Increased Alkaline Phosphatase Activity Research Articles

Effect of Incorporation of Zeolite Containing Silver-Zinc Nanoparticles into Mineral Trioxide Aggregate on Odontogenic Activity of Human Dental Pulp Stem Cells.

Statement of the Problem: The stimulation of odontogenic activity is considered an essential property for biomaterials used in vital pulp therapy.Purpose: The present study aimed to evaluate the effect of the incorporation of zeolite containing silver-zinc nanoparticles (Ze-Ag-Zn) into Angelous mineral trioxide aggregate (AMTA) on the odontogenic activity of human dental pulp stem cells (HDPSCs).Materials and Method: In this in vitro study, HDPSCs were treated with 2% wt of synthesized Ze-Ag-Zn particles+AMTA, AMTA and Ze-Ag-Zn disks. The negative control cells did not receive any treatment. Then, cell viability was measured using the MTT assay after 7 and 14 days of the treatment course. The alkaline phosphatase (ALP) activity and calcium ion level were also measured in the supernatant culture media using auto-analyzer kits. The obtained data were analyzed using one-way ANOVA and Student t-test where appropriate. The level of the statistical significance was set at p< 0.05.Results: The results indicated that HDPSCs treated with AMTA and AMTA+Ze-Ag-Zn particles did not show any significant cell death compared with the control cells after 14 days of the treatment course while the ALP activity and calcium ion levels were significantly (p< 0.05) elevated. Also, the addition of AMTA particles to the cell culture media resulted in increased ALP activity and calcium ion level compared with HDPSCs treated with AMTA+ Ze-Ag-Zn particles on day 7 of the treatment course (p< 0.05).Conclusion: It seems that the incorporation of Ze-Ag-Zn particles into AMTA did not have any significant positive effect on the biomineralization properties of AMTA.

Radiation Induces Valvular Interstitial Cell Calcific Response in an in vitro Model of Calcific Aortic Valve Disease

Background: Mediastinal ionizing radiotherapy is associated with an increased risk of valvular disease, which demonstrates pathological hallmarks similar to calcific aortic valve disease (CAVD). Despite advances in radiotherapy techniques, the prevalence of comorbidities such as radiation-associated valvular disease is still increasing due to improved survival of patients receiving radiotherapy. However, the mechanisms of radiation-associated valvular disease are largely unknown. CAVD is considered to be an actively regulated disease process, mainly controlled by valvular interstitial cells (VICs). We hypothesize that radiation exposure catalyzes the calcific response of VICs and, therefore, contributes to the development of radiation-associated valvular disease.Methods and Results: To delineate the relationship between radiation and VIC behavior (morphology, calcification, and matrix turnover), two different in vitro models were established: (1) VICs were cultured two-dimensional (2D) on coverslips in control medium (CM) or osteogenic medium (OM) and irradiated with 0, 2, 4, 8, or 16 Gray (Gy); and (2) three-dimensional (3D) hydrogel system was designed, loaded with VICs and exposed to 0, 4, or 16 Gy of radiation. In both models, a dose-dependent decrease in cell viability and proliferation was observed in CM and OM. Radiation exposure caused myofibroblast-like morphological changes and differentiation of VICs, as characterized by decreased αSMA expression. Calcification, as defined by increased alkaline phosphatase activity, was mostly present in the 2D irradiated VICs exposed to 4 Gy, while after exposure to higher doses VICs acquired a unique giant fibroblast-like cell morphology. Finally, matrix turnover was significantly affected by radiation exposure in the 3D irradiated VICs, as shown by decreased collagen staining and increased MMP-2 and MMP-9 activity.Conclusions: The presented work demonstrates that radiation exposure enhances the calcific response in VICs, a hallmark of CAVD. In addition, high radiation exposure induces differentiation of VICs into a terminally differentiated giant-cell fibroblast. Further studies are essential to elucidate the underlying mechanisms of these radiation-induced valvular changes.

