Higher Alkaline Phosphatase Activity Research Articles

Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

Bioprinting Macroporous Hydrogel with Aqueous Two‐Phase Emulsion‐Based Bioink: In Vitro Mineralization and Differentiation Empowered by Phosphorylated Cellulose Nanofibrils

AbstractAqueous two‐phase emulsion (ATPE)‐based bioinks, a creative innovation for bioprinting, enable the fabrication of complex 3D cell‐laden hydrogels with macroporous structure, which promote cellular activities within the scaffold. However, these bioinks intrinsically lack stability and specific biofunctionality, potentially limiting their application for targeted tissue engineering. This study proposes a new perspective by introducing less than 0.1% phosphorylated cellulose nanofibrils (pCNF), a 1D nanofibril top‐down produced from natural biomasses, into a dextran/methacrylated gelatin (GelMA)‐based ATPE system for extrusion‐based bioprinting of preosteoblastic cells, aiming to fabricate macroporous hydrogels with osteogenic differentiation potential. The pCNF that is selectively partitioned in the GelMA phase can not only improve the emulsion stability and alter the rheological behaviors of the ATPE‐based bioink, but also enhance the damping capacity and mineralization ability of the crosslinked hydrogels. Furthermore, macroporous hydrogels with pCNF demonstrate increased cell activity and higher viability in post‐printing, along with higher alkaline phosphatase activity and osteoblastic gene expression. Importantly, the organized interfaces within the hydrogel facilitate the formation of macroscopic biomineralized nodules in vitro. The incorporation of multifunctional pCNF in the ATPE system significantly boosts the physiochemical and biological performance of the macropore‐forming bioink, transforming them into a suitable platform for engineering in vitro bone models.

Fabrication of vascularized tissue-engineered bone models using triaxial bioprinting.

Bone tissue is a highly vascularized tissue. When constructing tissue-engineered bone models, both the osteogenic and angiogenic capabilities of the construct should be carefully considered. However, fabricating a vascularized tissue-engineered bone to promote vascular formation and bone generation, while simultaneously establishing nutrition channels to facilitate nutrient exchange within the constructs, remains a significant challenge. Triaxial bioprinting, which not only allows the independent encapsulation of different cell types while simultaneously forming nutrient channels, could potentially emerge as a strategy for fabricating vascularized tissue-engineered bone. Moreover, bioinks should also be applied in combination to promote both osteogenesis and angiogenesis. In this study, employing triaxial bioprinting, we used a blend bioink of gelatin methacryloyl (GelMA), sodium alginate (Alg), and different concentrations of nano beta-tricalcium phosphate (nano β-TCP) encapsulated MC3T3-E1 preosteoblasts as the outer layer, a mixed bioink of GelMA and Alg loaded with human umbilical vein endothelial cells (HUVEC) as the middle layer, and gelatin as a sacrificial material to form nutrient channels in the inner layer to fabricate vascularized bone constructs simulating the microenvironment for bone and vascular tissues. The results showed that the addition of nano β-TCP could adjust the mechanical, swelling, and degradation properties of the constructs. Biological assessments revealed the cell viability of constructs containing different concentrations of nano β-TCP was higher than 90% on day 7, The cell-laden constructs containing 3% (w/v) nano β-TCP exhibited better osteogenic (higher Alkaline phosphatase activity and larger Osteocalcin positive area) and angiogenic (the gradual increased CD31 positive area) potential. Therefore, using triaxial bioprinting technology and employing GelMA, Alg, and nano β-TCP as bioink components could fabricate vascularized bone tissue constructs, offering a novel strategy for vascularized bone tissue engineering.

3D printed PLGA/MgO/PDA composite scaffold by low-temperature deposition manufacturing for bone tissue engineering applications

