation of mineral hydroxyapatite and ALP activity. There was no change in the immunocytochemical localization of stem cell markers. Expression of CYP24A1 was higher in all groups receiving 5 nM 1,25D. There were tendencies for higher mRNA abundance of THY1, BGLAP, and NIPA1 in cells receiving 0.3 mM Mg2+ and 5 nM 1,25D. In conclusion, low levels of Mg2+ greatly enhanced the deposition of bone hydroxyapatite matrix. The effect of Mg2+ was not modulated by 1,25D, although the expression of certain genes (including BGLAP) tended to be increased by the combination of low Mg2+ and high 1,25D concentrations.

Mineralization of Bone Extracellular Matrix-like Scaffolds Fabricated as Silk Sericin-Functionalized Dense Collagen-Fibrin Hybrid Hydrogels.

The design of hydrogels that combine both the biochemical cues needed to direct seeded cellular functions and mineralization to provide the structural and mechanical properties approaching those of mineralized native bone extracellular matrix (ECM) represents a significant challenge in bone tissue engineering. While fibrous hydrogels constituting of collagen or fibrin (and their hybrids) can be considered as scaffolds that mimic to some degree native bone ECM, their insufficient mechanical properties limit their application. In the present study, an automated gel aspiration-ejection (automated GAE) method was used to generate collagen-fibrin hybrid gel scaffolds with micro-architectures and mechanical properties approaching those of native bone ECM. Moreover, the functionalization of these hybrid scaffolds with negatively charged silk sericin accelerated their mineralization under acellular conditions in simulated body fluid and modulated the proliferation and osteoblastic differentiation of seeded MC3T3-E1 pre-osteoblastic cells. In the latter case, alkaline phosphatase activity measurements indicated that the hybrid gel scaffolds with seeded cells showed accelerated osteoblastic differentiation, which in turn led to increased matrix mineralization. In summary, the design of dense collagen-fibrin hybrid gels through an automated GAE process can provide a route to tailoring specific biochemical and mechanical properties to different types of bone ECM-like scaffolds, and can provide a model to better understand cell-matrix interactions in vitro for bioengineering purposes.

Di-(2-ethylhexyl) phthalate impairs angiogenesis and hematopoiesis via suppressing VEGF signaling in zebrafish

Di-(2-ethylhexyl) phthalate (DEHP) is among the most widely used plasticizers in plastic production, which has been detected in various environments. However, DEHP safety remains poorly known. Using zebrafish models, the effects of DEHP on the angiogenesis and hematopoiesis, and the underlying mechanism, were studied. Transgenic zebrafish embryos with specific fluorescence of vascular endothelial cells, myeloid cells, or hematopoietic stem cells were exposed to 0, 100, 150, 200, or 250 nM of DEHP for 22, 46 or 70 h, followed by fluorescence observation, endogenous alkaline phosphatase activity measurement, erythrocyte staining, and gene expression analysis by quantitative PCR and whole mount in situ hybridization. High DEHP concentrations decreased the sprouting rate, average diameter, and length, and the expansion area of the vessels lowered the EAP activity and suppressed the vascular endothelial growth factor (vegf) and hematopoietic marker genes, including c-myb, hbae1, hbbe1, and lyz expressions. DEHP treatment also decreased the number of hematopoietic stem cells, erythrocytes, and myeloid cells at 24 and 72 hpf. These DEHP-induced angiogenetic and hematopoietic defects might be alleviated by vegf overexpression. Our results reveal a plausible mechanistic link between DEHP exposure-induced embryonic angiogenetic defect and hematopoietic impairment.

Comparison of Analytical Values after Changing to the International Standardized Method for Lactate Dehydrogenase and Alkaline Phosphatase Measurements in Mouse and Rat.