Octacalcium Phosphate Bone Substitute (Bontree®): From Basic Research to Clinical Case Study

Bone grafts used in alveolar bone regeneration can be categorized into autografts, allografts, xenografts, and synthetic bones, depending on their origin. The purpose of this study was to evaluate the effect of a commercialized octacalcium phosphate (OCP)-based synthetic bone substitute material (Bontree®) in vitro, in vivo, and in clinical cases. Material characterization of Bontree® granules (0.5 mm and 1.0 mm) using scanning electron microscopy and X-ray diffraction showed that both 0.5 mm and 1.0 mm Bontree® granules were uniformly composed mainly of OCP. The receptor activator of NF-κB ligand (RANKL) and alkaline phosphatase (ALP) activities of MG63 cells were assessed and used to compare Bontree® with a commercial biphasic calcium phosphate ceramic (MBCP+TM). Compared with MBCP+TM, Bontree® suppressed RANKL and increased ALP activity. A rabbit tibia model used to examine the effects of granule size of Bontree® grafts showed that 1.0 mm Bontree® granules had a higher new bone formation ability than 0.5 mm Bontree® granules. Three clinical cases using Bontree® for ridge or sinus augmentation are described. All eight implants in the three patients showed a 100% success rate after 1 year of functional loading. This basic research and clinical application demonstrated the safety and efficacy of Bontree® for bone regeneration.

Morphological, mechanical, and in-vitro bioactivity of gelatine/collagen/hydroxyapatite based scaffolds prepared by unidirectional freeze-casting

The fabrication of biomaterials to be used in segmental bone defects, mimicking the bone's organic-inorganic architecture and mechanical properties to induce osteogenesis, persists as a key challenge. The purpose of this study was to elucidate the effect of a lightweight, morphologically graded, and multiphase self-standing scaffold structure prepared from a combination of gelatine (Gel), collagen type 1 (Col) and/or hydroxyapatite (HAP) nanoparticles by a unidirectional freeze-casting process at different temperatures (−20, −40, −60 °C), followed by carbodiimide induced cross-linking, on their in-vitro mechanical stability and bioactive properties. In addition, the rheological study of differently formulated Gel solutions has been performed to determine the effect of Col and HAP content on their microstructural arrangement, which, together with the freezing kinetic, affects Gel/Col orientation and cross-linking, and, thus, the scaffold's mechanical strength and stability.A bone-like anisotropic, interconnected, and graded porosity (from 120 to a few μm) scaffold structure with up to 30% total porosity and ~61 μm average pores' diameter is obtained by using a higher Col content (Col: Gel = 2:5) and freezing temperature (−20 °C) while forming a few μm thick close-to-parallel lamellae, separated with a 10–100 μm space when prepared at −60 °C. Such a structure influenced in-vitro stability strongly (lower swelling without weight loss), being accompanied with a ~76% increase of compression strength (to 37 kPa) and ~67% decrease of elastic modulus (to 17 kPa) when prepared with HAP and incubated in HBSS for 7 days. On the other hand, a significant reduction of both strength (~78%, to 15 kPa) and elasticity (~95%, to 5 kPa) was noted for a scaffold prepared with HAP at −60 °C, being related to faster degradation and the formation of a highly opened structure on the bottom, required to stimulate the bone ingrowth, while a more closed network structure on the top to adhere with the surrounding soft tissue. None of the scaffolds induced cytotoxicity to human bone-derived osteoblasts, even after 19 days of incubation, but rather improved their viability while promoting cells' adhesions, proliferation, and differentiation, being supported with an increased alkaline phosphatase activity and rod-like CaP formation.

Novel Aptamer-Based Small-Molecule Drug Screening Assay to Identify Potential Sclerostin Inhibitors against Osteoporosis.

A novel aptamer-based competitive drug screening platform for osteoporosis was devised in which fluorescence-labeled, sclerostin-specific aptamers compete with compounds from selected chemical libraries for the binding of immobilized recombinant human sclerostin to achieve high-throughput screening for potential small-molecule sclerostin inhibitors and to facilitate drug repurposing and drug discovery. Of the 96 selected inhibitors and FDA-approved drugs, six were shown to result in a significant decrease in the fluorescence intensity of the aptamer, suggesting a higher affinity toward sclerostin compared with that of the aptamer. The targets of these potential sclerostin inhibitors were correlated to lipid or bone metabolism, and several of the compounds have already been shown to be potential osteogenic activators, indicating that the aptamer-based competitive drug screening assay offered a potentially reliable strategy for the discovery of target-specific new drugs. The six potential sclerostin inhibitors suppressed the level of both intracellular and/or extracellular sclerostin in mouse osteocyte IDG-SW3 and increased alkaline phosphatase activity in IDG-SW3 cells, human bone marrow-derived mesenchymal stem cells and human fetal osteoblasts hFOB1.19. Potential small-molecule drug candidates obtained in this study are expected to provide new therapeutics for osteoporosis as well as insights into the structure–activity relationship of sclerostin inhibitors for rational drug design.