IntroductionBones are easily damaged. Biomimetic scaffolds are involved in tissue engineering. This study explored polydopamine (PDA)-coated poly lactic-co-glycolic acid (PLGA)-magnesium oxide (MgO) scaffold properties and its effects on bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation. MethodsPLGA/MgO scaffolds were prepared by low-temperature 3D printing technology and PDA coatings were prepared by immersion method. Scaffold structure was observed by scanning electron microscopy with an energy dispersive spectrometer (SEM-EDS), fourier transform infrared spectrometer (FTIR). Scaffold hydrophilicity, compressive/elastic modulus, and degradation rates were analyzed by water contact angle measurement, mechanical tests, and simulated-body fluid immersion. Rat BMSCs were cultured in scaffold extract. Cell activity on days 1, 3, and 7 was detected by MTT. Cells were induced by osteogenic differentiation, followed by evaluation of alkaline phosphatase (ALP) activity on days 3, 7, and 14 of induction and Osteocalcin, Osteocalcin, and Collagen I expressions. ResultsThe prepared PLGA/MgO scaffolds had dense microparticles. With the increase of MgO contents, the hydrophilicity was enhanced, scaffold degradation rate was accelerated, magnesium ion release rate and scaffold extract pH value were increased, and cytotoxicity was less when magnesium mass ratio was less than 10%. Compared with other scaffolds, compressive and elastic modulus of PLGA/MgO (10%) scaffolds were increased; BMSCs incubated with PLGA/MgO (10%) scaffold extract had higher ALP activity and Osteocalcin, Osteopontin, and Collagen I expressions. PDA coating was prepared in PLGA/MgO (10%) scaffolds and the mechanical properties were not affected. PLGA/MgO (10%)/PDA scaffolds had better hydrophilicity and biocompatibility and promoted BMSC osteogenic differentiation. ConclusionLow-temperature 3D printing PLGA/MgO (10%)/PDA scaffolds had good hydrophilicity and biocompatibility, and were conducive to BMSC osteogenic differentiation.

Biocompatibility and Osteo/Odontogenic Potential of Various Bioactive Root-End Filling Materials

This in vitro study aimed to investigate if different bioactive root-end filling materials can promote osteo/odontogenic differentiation of mesenchymal stem cells (MSCs) and support their viability. MSCs from porcine tooth germs were isolated. Cells were exposed to extracts from MTA Angelus, BIOfactor MTA, Medcem MTA, Well-Root ST, and Pure Portland Cement for 7 days. Viability was determined with MTS and live/dead assay. Osteo/odontogenic differentiation was evaluated with alkaline phosphatase (ALP) activity and quantitative real-time PCR (RUNX2, DMP-1, and DSPP genes) which were compared with osteo/odontogenic-induced MSCs and non-treated cells. All the tested materials supported cell proliferation and cells maintained their viability after 7 days. Osteo/odontogenic differentiation of MSCs was promoted by the tested materials in varying levels as demonstrated by increased ALP activity and upregulation of related gene markers in comparison to the control group. Pure Portland Cement demonstrated a continuous high ALP activity on day 7, showing the highest value among all materials and significantly increased in comparison to the control group (p &lt; 0.001). Significant RUNX2 expression and high ALP activity (p &lt; 0.001) similar to that of osteogenically induced cells was detected for Pure Portland Cement after 7 days. Tested MTA-based cement materials are biocompatible and induce osteo/odontogenic differentiation in vitro. MTA materials performed similarly to Pure Portland Cement regarding osteo/odontogenic differentiation.

Features of the biochemical composition of blood in calves with different dynamics of live weight gain

The biochemical markers of blood associated with the rate of body weight gain were studied in the work. For experimental work, 30 calves on the 1st and 14th day after birth received venous blood and conducted laboratory tests. At the time of birth, as well as after 6 months of cultivation, the live weight of young an-imals was determined and the average daily increase was calculated. It was found that on the first day after birth in intensively growing animals, the activity of AlAT was lower by 56.6% (P?0.01), and AsAT by 45.4% (P?0.05), in relation to the indicators of young animals with a low rate of weight gain. Similarly, calves with intensive growth had a higher concentration of VSNMM in plasma by 30.4...48.4% (P?0.001) and 37.1...48.6% (P?0.01) in blood. On day 14, the biochemical profile of calves with low growth rates was characterized by a higher activity of alkaline phosphatase (by 27.4...46.6%; P&lt;0.05), whereas in intensively growing young calves a higher level of calcium was observed (by 5.9%; P&lt;0.05). The analysis of correlations showed that on the first day after birth, there was a pronounced relationship between the growth rate in relation to the activity of AsAT (-0.510) and AlAT (-0.497), iron concentration (-0.650) and VSNMM in blood plasma (0.626). Subsequently, the connection of these markers weakened, and the growth rate of calves was more dependent on the activity of alkaline phosphatase. So on the 14th day of postnatal ontogenesis, the correlation between the increase in live weight and enzyme activity was -0.744. Thus, it can be concluded that the activity of alkaline phosphatase is the most promising biochemical marker associated with the dynamics of body weight gain.