Since April 2020, the method for lactate dehydrogenase (LD) and alkaline phosphatase (ALP) activity measurements in Japan has been switched from the Japan Society of Clinical Chemistry (JSCC) reference method, which is only used in Japan, to the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method. However, in some species, the relationship between the blood values of both enzymes measured by the two methods remains unclear. Hence, values measured by these two methods cannot be used interchangeably. Therefore, this relationship was examined in ICR mice and Wistar/ST rats. The LD and ALP values obtained by both methods were plotted on scatter graphs, and regression equations were obtained. To compare the JSCC (x) and IFCC (y) methods, regression equations were generated for LD values in non-hemolytic samples as follows: y = 0.954x - 4.008 for ICR mice and y = 0.963x - 6.324 for Wistar/ST rats. The conversion factors from the JSCC to the IFCC methods were 0.954 (mice) and 0.963 (rats). The conversion coefficients from the IFCC to the JSCC methods were 1.048 (mice) and 1.088 (rats). For ALP values in fasted mouse and rat samples, the regression equations were y = 0.336x - 2.247 and y = 0.314x - 17.626, respectively. The conversion factors from the JSCC to the IFCC methods were 0.336 (mice) and 0.314 (rats). The conversion coefficients from the IFCC to the JSCC methods were 2.978 (mice) and 3.188 (rats). These conversion factors can be used for the mutual conversion of both measured values during the transition period from the JSCC to the IFCC method. However, it should be noted that the conversion coefficients for both LD and ALP were affected by isozyme composition.

A smart magnetic hydrogel containing exosome promotes osteogenic commitment of human adipose-derived mesenchymal stem cells.

Objective(s):Exosomes, as nano-sized extracellular vehicles acting as cell-to-cell communicators, are novel promising therapeutics in the area of bone tissue engineering. Moreover, magnetic nanoparticles, whose integration with other appropriate components is viewed as an intriguing approach to strengthen bone tissue engineering efficacy. We investigated the effect of magnetic enriched with exosomes on osteogenic differentiation.Materials and Methods:Exosomes were isolated from human adipose-derived mesenchymal stem cells by Exo-spin™ kit (MSC-EX). Alginate (Alg) scaffold containing 1% (w/w) cobalt ferrite nanoparticles (CoFe2O4) was produced. MSC-EX were gently loaded onto Alg and Alg-cobalt ferrite (Alg-CF) scaffolds yielding Alg-EX and Alg-CF-EX scaffolds. The effects of MSC-Ex and magnetic hydrogel composite under an external static magnetic field (SMF) on proliferation and differentiation of MSCs were evaluated by alkaline phosphatase (ALP) activity measurement, alizarin red staining, and energy dispersive X-ray (EDX) analysis.Results:Our results showed that Alg and Alg-CF scaffolds were not only cytotoxic but also supported AdMSCs proliferation. MSC-EX loading of the scaffolds enhanced AdMSCs proliferation significantly. According to the results, Alg-CF-EX scaffolds under magnetic stimulation exhibited the most potent effect on osteogenic differentiation of cultured AdMSCs as evidenced by higher ALP activity and mineralization.Conclusion:We provided evidence that the combination of Alg hydrogel, CFNPs, and MSC-EX resulted in the construction of a bone tissue-engineering scaffold that highly supports the osteogenic commitment of MSCs.

An amelogenin-based peptide hydrogel promoted the odontogenic differentiation of human dental pulp cells

Amelogenin can induce odontogenic differentiation of human dental pulp cells (HDPCs), which has great potential and advantages in dentine-pulp complex regeneration. However, the unstability of amelogenin limits its further application. This study constructed amelogenin self-assembling peptide hydrogels (L-gel or D-gel) by heating-cooling technique, investigated the effects of these hydrogels on the odontogenic differentiation of HDPCs and explored the underneath mechanism. The critical aggregation concentration, conformation, morphology, mechanical property and biological stability of the hydrogels were characterized, respectively. The effects of the hydrogels on the odontogenic differentiation of HDPCs were evaluated via alkaline phosphatase activity measurement, quantitative reverse transcription polymerase chain reaction, western blot, Alizarin red staining and scanning electron microscope. The mechanism was explored via signaling pathway experiments. Results showed that both the L-gel and D-gel stimulated the odontogenic differentiation of HDPCs on both Day 7 and Day 14, while the D-gel showed the highest enhancement effects. Meanwhile, the D-gel promoted calcium accumulation and mineralized matrix deposition on Day 21. The D-gel activated MAPK-ERK1/2 pathways in HDPCs and induced the odontogenic differentiation via ERK1/2 and transforming growth factor/smad pathways. Overall, our study demonstrated that the amelogenin peptide hydrogel stimulated the odontogenic differentiation and enhanced mineralization, which held big potential in the dentine-pulp complex regeneration.