Contrasting effects of carbon source recalcitrance on soil phosphorus availability and communities of phosphorus solubilizing microorganisms

Carbon (C) additions to soil interact through chemical and microbiological processes to cause changes in soil phosphorus (P) availability. However, the response of soil P transformations and relevant microbial communities to C additions having different degrees of recalcitrance remains uncertain. We studied the effects of glucose, hemicellulose and lignin addition on soil P availability, P transformation processes and relevant microbial activity and communities in a P-deficient flooded soil. Lignin significantly increased soil available P concentrations, which was attributed to chemical release of inorganic P and increased alkaline phosphatase activity. Glucose and hemicellulose additions stimulated microbial metabolism of C thereby enhancing microbial demand for P, with increased soil P availability especially in the early incubation period. Glucose or hemicellulose addition changed soil microbial diversity and community composition, leading to enhanced growth and interactions of P solubilizing microorganisms such as Desulfitobacterium, Bacillus and Desulfosporosinus. Our results infer the importance of pH alteration and competitive sorption between PO4 and functional groups of recalcitrant C (e.g., lignin) with Fe/Al (hydr) oxides in regulating soil P availability. Further, the microbial response to labile C additions led to increased P availability in the P-deficient soil. This study provides important mechanistic information to guide microbially-regulated soil P management in agricultural ecosystems.

Efficiency of fatty acid-enriched dipteran-based meal on husbandry, digestive activity and immunological responses of Nile tilapia Oreochromis niloticus juveniles

This study aimed to compare the enrichment capacity of polyunsaturated fatty acids (PUFA) and the long chain polyunsaturated fatty acids (LC-PUFA) of two dipteran species, Hermetia illucens - black soldier fly (BSF) - and a blowfly, Chrysomya putoria (CP), and to test its influence on growth, digestive activities and immune responses of Nile tilapia. Two types of enriched insect larval meal were produced using larvae cultured either on vegetable substrates (VGS) to formulate two diets rich in linolenic acid (ALA) (BSF/VGD and CP/VGD), or on fish offal substrates (FOS), in order to produce two diets rich in ALA and eicosapentaenoic acid (EPA) (BSF/FOD) or in ALA + EPA and docosahexaenoic acid (DHA) (CP/FOD). These four insect-based diets containing only palm oil as a lipid source were compared to a control diet based on fish meal (FM) and fish oil (FO). After 60 days of feeding, ALA or DHA muscle content of fish fed BSF/VGD or CP/FOD diet was comparable to that of the FMFO diet, and all insect diets increased the EPA muscle levels, except for a reduction by the BSF/VGD one. The CP/FOD diet induced similar fish growth, feed efficiency and protein efficiency ratio compared to the control FMFO diet, while a decrease was observed in fish fed other insect diets. Only BSF/VGD led to a decrease in protein and lipid digestibility. CP or BSF larval meal significantly increased alkaline phosphatase activity regardless of fatty acid (FA) enrichment. The expression level of fads2, fads6 and elovl5 was significantly higher in fish fed the BSF/VGD diet compared to fish fed the FMFO diet. FA-enriched insect diets increased some immune variables such as lysozyme, peroxidase and ACH50 values of fish fed CP/FOD, CP/VGD or BSF/VGD diets. Moreover, the expression level of β-defensin-1 and mhcII genes were significantly higher in fish fed the BSF/FOD diet than the FMFO diet. Also, the expression of the pro-inflammatory gene il-1-β was significantly higher in fish fed FMFO diet than in those fed CP/FOD diet, but comparable to fish fed all other diets. No significant effects were observed for the other tested genes. The results showed a better efficiency in LC-PUFA enrichment of the CP larvae compared to BSF ones, resulting in a higher stimulation of the fish nutrient utilization processes and therefore, a higher growth capacity. Nonetheless, all dipteran larval meal stimulated the immune status whatever the insect species or dietary essential fatty acids.