Site-specific periosteal cells with distinct osteogenic and angiogenic characteristics.

This study aimed to investigate the site-specific characteristics of rat mandible periosteal cells (MPCs) and tibia periosteal cells (TPCs) to assess the potential application of periosteal cells (PCs) in bone tissue engineering (BTE). MPCs and TPCs were isolated and characterized. The potential of proliferation, migration, osteogenesis and adipogenesis of MPCs and TPCs were evaluated by CCK-8, scratch assay, Transwell assay, alkaline phosphatase staining and activity, Alizarin Red S staining, RT‒qPCR, and Western blot (WB) assays, respectively. Then, these cells were cocultured with human umbilical vein endothelial cells (HUVECs) to investigate their angiogenic capacity, which was assessed by scratch assay, Transwell assay, Matrigel tube formation assay, RT‒qPCR, and WB assays. MPCs exhibited higher osteogenic potential, higher alkaline phosphatase activity, and more mineralized nodule formation, while TPCs showed a greater capability for proliferation, migration, and adipogenesis. MPCs showed higher expression of angiogenic factors, and the conditioned medium of MPCs accelerated the migration of HUVECs, while MPC- conditioned medium induced the formation of more tubular structure in HUVECs in vitro. These data suggest that compared to TPCs, MPCs exert more consequential proangiogenic effects on HUVECs. PCs possess skeletal site-specific differences in biological characteristics. MPCs exhibit more eminent osteogenic and angiogenic potentials, which highlights the potential application of MPCs for BTE. Autologous bone grafting as the main modality for maxillofacial bone defect repair has many limitations. Constituting an important cell type in bone repair and regeneration, MPCs show greater potential for application in BTE, which provides a promising treatment option for maxillofacial bone defect repair.

Improving bone regeneration with electrospun antibacterial polycaprolactone/collagen/polyvinyl pyrrolidone scaffolds coated with hydroxyapatite and cephalexin delivery capability

Electrospinning is a facile popular method for the creation of nano-micro fibers tissue engineering scaffolds. Here, polycaprolactone (PCL)/collagen (COL): polyvinyl pyrrolidone (PVP) scaffolds (PCL/COL: PVP) were fabricated for bone regeneration. Various concentrations of Cephalexin (CEF) (0.5, 1, 1.5 wt. %) were added to PCL/COL: PVP scaffold to provide an antibacterial scaffold, and different concentrations of hydroxyapatite (HA) (1, 2, 5 wt. %) was electrospray on the surface of the scaffolds. The PCL/COL: PVP scaffold contained 1.5% CEF and coated with 2% HA was introduced as the best sample and in-vitro tests were performed on this scaffold based on the antibacterial and MTT test results. Morphology observations demonstrated a bead-free uniform combined nano-micro fibrous structure. Fourier transform infrared spectroscopy and X-ray diffraction tests confirmed the successful formation of the scaffolds and the wettability, swelling, and biodegradability evaluations of the scaffolds confirmed the hydrophilicity nature of the scaffold with high swelling properties and suitable biodegradation ratio. The scaffolds supported cell adhesion and represented high alkaline phosphatase activity. CEF loading led to antibacterial properties of the designed scaffolds and showed a suitable sustained release rate within 48 h. It seems that the electrospun PCL/COL: PVP scaffold loaded with 1.5% CEF and coated with 2% HA can be useful for bone regeneration applications that need further evaluation in the near future.

Exploring the Phosphate Solubilizing Potential and Acid-Alkaline Phosphatase Activity of Two Multitrait Heavy Metal Tolerant Bacterial Strains Isolated from North Gujarat, India