CircRNA hsa_circ_0001421 promotes the osteoblast differentiation of human adipose mesenchymal stem cells through the miR-608/SP7 axis

Human adipose mesenchymal stem cells (hAMSCs) can differentiate into osteoblasts, which can enhance bone mass and bone formation. Circular RNA plays an important role in the osteoblast differentiation of mesenchymal stem cells, but the mechanism remains unclear. We used hAMSCs to study the role of circRNA hsa_circ_0001421/miR-608/SP7 axis in osteoblast differentiation. qRT-PCR, western blotting, alkaline phosphatase (ALP) activity measurement, and alizarin red staining (ARS) were used to evaluate the osteoblast differentiation ability of hAMSCs. Dual luciferase reporter gene assay to analyze the relationship of miR-608 with circRNA hsa_circ_0001421 and SP7. Our results showed that circRNA hsa_circ_0001421 could promote the osteoblast differentiation of hAMSCs. qRT-PCR and western blotting showed that the expression of osteogenic genes (ALP, RUNX2, and OCN) was upregulated, and ALP activity and ARS were significantly increased. The dual luciferase reporter gene assay showed that miR-608 competitively binds with circRNA hsa_circ_0001421 and SP7. MiR-608 inhibited the expression of SP7 and repressed the osteoblast differentiation of hAMSCs, whereas circRNA hsa_circ_0001421 inhibited the expression of miR-608 and reversed the downregulation of SP7 by miR-608. Taken together, the circRNA hsa_circ_0001421/miR-608/SP7 axis at least partially regulates the osteoblast differentiation of hAMSCs, which may be a potential target of hAMSCs for the treatment of osteoporosis.

The role of ZBTB16 in odontogenic differentiation of dental pulp stem cells

ObjectiveOdontogenic differentiation of dental pulp stem cells (DPSCs) is highly controlled by the activation of several transcription factors. The zinc finger and BTB domain-containing 16 (ZBTB16) gene encodes a BTB/POZ domain and zinc finger containing transcription factors and is involved in several biological processes, but little is known about its role in odontogenic differentiation. The main goal of the current study was to determine the role of ZBTB16 in odontogenic differentiation of DPSCs. DesignZBTB16, runt-related transcription factor 2 (RUNX2), and osterix (OSX) were silenced via small-hairpin RNA (shRNA) lentivirus. The odontoblastic differentiation of DPSCs was detected by alkaline phosphatase (ALP) staining, activity measurement, and alizarin red S staining in vitro. The gene and protein expression levels were assessed by RT-qPCR and western blotting. Further, an ectopic implantation experiment was performed to explore the role of ZBTB16 in mineralization regulation in vivo followed by histological examination. ResultsThe silencing of ZBTB16 attenuated ALP activity and mineralized nodules formation by DPSCs. In addition, knockdown of ZBTB16 impaired the expression of markers involved in odontogenic differentiation, including dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, and collagen 1 in vitro and vivo. Silencing the OSX gene suppressed ZBTB16 expression and, in turn, OSX expression decreased after ZBTB16 knockdown. However, shRUNX2 did not suppress ZBTB16 expression and shZBTB16 did not affect RUNX2 expression. ConclusionsZBTB16 may play an important role in modulating the odontoblastic differentiation of DPSCs and act as a regulator of OSX in a possible feed-back cycle independent of RUNX2.

Hydrogenated TiO2 Nanotubes Regulate Osseointegration by Influencing Macrophage Polarization in the Osteogenic Environment

The immunomodulatory role of monocytes is essential for tissue healing and can influence the osseointegration of implanted materials. Properties such as the surface structure, hydrophilicity, and roughness of the implanted materials can modulate monocyte–macrophage function. In this study, we characterized material‐hydrogenated TiO2 nanotubes (H‐TNT) with superhydrophilic surfaces to investigate the effect of H‐TNT on macrophage polarization and osseointegration. H‐TNT were prepared by anodic oxidation and hydrogenation and used in the experimental group, while TNT and smooth pure Ti were employed in the control groups. RAW264.7 cells were selected to observe the immunomodulatory effect of these samples. The cell morphology was observed via scanning electron microscopy, and cytokine expression was detected using an enzyme‐linked immunosorbent assay and immunofluorescence staining. After 24 hr of cultivation, the macrophage‐conditioned medium was collected and used for indirect coculture with MC3T3‐E1 cells. The morphology of MC3T3‐E1 cells was observed using fluorescence staining. Cell adhesion and proliferation were measured using the Cell Counting Kit‐8 assay. Alkaline phosphatase activity measurement, alizarin red staining, calcium quantification, and reverse transcription polymerase chain reaction were performed to assess the osteogenic differentiation of MC3T3‐E1 cells. The results showed that H‐TNT promoted the M2‐type polarization of macrophages, which in turn influenced the adhesion, proliferation, and osteogenic differentiation of MC3T3‐E1 cells. These materials can serve as useful candidates for bone implants because they activate macrophages to produce a favorable osteoimmunomodulatory microenvironment.