Compounds Isolated from Wikstroemia taiwanensis Regulate Bone Remodeling by Modulating Osteoblast and Osteoclast Activities.

Bone remodeling, a dynamic process in which bone formation by osteoblast is preceded by bone resorption by osteoclast, is a vital physiological process for maintaining bone mass and strength, imbalances in which could precipitate osteoporosis. Due to the unilateral mechanism of the existing bone remodeling drugs, identifying compounds that could regulate the balance between osteoclast and osteoblast could improve the treatment of osteoporosis. Here, we show that compounds isolated from Wikstroemia taiwanensis modulate osteoclast and osteoblast activities. Specifically, astragalin (1) and kaempferol 3-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (2), besides increasing mineral deposition, increased alkaline phosphatase activity (137.2% for 1 and 115.8% for 2) and ESR-α expression (112.8% for 1 and 122.5% for 2) in primary human osteoblasts. In contrast, compounds 1, 2, 3, and 5 inhibited tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor-κB ligand-induced osteoclasts by 40.8, 17.1, 25.9, and 14.5% and also decreased the number of TRAP-positive cells by 51.6, 26.8, 20.5, and 18.6%, respectively. Our findings, therefore, showed that compounds isolated from W. taiwanensis could increase osteoblast activity while simultaneously decreasing osteoclast activity, and hence, warrant further evaluation for development as anti-osteoporosis agents.

Curculigoside Protects against Titanium Particle-Induced Osteolysis through the Enhancement of Osteoblast Differentiation and Reduction of Osteoclast Formation.

Wear particle-induced periprosthetic osteolysis is mainly responsible for joint replacement failure and revision surgery. Curculigoside is reported to have bone-protective potential, but whether curculigoside attenuates wear particle-induced osteolysis remains unclear. In this study, titanium particles (Ti) were used to stimulate osteoblastic MC3T3-E1 cells in the presence or absence of curculigoside, to determine their effect on osteoblast differentiation. Rat osteoclastic bone marrow stromal cells (BMSCs) were cocultured with Ti in the presence or absence of curculigoside, to evaluate its effect on osteoclast formation in vitro. Ti was also used to stimulate mouse calvaria to induce an osteolysis model, and curculigoside was administrated to evaluate its effect in the osteolysis model by micro-CT imaging and histopathological analyses. As the results indicated, in MC3T3-E1 cells, curculigoside treatment attenuated the Ti-induced inhibition on cell differentiation and apoptosis, increased alkaline phosphatase activity (ALP) and cell mineralization, and inhibited TNF-α, IL-1β, and IL-6 production and ROS generation. In BMSCs, curculigoside treatment suppressed the Ti-induced cell formation and suppressed the TNF-α, IL-1β, and IL-6 production and F-actin ring formation. In vivo, curculigoside attenuated Ti-induced bone loss and histological damage in murine calvaria. Curculigoside treatment also reversed the RANK/RANKL/OPG and NF-κB signaling pathways, by suppressing the RANKL and NF-κB expression, while activating the OPG expression. Our study demonstrated that curculigoside treatment was able to attenuate wear particle-induced periprosthetic osteolysis in in vivo and in vitro experiments, promoted osteoblastic MC3T3-E1 cell differentiation, and inhibited osteoclast BMSC formation. It suggests that curculigoside may be a potential pharmaceutical agent for wear particle-stimulated osteolysis therapy.

Effect of crude aqueous leaves extract of Bryophyllum Pinnatum on antioxidant status, blood glucose, lipid profile, liver and renal function indices in albino rats