A study was conducted to isolate and characterize phosphate solubilizing bacteria (PSB) from soils of various locations in North Gujarat. A total of 40 PSB isolates were obtained, exhibiting a phosphate solubilizing index (PSI) ranging from 2.11 to 4.33. Interestingly, among 40 PSB isolates, a subset of 16 displayed an additional trait of potassium solubilization, as evidenced by their potassium solubilizing index (KSI) values falling within the range of 2.10 to 3.10. These 16 PSB isolates were screened for tolerance to heavy metal stressors, namely CdCl2, HgCl2, Pb(NO3)2, and ZnSO4, spanning a concentration gradient from 0.1 mM to 10 mM. Remarkably, among the tested isolates, two, namely, D4 and G2, exhibited exceptional resilience against the heavy metal stressors, as indicated by their minimum inhibitory concentration (MIC). Specifically, isolate D4 showed MIC values of 5 mM, 0.2 mM, 18 mM, and 12 mM, respectively, against CdCl2, HgCl2, Pb(NO3)2, and ZnSO4. For isolate G2, the corresponding MIC values were 6 mM, 0.2 mM, 21 mM, and 11 mM. The bacterial isolates were molecularly identified as strains Klebsiella pneumoniae PHmD4 (abbreviated as D4) and Enterobacter chuandaensis PHmG2 (abbreviated as G2), based on sequence homology obtained from 16S rDNA gene sequencing and subsequent BLAST analysis. In Pikovskaya’s broth, K. pneumoniae PHmD4 and E. chuandaensis PHmG2 had phosphate solubilization values of 4.30 μ moles mL⁻¹ and 3.51 μmoles mL⁻¹, respectively, with terminal pH values of 3.06 and 4.07. Enzymatic activity assays conducted after 96 hours of incubation in Pikovskaya’s broth revealed that the highest acid phosphatase activity was 21.62 U mL⁻¹ for K. pneumoniae PHmD4 and 21.09 U mL⁻¹ for E. chuandaensis PHmG2. The highest alkaline phosphatase activity was 5.85 U mL⁻¹ and 6.38 U mL⁻¹, respectively. In the tomato seed germination study, both non-bacterized and bacterized tomato seeds achieved a 100% germination rate. However, increasing metal concentrations diminished the germination percentage of heavy metal-treated seeds. Interestingly, bacterization with K. pneumoniae PHmD4 and E. chuandaensis PHmG2 ameliorated germination under cadmium stress but had no mitigative effect under mercury and zinc stress conditions.

Feeding spent hemp biomass to lactating dairy cows: Effects on performance, milk components and quality, blood parameters, and nitrogen metabolism

The legalization of industrial hemp by the 2018 Farm Bill in the United States has driven a sharp increase in its cultivation, including for cannabinoid extraction. Spent hemp biomass (SHB), produced from the extraction of cannabinoids, can potentially be used as feed for dairy cows; however, it is still illegal to do so in the United States, according to the US Food and Drug Administration Center for Veterinary Medicine, due to the presence of cannabinoids and the lack of data on the effect on animals. To assess the safety of this byproduct as feed for dairy cows, late-lactation Jersey cows (245 ± 37 d in milk; 483 ± 38 kg body weight; 10 multiparous and 8 primiparous) received a basal total mixed ration (TMR) diet plus 13% alfalfa pellet (CON) or 13% pelleted SHB for 4 wk (intervention period [IP]) followed by 4 wk of withdrawal period (WP), where all cows received only the basal TMR during WP. The dry matter intake (DMI), body weight, body condition score, milk yield, milk components, and fatty acid profile, blood parameters, N metabolism, methane emission, and activity were measured. Results indicated that feeding SHB decreased DMI mainly due to the low palatability of the SHB pellet, as the cows consumed only 7.4% of the total TMR with 13.0% SHB pellet offered in the ration. However, milk yield was not affected during the IP and was higher than CON during the WP, leading to higher milk yield/DMI. Milk components were not affected, except for a tendency in decreased fat percentage. Milk fat produced by cows fed SHB had a higher proportion of oleate and bacteria-derived fatty acids than CON. The activity of the cows was not affected, except for a shorter overall lying time in SHB versus CON cows during the IP. Blood parameters related to immune function were not affected. Compared with CON, cows fed SHB had a lower cholesterol concentration during the whole experiment and higher β-hydroxybutyric acid during the WP, while a likely low-grade inflammation during the IP was indicated by higher ceruloplasmin and reactive oxidative metabolites. Other parameters related to liver health and inflammatory response were unaffected, except for a tendency for higher activity of alkaline phosphatase during IP and a lower activity of gamma-glutamyl transferase during WP in the SHB group versus CON. The bilirubin concentration was increased in cows fed SHB, suggesting a possible decrease in the clearance ability of the liver. Digestibility of the dry matter and protein and methane emission were not affected by feeding SHB. The urea, purine derivatives, and creatinine concentration in urine was unaffected, but cows fed SHB had higher N use efficiency and lower urine volume. Altogether, our data revealed a relatively low palatability of SHB affecting DMI with minimal biological effects, except for a likely low-grade inflammation, a higher N use efficiency, and a possible decrease in liver clearance. Overall, the data support the use of SHB as a safe feed ingredient for lactating dairy cows.