Hollow pollen grains as scaffolding building blocks in bone tissue engineering.

Introduction: The current study, for the first time, suggests nature-made pollen grains (PGs) of Pistacia vera L. as a potential candidate for using as scaffolding building blocks with encapsulation capability of bioactive compounds, such as bone morphogenetic protein 4 (BMP4). Methods: A modified method using KOH (5%, 25ºC) was developed to produce nonallergic hollow pollen grains (HPGs), confirmed by energy dispersive X-ray (EDX) analysis, field emission scanning electron microscopy (FESEM), and DNA and protein staining techniques. The in-vitro study was conducted on human adipose-derived mesenchymal stem cells (hAD-MSCs) to investigate the applicability of HPGs as bone scaffolding building blocks. Cytocompability was evaluated by FESEM, MTT assay, and gene expression analysis of apoptotic markers (BAX and BCL2). The osteoconductive potential of HPGs was assessed by alkaline phosphatase (ALP) activity measurement and gene expression analysis of osteogenic markers (RUNX2 and osteocalcin). Results: Findings demonstrated that HPGs can be considered as biocompatible compounds increasing the metabolic activities of the cells. Further, the bioactive nature of HPGs resulted in suitable cellular adhesion properties, required for a potent scaffold. The investigation of apoptotic gene expression indicated a reduced BAX/BCL2 ratio reflecting the protective effect of HPGs on hAD-MSCs. The increased ALP activity and expression of osteogenic genes displayed the osteoconductive property of HPGs. Moreover, the incorporation of BMP4 in HPGs initiated a synergistic effect on osteoblast maturation. Conclusion: Owing to the unique compositional and surface nanotopographical features of the Pistacia vera L. HPG, this microscale architecture provides a favorable microenvironment for the bottom-up remodeling of bone.

Sevelamer Attenuates Bioprosthetic Heart Valve Calcification.

Objective: Sevelamer hydrochloride is a phosphate binder used to treat hyperphosphatemia in chronic kidney disease (CKD) patients that can reduce valvular and vascular calcification. The aim of this study was to examine the effects of sevelamer treatment on calcification in bioprosthetic heart valves (BHVs).Methods: Wister rats were randomly divided into three groups according to sevelamer intake and implantation (sham–sham operation; implant–implantation and normal diet, implant+S implantation, and sevelamer diet). Two kinds of BHVs—bovine pericardium treated with glutaraldehyde (GLUT) or non-GLUT techniques—were implanted in rat dorsal subcutis at 4 weeks. After implantation, sevelamer was administered to the implant+S group. The animals were executed at days 0 (immediately after implantation), 7, 14, 28, and 56. Calcium levels were determined by atomic absorption spectroscopy and von Kossa staining. Serum biochemistry analysis, Western blotting, real-time quantitative polymerase chain reaction, alkaline phosphatase activity measurement, histopathologic analysis, immunohistochemistry, and enzyme-linked immunosorbent assay were conducted to identify the anti-calcification mechanism of sevelamer.Results: Non-GLUT crosslinking attenuates BHV calcification. Serum phosphate and calcium remained unreactive to sevelamer after a 14-day treatment. However, the mean calcium level in the implant+S group was significantly decreased after 56 days. In addition, the PTH level, inflammatory cell infiltration, system and local inflammation, and expression of Bmp2, Runx2, Alp, IL-1β, IL-6, and TNF-α were significantly reduced in the implant+S group.Conclusion: Sevelamer treatment significantly attenuated the calcification of BHVs and had anti-inflammation effects that were independent from serum calcium and phosphate regulation. Thus, sevelamer treatment might be helpful to improve the longevity of BHVs.