Bryophyllum pinnatum is an important enthnomedicinal plant. The study assessed the effect of crude aqueous leaves extract of Bryophyllum pinnatum (CALEBP) on fasting plasma glucose (FPG), antioxidant status, lipid profile, liver and renal function indices in albino rats. The rats were housed under standard laboratory conditions (12h light: 12h dark photoperiod), 23± 2 ºC and were given rat pellets and tap water ad libitum. Twenty four rats weighing 190-232g were randomized into four groups (A-D) of six rats each. Group A (control) received normal feed and water only. Groups B, C and D received orally 180, 360 and 540 mg/kg body weight respectively of CALEBP for 28 days. Serum aminotransferases, alkaline phosphatase (ALP), superoxide dismutase, catalase, FPG, lipid profile, urea, creatinine, bilirubin, proteins, malondialdehyde, glutathione (GSH) and total antioxidant capacity (TAC) and electrolytes were assessed by standard methods. Data were analyzed using one-way analysis of variance and p&lt;0.05 was considered statistically significant. Groups C and D had significantly lower FPG (p = 0.030; p = 0.01) and higher ALP (p = 0.01; p = 0.001) compared to the controls. Group D had significantly lower creatinine (p = 0.03) and K+ (p = 0.02) compared to control. Group B, C and D had significantly lower GSH (p = 0.020, p = 0.000 and p = 0.000) while group B had significantly higher TAC (p = 0.04) compared to the controls. Dosage of extracts correlated positively with ALP (r = 0.705, p = 0.000) and negatively with FPG (r = -0.603, p = 0.002), K+ (r = -0.563, p = 0.004), creatinine (r = -0.464, p = 0.022) and GSH (r = -0.786, p = 0.000). Bryophyllum pinnatum aqueous leaves extract could lower blood glucose, potassium and creatinine levels and may increase ALP activity and GSH depletion in high doses.&#x0D; Keywords: Bryophyllum pinnatum, aqueous leaf extract, dosage, effect assessment, Albino rats

English

BACKGROUND Alcoholism remains to be the major cause of morbidity and mortality throughout the world. Consuming alcohol is the potent etiological factor for the development of alcoholic liver diseases (ALD), ranging from fatty liver to hepatocellular carcinoma with varying rates of development in both genders depending on the quality, quantity, and duration of the drink. Zinc deficiency has been documented with the progression of alcoholic liver disease. It is also a well-known fact that zinc is a co-factor for enzyme alkaline phosphatase. This study aims to assess the zinc status and alkaline phosphatase activity in patients with various stages of alcoholic liver disease, correlate zinc with alkaline phosphatase activity, albumin, gamma glutamyl transferase activity, MELD score and duration of alcohol intake and analysing the need for evaluating zinc in these patients. METHODS This comparative observational study involves group I healthy controls and group II patients diagnosed to have ethanol related decompensated liver disease with or without portal hypertension for more than three years from the Department of Medical Gastroenterology, Government Medical College Hospital. 5 ml of venous blood in fasting state was collected from both groups and assayed for serum zinc, and serum alkaline phosphatase activity. The data was statistically analysed. RESULTS The study results demonstrate that higher percentage of patients with alcoholic liver disease have low serum zinc levels than healthy controls. Zinc when compared with variables like serum albumin, duration of alcohol intake, MELD score, serum gamma glutamyl transferase and alkaline phosphatase in the case and control groups were found to be statistically significant. CONCLUSIONS There is decrease in serum zinc level and increased alkaline phosphatase activity in patients with alcoholic liver disease. The statistically significant data is a strong rationale for evaluating the zinc status and thereby supplementing zinc to patients with alcoholic liver disease. KEYWORDS Alcoholic Liver Disease, Zinc, Alkaline Phosphatase, MELD Score

Long noncoding RNA AC092155 facilitates osteogenic differentiation of adipose-derived stem cells through the miR-143-3p/STMN1 axis.

Numerous studies have demonstrated that long noncoding RNAs (lncRNAs) induce osteogenesis in adipose-derived stem cells (ADSCs). This study aimed to explore the role of lncRNAs AC092155 in promoting osteogenic differentiation of ADSCs. MicroRNA (miRNA) and lncRNA sequencing were performed in ADSCs that underwent normal or osteogenic induction. Differentially expressed miRNAs and lncRNAs were identified using R software. The relative expression levels of lncRNA AC092155, miR-143-3p, and STMN1 during the process of osteogenic induction were determined by real-time polymerase chain reaction (RT-PCR). ADSCs were then transfected with agomiR-143-3p and pcDNA3.1-sh-lncRNA AC092155. Alkaline phosphatase (ALP) and alizarin red staining (ARS) were used to confirm the regulatory function of the lncRNA AC092155/miR-143-3p/STMN1 axis in osteogenic differentiation of ADSCs. lncRNA AC092155 was significantly upregulated in ADSCs following induction in the osteogenic medium. lncRNA AC092155 and STMN1 mimics increase the markers of osteogenic differentiation in the early and late phases, which was reflected in increased ALP activity as well as the higher deposition of calcium nodules. An miR-143-3p mimic showed the opposite effect. Luciferase reporter gene analysis demonstrated that lncRNA AC092155 directly targets miR-143-3p. Moreover, the lncRNA AC092155/miR-143-3p/STMN1 regulatory axis was found to activate the Wnt/β-catenin signaling pathway. lncRNA AC092155 contributes to the osteogenic differentiation of ADSCs. The lncRNA AC092155/miR-143-3p/STMN1 axis may be a new therapeutic target for bone-related diseases.