Using Ribonucleoprotein-based CRISPR/Cas9 to Edit Single Nucleotide on Human Induced Pluripotent Stem Cells to Model Type 3 Long QT Syndrome (SCN5A±)

Human induced pluripotent stem cells (hiPSCs) have been widely used in cardiac disease modelling, drug discovery, and regenerative medicine as they can be differentiated into patient-specific cardiomyocytes. Long QT syndrome type 3 (LQT3) is one of the more malignant congenital long QT syndrome (LQTS) variants with an SCN5A gain-of-function effect on the gated sodium channel. Moreover, the predominant pathogenic variants in LQTS genes are single nucleotide substitutions (missense) and small insertion/deletions (INDEL). CRISPR/Cas9 genome editing has been utilised to create isogenic hiPSCs to control for an identical genetic background and to isolate the pathogenicity of a single nucleotide change. In this study, we described an optimized and rapid protocol to introduce a heterozygous LQT3-specific variant into healthy control hiPSCs using ribonucleoprotein (RNP) and single-stranded oligonucleotide (ssODN). Based on this protocol, we successfully screened hiPSCs carrying a heterozygous LQT3 pathogenic variant (SCN5A±) with high efficiency (6 out of 69) and confirmed no off-target effect, normal karyotype, high alkaline phosphatase activity, unaffected pluripotency, and in vitro embryonic body formation capacity within 2 weeks. In addition, we also provide protocols to robustly differentiate hiPSCs into cardiomyocytes and evaluate the electrophysiological characteristics using Multi-electrode Array. This protocol is also applicable to introduce and/or correct other disease-specific variants into hiPSCs for future pharmacological screening and gene therapeutic development.Graphical

Establishment and functional characterization of immortalized rabbit dermal papilla cell lines

Hair follicle (HF) undergo periodic growth and development in mammals, which regulated by dermal papilla cells (DPCs) are reported to play an important role in HF morphogenesis and development. However, primary DPCs have low proliferative activity, age quickly, and fresh cell isolation is both time-consuming and laborious. In this study, we introduced the SV40 large T antigen (SV40T) into dissociated early passage rabbit vibrissae DPCs with lentiviral vectors and established seven immortalized DPC lines (R-1, R-2, R-3, R-4, R-5, R-6 and R-7). These cell lines displayed early passage morphology and high alkaline phosphatase activity. RT-PCR and immunofluorescence staining showed that all the immortalized cell lines expressed the DPC markers (α-SMA, IGF1, ALPL, FGF2, BMP2 and TGFβ2), but α-SMA was only expressed well in R-3, R-4, and R-7. Furthermore, it was found that R-7 was the only line to survive beyond 50 passages. Compared to melanoma cells, R-7 did not undergo malignant transformation. Karyotyping and cell growth viability analysis illustrated that the R-7 cell line preserved the basic characteristics of primary DPCs. The R-7 DPCs established have potential application for future hair research. The study provides the theoretical basis in the cell research of HF growth and development.

Titanium surfaces with biomimetic topography and copper incorporation to modulate behaviors of stem cells and oral bacteria.