Biochemical inhibition of acid phosphatase activity in two mountain spruce forest soils

The product inhibition of the 4-methylumbelliferyl phosphate (MUB-P) decay rate, a measure of potential acid phosphatase activity, has not been considered in most of the published kinetic studies. The aim of this study was to determine the type and strength of the product inhibition in order to better define reaction conditions at which the acid phosphatase activity assay produces unbiased results. The MUB-P decay rate was measured in the forest floor and topsoil organic horizons of two spruce forest catchments at different initial MUB-P and P-PO43− concentrations. The type and strength of the inhibition was analyzed by non-linear regression. We found that MUB-P decay was competitively inhibited by the P-PO43− but also by dissolved organic P. By using estimated kinetic parameters, we calculated an underestimation of potential acid phosphatase activity by up to ~ 20% at concentrations of added MUB-P as high as 300 µmol g−1 dry weight and an incubation time one hour due to background inorganic and organic P concentrations in range of units of µmol per gram of dry soil. We discuss approaches that can be used to minimize the underestimation and show that the previously defined recommendations might not be always sufficient to avoid the bias. Therefore, we recommend to analyze progress curves at a wide range of initial MUB-P concentrations using non-linear methods when the enzyme assay is optimized for a given soil. When changes in inorganic and/or organic P concentrations are expected, we further recommend measuring background inorganic and organic P concentrations.

Nitrous oxide fluxes from long-term limed soils following P and glucose addition: Nonlinear response to liming rates and interaction from added P

Liming of acidic soils to regulate pH for crop growth may decrease emissions of nitrous oxide (N2O) due to direct effects of pH on the synthesis of N2O reductases by denitrifying bacteria. However, liming also changes general pH-dependent soil properties, including availability of phosphorus (P), with a feedback on N2O fluxes that remains largely unknown. Here we used a mesocosm approach to study the combined role of liming and P in regulating N2O fluxes from denitrification in an arable coarse sandy soil where N2O emissions under field condition coincided with rainfall events and irrigation, which facilitated anoxia. Soils from three long-term liming treatments (0, 4, and 12 Mg ha-1) with resulting pH(CaCl2) of 3.6, 4.7 and 6.3 were incubated at original bulk density first at 60% water filled pore space (WFPS) and successively at 75% WFPS with added nitrate, inorganic P (0 and 10 μg P g-1 soil) and glucose as labile carbon. N2O fluxes were measured during 28 days and were supplemented with measurements of CO2 fluxes, microbial biomass, potential denitrification, and acid phosphatase activity. The results showed a nonlinear response of N2O fluxes to liming rates, with highest fluxes at the intermediate liming level (4 Mg ha-1). Furthermore, inorganic P stimulated N2O fluxes only at the intermediate liming level. Assays of potential denitrification indicated that the N2O/(N2O + N2) product ratio decreased consistently with increasing liming rates, but total N2O fluxes responded nonlinearly likely due to combined effects on N2O/(N2O + N2) product ratios and total denitrification rates. The results suggest that liming and P addition interact on microbial properties and N2O emissions from acidic arable soils and may not follow linear trends. This makes it uncertain to predict and model the resulting net effect, which may depend on the actual pH range and P availability from the unlimed to the limed treatments.

Turn-on fluorescence measurement of acid phosphatase activity through an aggregation-induced emission of thiolate-protected gold nanoclusters

A fluorescence method was developed for a turn-on measurement of acid phosphatase (ACP) activity. It was found that cerous ion (Ce3+) could lead to an enhancement of glutathione protected gold nanocluster fluorescence through an aggregation-induced-emission (AIE) process, while its higher valent counterpart ceric ion (Ce4+) could not. In a weakly acidic environment, ACP catalyzed the dephosphorylation of a phosphate ester of ascorbic acid, with a generation of ascorbic acid (AA). AA reduced Ce4+ into Ce3+, which subsequently enhanced the nanocluster fluorescence. This kind of turn-on fluorescence linearly related to the ACP activity in the range of 0.005–2.4 U/L, with a limit of detection as 0.001 U/L. Human serum samples were measured after a trichloroacetic acid treatment and a simple dilution. The whole analyses were accomplished in 1.5 h with results in good accordance with a reference method.