In vitro osteogenic induction of human adipose stem cells co-treated with betaine/osteogenesis differentiation medium.

Human adipose-derived stem cells (hADSCs) are widely used in regenerative medicine and affected by many biochemical and biophysical stimuli in vivo. Betaine has been reported to be a type of osteogenic stimulating biochemical factor. This study aimed to investigate the effects of betaine; on osteogenic differentiation of cultured hADSCs in osteogenesis differentiation medium. Mesenchymal stem cells were extracted from women undergoing liposuction after obtaining written consent and cultured in vitro. The cells at passage 4 were confirmed by flow cytometry and differentiated into osteocytes and adipocytes. Experimental groups were the cells cultured in osteogenesis differentiation medium (control), cultured in α-MEM and 10% serum-containing Betaine (BET) ,and cultured in osteogenesis differentiation medium containing 10 mM Betaine (OD+BET). After 14 and 21 days of treatment, osteogenic differentiation and the expression of RUNX2 and OCN genes were assessed by qualitative and quantitative Alizarin red staining and real-time PCR. There were significant increases in the calcium matrix deposits, alkaline phosphatase activity ,and expression of RUNX2 and OCN genes in the OD+BET group compared to the BET group. At the end of day 14, the calcium matrix formation was significantly decreased the in BET group compared to the control. Treatment of hADSCs with Betaine, and osteogenesis differentiation medium leads to increased alkaline phosphatase activity, matrix calcium deposits and expression of RUNX2 and OCN genes and finally stimulated osteogenesis. This kind of treatment could be used to support bone regeneration in the future of tissue engineering.

Knockdown of hsa_circ_0001275 reverses dexamethasone-induced osteoblast growth inhibition via mediation of miR-377/CDKN1B axis.

BackgroundOsteoporosis affects the quality of life among middle-aged and elderly individuals. In addition, dysfunction of osteoblasts can lead to the progression of osteoporosis. Circular (circ)RNAs are involved in various types of diseases, including osteoporosis. Moreover, it has been reported that hsa_circ_0001275 expression is upregulated in osteoporosis. However, the effects of hsa_circ_0001275 on the growth of osteoblasts remain unclear.MethodsIn the present study, the gene and protein expression levels in hFOB1.19 cells were detected via reverse transcription-quantitative (RT-qPCR) and western blot analyses, respectively. In addition, alkaline phosphatase (ALP) activity and calcium nodules were examined by ALP and alizarin red staining, respectively. Cell proliferation was measured using the Cell Counting Kit-8 assay. Cell apoptosis and cell cycle were analyzed by flow cytometry. Furthermore, dual luciferase reporter and RNA pull-down assay were used to confirm the association among hsa_circ_0001275, microRNA (miR)-377 and CDKN1B.ResultsDEX-induced hFOB1.19 cell growth inhibition was significantly reversed by silencing hsa_circ_0001275. Moreover, DEX significantly increased ALP activity and calcium nodules in hFOB1.19 cells, while this effect was significantly reversed in the presence of hsa_circ_0001275 small interfering RNA. In addition, miR-377 was sponged by hsa_circ_0001275 and CDKN1B was directly targeted by miR-377 in hFOB1.19 cells. Furthermore, the therapeutic effect of hsa_circ_0001275 knockdown on osteoporosis was notably reversed by miR-377 antagomir.ConclusionThe data demonstrated that knockdown of hsa_circ_0001275 reversed DEX-induced osteoblast growth inhibition via activation of the miR-377/CDKN1B axis. Therefore, this study might shed new lights on the treatment of osteoporosis.