Purpose: Insufficient osseointegration and implant-associated infection are major factors in the failure of Ti-based implants, thus spurring scientists to develop multifunctional coatings that are better suited for clinical requirements. Here, a new biomimetic micro/nanoscale topography coating combined with antibacterial copper was simultaneously designed for Ti-based implant surfaces by adopting a hybrid approach combining plasma electrolytic oxidation and hydrothermal treatment. Results: The biological interactions between this biofunctionalized material interface and stem cells promoted cellular adhesion and spreading during initial attachment and supported cellular proliferation for favorable biocompatibility. Bone marrow mesenchymal stem cells (BMMSCs) on the coating displayed enhanced cellular mineral deposition ability, higher alkaline phosphatase activity, and upregulated expression of osteogenic-related markers without the addition of osteoinductive chemical factors, which improved osseointegration. More interestingly, this new coating reduced the viability of oral pathogens (Fusobacterium nucleatum and Porphyromonas gingivalis)-the primary causes of implant-associated infections as indicated by damage of cellular structures and decreased population. This is the first study investigating the antibacterial property of dental implants modified by a hybrid approach against oral pathogens to better mimic the oral environment. Conclusion: These findings suggest that biofunctionalization of the implant coating by surface modification methods and the incorporation of antibacterial copper (Cu) offer superior osteogenesis capability and effective antibacterial activity, respectively. These strategies have great value in orthopedic and dental implant applications.

ACTIVITY OF LIVER FUNCTION ENZYME AND ANTIBACTERIAL SUSCEPTIBILITY OF ANNONA SENEGALENSIS LEAF EXTRACT

In ethnomedicine, various natural products have been implicated in the treatment of several diseases. Annona senegalensis has been identified as one of the plants that have the potential to cure ailments arising from microbial infections. This study however was carried out to investigate the liver function enzyme activity and in vitro antibacterial susceptibility of the 50% ethanol-methanol leaf extract of A. senegalensis. The effect of the administration of 100 mg/kg body weight of A. senegalensis on serum enzyme parameters and antibacterial susceptibility was investigated in albino rats. Thirty-three albino rats with average weights of 200 g were divided into two groups. Group 1 contained 30 rats and was treated with 20 mg/mL of A. senegalensis leaf extract while Group 2 contained three rats and served as the control. The treatment lasted for 20 consecutive days while the rats in Group 1 were daily administered with the leaf extract of A. senegalensis. Data were analyzed and presented using a descriptive analysis from Microsoft Excel 2016 version. The results obtained from administration of the leaf extract of A. senegalensis on serum enzymes parameters show a significant increase in serum enzyme activity when compared to the control. This indicates that the leaf extracts of A. senegalensis have significant effects on the serum enzymes parameters. The in vitro antibacterial susceptibility of the leaf extracts relative to the drugs (Ampicillin and Ciprofloxacin) revealed that the leaf extract of the plant holds much promise in antibacterial property when combined with antibiotics used in this study. This research revealed that there is high activity of alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase in the serum following the administration of 100 mg/kg body weight of 50% ethanol-methanol leaf extract of A. senegalensis. The antibacterial susceptibility test of the leaf extract carried out in vitro also revealed the plant antibacterial property. The combination of A. senegalensis leaf extract with antibiotic drugs, especially the one used in this study hold much promise in its efficacy as revealed in its synergistic interaction.

Cascade-Activated AIEgen-Peptide Probe for Noninvasively Monitoring Chymotrypsin-like Activity of Proteasomes in Cancer Cells.

Noninvasive monitoring of chymotrypsin-like (ChT-L) activity of proteasomes is of great significance for the diagnosis and prognosis of various cancers. However, commercially available proteasome probes usually lack adequate cancer-cell selectivity. To noninvasively monitor ChT-L activity of proteasomes in living cells, we rationally designed a cascade-activated AIEgen-peptide probe (abbreviated as TPE-1p), which self-assembled in aqueous solution to exhibit bright fluorescence in response to sequential treatment of alkaline phosphatase (ALP) and ChT-L. Transmission electron microscopy, enzymatic kinetics, and in vitro fluorescence experiments validated that TPE-1p was efficiently dephosphorylated by ALP to generate TPE-1, which was recognized by ChT-L in the proteasome, and transformed to form nanofibers with strong fluorescence signals. Cell imaging experiments revealed that bright blue fluorescence was observed in TPE-1p-treated HeLa cells, whereas NIH3T3 and HepG2 cells showed less fluorescence at the same condition. The enhanced fluorescence signals in HeLa cells were attributed to the high activities of endogenous ALP and ChT-L. Moreover, TPE-1p was utilized to noninvasively assess the inhibition efficiency of a ChT-L inhibitor (bortezomib, abbreviated as Btz) in HeLa cells. Significant correlation was found between the fluorescence signals of TPE and the viabilities of Btz-treated cells in concentration ranges from 0 to 1 μM, indicating that TPE-1p could be employed to predict the activity of ChT-L inhibitors. The design of the cascade-activated AIEgen-peptide probe provides a viable approach for noninvasively monitoring the ChT-L activity of proteasomes in living cells, which facilitates high-throughput screening of ChT-L inhibitors in cancer therapy.

The Combined Effects of Hydraulic Calcium Silicate Cement and Enamel Matrix Derivative Regarding Osteogenic and Dentinogenic Differentiation on Human Dental Pulp Stem Cells.

The ideal treatment option for immature necrotic permanent teeth is regeneration of the pulp-dentin complex. Mineral trioxide aggregate (MTA), the conventional cement used for regenerative endodontic procedures, induces hard tissue repair. Various hydraulic calcium silicate cements (HCSCs) and enamel matrix derivative (EMD) also promote osteoblast proliferation. The purpose of the present study was to determine the osteogenic and dentinogenic potential of commercially distributed MTA and HCSCs when applied in combination with Emdogain gel on human dental pulp stem cells (hDPSCs). The presence of Emdogain resulted in greater cell viability, and higher alkaline phosphatase activity was detected in the Emdogain-supplemented groups in the early days of cell culture. On qRT-PCR, the groups treated, respectively, with Biodentine and Endocem MTA Premixed in the presence of Emdogain showed an increased expression of the dentin formation marker DSPP, and the group treated with Endocem MTA Premixed in the presence of Emdogain showed an upregulated expression of the bone formation markers OSX and RUNX2. In an Alizarin Red-S staining assay, all of the experimental groups exhibited a greater formation of calcium nodules when treated in combination with Emdogain. Overall, the cytotoxicity and osteogenic/odontogenic potential of HCSCs were similar to that of ProRoot MTA. The addition of the EMD increased the osteogenic and dentinogenic differentiation markers.

Milk osteopontin promotes intestinal development by up-regulating the expression of integrin αvβ3 and CD44.

Osteopontin (OPN) is a pleiotropic protein involved in numerous biological processes such as cell proliferation and differentiation. Since OPN is abundantly present in milk and is known to be relatively resistant to in vitro gastrointestinal digestion, the current study aimed to investigate the roles of oral intake of milk OPN in intestinal development using an established OPN knockout (KO, OPN-/- ) mouse model, in which wild-type (WT, OPN+/+ ) mouse pups were nursed by either WT (OPN+/+ OPN+ group) or OPN KO dams (OPN+/+ OPN- group; +/+ indicates genotype and - indicates milk without OPN), receiving milk with or without OPN from postnatal days 0 to 21 (P0-P21). Our results showed that milk OPN is resistant to in vivo digestion. Compared to OPN+/+ OPN- pups, OPN+/+ OPN+ pups at P4 and P6 had significantly longer small intestines, at P10 and P20 had larger inner jejunum surfaces, and at P30 exhibited more mature/differentiated intestines, as revealed by higher activities of alkaline phosphatase in brush border and more goblet cells, enteroendocrine cells, and Paneth cells. qRT-PCR and immunoblotting results showed that milk OPN increased the expression of integrin αv, integrin β3, and CD44 in jejunum of mouse pups (P10, P20, and P30). Immunohistochemistry analysis showed that both integrin αvβ3 and CD44 are localized in jejunum crypts. In addition, milk OPN increased the phosphorylation/activation of the ERK, PI3K/Akt, Wnt, and FAK signaling pathways. In summary, oral intake of milk OPN in early life promotes intestinal proliferation and differentiation by upregulating the expression of integrin αvβ3 and CD44 and thus regulates OPN-integrin αvβ3 and OPN-CD44 mediated cellular signaling pathways.

Elastin-like recombinamer-mediated hierarchical mineralization coatings on Zr-16Nb-xTi (x = 4,16 wt%) alloy surfaces improve biocompatibility