Effects of NSAIDs on pre-osteoblast viability and osteogenic differentiation.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of a variety of musculoskeletal conditions, injuries and after surgery for postoperative pain management. Their use has been associated with impaired bone healing, possibly due to a multifactorial function, which may include inhibition of osteoblast recruitment and differentiation. However, up to date, there is no consensus regarding the impact of NSAIDs on bone-healing. The aim of the current study was to investigate the effects of five NSAIDs on the cellular functions of mouse MC3T3-E1 pre-osteoblasts. Cells were treated with the non-selective COX inhibitors lornoxicam and diclofenac, the COX-2 selective inhibitors parecoxib, meloxicam and paracetamol, as well as steroidal prednisolone at different doses and exposure times. The PrestoBlue™ technique was used to measure cell viability, an enzymatic assay was employed for alkaline phosphatase (ALP) activity and alizarin red S mineral staining was used to determine osteogenic differentiation. All drugs had a negative impact on pre-osteoblast cell growth, with the exception of paracetamol. Lornoxicam, diclofenac and meloxicam reduced ALP activity, while the other NSAIDs had no effect and prednisolone strongly increased ALP activity. In contrast, calcium deposits were either unaffected or increased by NSAID treatments but were significantly decreased by prednisolone. These results provide evidence that NSAIDs may adversely affect the viability of mouse pre-osteoblast cells but their actions on the osteogenic differentiation are drug-specific. The direct comparison of the effects of different NSAIDs and prednisolone on pre-osteoblasts may serve to place some NSAIDs in a preferential position for analgesic and anti-inflammatory therapy during bone repair.

Rifampicin induces the bone form of alkaline phosphatase in humans.

Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that regulates drug metabolism in the liver and intestine. In our clinical trials on healthy volunteers to discover novel metabolic functions of PXR activation, we observed that rifampicin, a well-established ligand for human PXR, 600mg daily for a week, increased the plasma alkaline phosphatase (ALP) significantly compared with the placebo. Further analysis with lectin affinity electrophoresis revealed that especially the bone form of ALP was elevated. To investigate the mechanism(s) of bone ALP induction, we employed osteoblast lineage differentiated from human primary bone marrow-derived mesenchymal stromal cells. Rifampicin treatment increased ALP activity and mRNA level of bone biomarker genes (ALP, MGP, OPN and OPG). PXR expression was detected in the cells, but the expression was very low compared with the human liver. To further investigate the potential role of PXR in the ALP induction, we treated mice and rats with a rodent PXR ligand pregnenolone 16α-carbonitrile (PCN). However, PCN treatment did not increase plasma ALP activity or bone ALP mRNA expression. In conclusion, rifampicin treatment induces the bone form of ALP in the serum of healthy human volunteers. Further studies are required to establish the mechanism of this novel finding.

Axial mechanical loading to ex vivo mouse long bone regulates endochondral ossification and endosteal mineralization through activation of the BMP-Smad pathway during postnatal growth

Mechanical loading contributes to bone development, growth, and metabolism. However, the mechanisms underlying long bone mineralization via changes in loading during the growth period are unclear. The aim of the present study was to investigate the regulatory mechanisms underlying endochondral ossification and endosteal mineralization by developing an ex vivo organ culture model with cyclic axial mechanical loads. The metacarpal bones of 3-week-old C57BL/6 mice were exposed to mechanical loading (0, 7.8, and 78 mN) for 1 h/day for 4 days. Histomorphometry revealed that axial mechanical loading regulated the thickness of the calcified zone in the growth plate and endosteal mineralization in the diaphysis in a load-dependent manner. Mechanical loading also resulted in load-dependent upregulation of endochondral ossification and bone mineralization-related genes, including bone morphogenetic protein 2 (Bmp2). Recombinant human BMP-2 administration caused similar changes in tissue structures. Conversely, inhibition of the BMP-Smad pathway diminished the stimulatory effects of mechanical loading and BMP-2 administration, suggesting that the effects of mechanical loading may be exerted through activation of the BMP-Smad pathway with the results of gene ontology and pathway analyses. Mechanical loading increased alkaline phosphatase activity and decreased carbonic anhydrase IX (Car9) mRNA expression, resulting in a significant pH increase in the culture supernatant. We hypothesize that, through activation of the BMP-Smad pathway, mechanical loading downregulates Car9, which may alkalize the local milieu, thereby inducing bone formation and long bone mineralization. Our results showed that cyclic axial mechanical loading increased endochondral ossification and endosteal mineralization in developing mouse long bones, which may have resulted from changes in the pH, ALP activity, and Pi/PPi of the extracellular environment. These findings advance our understanding of the regulation of mineralization mechanisms by mechanical loading mediated through activation of the BMP-Smad pathway.

Growth of Mesenchymal Stem Cells on Poly(3-Hydroxybutyrate) Scaffolds Loaded with Simvastatin.

We studied the effect of porous composite scaffolds based on poly(3-hydroxybutyrate) (PHB) loaded with simvastatin on the growth and differentiation of mesenchymal stem cells. The scaffolds have a suitable microstructure (porosity and pore size) and physicochemical properties to support the growth of mesenchymal stem cells. Scaffold loading with simvastatin suppressed cell growth and increased alkaline phosphatase activity, which can attest to their osteoinductive properties.

Highly osteogenic and mechanically strong nanofibrous scaffolds based on functionalized multi-walled carbon nanotubes-reinforced electrospun keratin/poly(ε-caprolactone)

Here, we fabricated the electrospun scaffolds (so called PCL/Kr and PCL/Kr/CNT) based on poly(ε-caprolactone) (PCL), keratin (Kr), and carboxylated multiwalled carbon nanotubes (CNTs-COOH). Our goal was to evaluate the effect of CNTs-COOH on the osteogenic differentiation and mechanical and physicochemical properties of the scaffolds. We found that the addition of CNTs-COOH to the PCL/Kr scaffold reduced the average fiber diameter from 123 to 55 nm. As a result, the specific surface area of the scaffolds increased from 6 m2/g for the PCL/Kr scaffold to 15 m2/g for the PCL/Kr/CNT scaffold. Also, the PCL/Kr/CNT scaffold showed significantly higher tensile strength (8 vs 3 MPa) and modulus (114 vs 43 MPa) compared to the PCL/Kr scaffold. The formation of hydroxyapatite on the scaffolds incubated in simulated body fluid indicated the excellent osteoconductivity of the CNTs-containing scaffold. The presence of CNTs-COOH in the PCL/Kr/CNT scaffold also improved the osteogenic differentiation of mesenchymal stem cells as confirmed by the increased alkaline phosphatase activity and mineral formation on the surface of the scaffold. The PCL/Kr/CNT scaffold developed here can provide a mechanically strong construct with improved osteogenic properties for a variety of hard tissue engineering applications.

Irradiation with red light-emitting diode enhances proliferation and osteogenic differentiation of periodontal ligament stem cells.

This study aimed to evaluate the effects of low-energy red light-emitting diode (LED) irradiation on the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). PDLSCs were derived from human periodontal ligament tissues of premolars and were irradiated with 0 (control group), 1, 3, or 5 J/cm2 red LED in osteogenic induction medium. Cell proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Osteogenic differentiation activity was evaluated by monitoring alkaline phosphatase (ALP) activity, alizarin red staining, and real-time polymerase chain reaction (RT-PCR) results. Osteoblast-associated proteins (Runx2, OCN, OPN, and BSP) were detected using western blotting. The results of the MTT assay indicated that PDLSCs in the irradiation groups exhibited a higher proliferation rate than those in the control group (P < 0.05). ALP results showed that after 7 days of illumination, only 5 J/cm2 promoted the expression of ALP of PDLSCs. However, after 14 days of illumination, the irradiation treatments did not increase ALP activity. The results of alizarin red staining showed that red LED promoted osteogenic differentiation of the PDLSCs. The real-time polymerase chain reaction (RT-PCR) results demonstrated that red LED upregulated the expression levels of osteogenic genes. Expression of the proteins BSP, OPN, OCN, and Runx2 in the irradiation groups was higher than that in the control group. Our results confirmed that low-energy red LED at 1, 3, and 5 J/cm2 promotes proliferation and osteogenic differentiation of PDLSCs.