The biocompatibility of biomedical materials is vital to their applicability and functionality. However, modifying surfaces for enhanced biocompatibility using traditional surface treatment techniques is challenging. We employed a mineralizing elastin-like recombinamer (ELR) self-assembling platform to mediate mineralization on Zr-16Nb-xTi (x = 4,16 wt%) alloy surfaces, resulting in the modification of surface morphology and bioactivity while improving the biocompatibility of the material. We modulated the level of nanocrystal organization by adjusting the cross-linker ratio. Nanoindentation tests revealed that the mineralized configuration had nonuniformity with respect to Young's modulus and hardness, with the center areas having higher values (5.626 ± 0.109 GPa and 0.264 ± 0.022 GPa) compared to the edges (4.282 ± 0.327 GPa and 0.143 ± 0.023 GPa). The Scratch test results indicated high bonding strength (2.668 ± 0.117 N) between the mineralized coating and the substrate. Mineralized Zr-16Nb-xTi (x = 4,16 wt%) alloys had higher viability compared to untreated alloys, which exhibited high cell viability (>100 %) after 5 days and high alkaline phosphatase activity after 7 days. Cell proliferation assays indicated that MG 63 cells grew faster on mineralized surfaces than on untreated surfaces. Scanning electron microscopy imaging confirmed that the cells adhered and spread well on mineralized surfaces. Furthermore, hemocompatibility test results revealed that all mineralized samples were non-hemolytic. Our results demonstrate the viability of employing the ELR mineralizing platform to improve alloy biocompatibility.

Platelet-rich Fibrin Promotes the Proliferation and Osteo-/odontoblastic Differentiation of Human Dental Pulp Stem Cells.

Pulp regeneration is a promising strategy that promotes the continued development of young permanent teeth with immature apical foramen. Platelet-rich fibrin (PRF) was found to stimulate the proliferation and differentiation of osteoblasts, but its effects on osteoblast/odontoblast differentiation of human dental pulp stem cells (hDPSCs) are unknown. The hDPSCs were isolated and identified using known surface markers by flow cytometry. The CCK-8 assay and the expression of Ki67 and PCNA were used to examine hDPSC proliferation. After 7 days of culture in an osteo-/odontoblastic induction medium with various concentrations of liquid PRF (0, 10% and 20%), the early stage of osteogenesis-intracellular alkaline phosphatase (ALP) was checked. After 21 days of culture, matrix mineralization was checked using Alizarin Red S and quantified. The mRNA and protein levels of osteo-/odontoblastic genes, including RUNX2, DSPP, DMP1 and BSP, were measured by qRT-PCR. The notch signal was checked by Western blot to analyze three key proteins (Notch 1, Jagged 1 and Hes 1). PRF-treated groups showed higher expression of Ki-67 and PCNA, higher ALP activity, and the higher dose showed a stronger induction. PRF promoted osteo-/odontoblastic differentiation of hDPSCs indicated by elevated protein levels and mRNA levels of the expression of osteo-/odontoblastic markers. The three key proteins in Notch signaling showed an increase compared with the control group and increased as the PRF concentration increased. PRF can promote the proliferation and osteo-/odontoblastic differentiation of hDPSC, which may be through the Notch signaling pathway.

Improving Phosphorus Availability and Wheat Yield in Saline Soil of the Lake Urmia Basin through Enriched Biochar and Microbial Inoculation

To reduce requirements for conventional chemical fertilizer and alleviate salinity stress in soils, a glasshouse experiment was conducted to assess the effects of enriched biochar on phosphatase activity, microbial respiration and wheat yield in non-saline and saline soils from the Lake Urmia basin (electrical conductivities 2 dS.m−1 and 15 dS.m−1, respectively). Nine treatments were tested: control, 1:1 mixture of apple and grape biochars (BC), phosphate solubilizing bacteria (PSB), BC plus PSB (BC-PSB), BC plus rock phosphate (BC-RP), BC enriched by rock phosphate and bacteria (BC-RP-PSB), BC enriched by rock phosphate and HCl (BC-RP-HCl) or H3PO4 (BC-RP-H3PO4) and chemical fertilizer (TSP). The addition of enriched biochar decreased the soil pH (by 0.5–0.9 units) and increased available phosphorus (&gt;7-fold). In both the saline and non-saline soils, the highest alkaline phosphatase activity was obtained for BC-H3PO4-RP and BC-HCl-RP. Wheat growth parameters were reclaimed after enriched biochar application, indicating superior dry matter yields compared to the control and non-enriched biochar treatments and significantly higher yields compared to TSP. Beneficial effects on soil pH, phosphatase activity, soil respiration and biomass yield demonstrated that enriched biochar could partly substitute chemical fertilizers and increase plant growth in salt stress conditions. However, further field studies are needed to understand the benefits of enriched biochar in different soils and climates.