Mendelian randomization to evaluate the causal relationship between liver enzymes and the risk of six specific bone and joint-related diseases.

Background: Studies of liver dysfunction in relation to bone and joint-related diseases are scarce, and its causality remains unclear. Our objective was to investigate whether serum liver enzymes are causally associated with bone and joint-related diseases using Mendelian randomization (MR) designs. Methods: Genetic data on serum liver enzymes (alkaline phosphatase (ALP); alanine transaminase (ALT); gamma-glutamyl transferase (GGT)) and six common bone and joint-related diseases (rheumatoid arthritis (RA), osteoporosis, osteoarthritis (OA), ankylosing spondylitis, psoriatic arthritis, and gout) were derived from independent genome-wide association studies of European ancestry. The inverse variance-weighted (IVW) method was applied for the main causal estimate. Complementary sensitivity analyses and reverse causal analyses were utilized to confirm the robustness of the results. Results: Using the IVW method, the positive causality between ALP and the risk of osteoporosis diagnosed by bone mineral density (BMD) at different sites was indicated (femoral neck, lumbar spine, and total body BMD, odds ratio (OR) [95% CI], 0.40 [0.23-0.69], 0.35 [0.19-0.67], and 0.33 [0.22-0.51], respectively). ALP was also linked to a higher risk of RA (OR [95% CI], 6.26 [1.69-23.51]). Evidence of potential harmful effects of higher levels of ALT on the risk of hip and knee OA was acquired (OR [95% CI], 2.48 [1.39-4.41] and 3.07 [1.49-6.30], respectively). No causal relationship was observed between GGT and these bone and joint-related diseases. The study also found that BMD were all negatively linked to ALP levels (OR [95% CI] for TBMD, FN-BMD, and LS-BMD: 0.993 [0.991-0.995], 0.993 [0.988-0.998], and 0.993 [0.989, 0.998], respectively) in the reverse causal analysis. The results were replicated via sensitivity analysis in the validation process. Conclusions: Our study revealed a significant association between liver function and bone and joint-related diseases.

Garlic extract enhances bioceramic bone scaffolds through upregulating ALP & BGLAP expression in hMSC-monocyte co-culture.

Bone homeostasis is predicated by osteoblast and osteoclast cell cycles where gene expressions are responsible for their differentiation from human mesenchymal stem cells (hMSC) and monocytes, respectively. The pro-osteogenic potential of an hMSC-monocyte co-culture can be measured through complementary DNA (mRNA synthesis) within the nucleus, known as quantitative polymerase chain reaction (qPCR). Through this technique, the effects of garlic extract (allicin) release from calcium phosphate bone scaffolds on gene expression of bone forming and bone remodeling cells was explored. Results show this complex biomaterial system enhances hMSC differentiation through the upregulation of bone-forming proteins. Osteoblastic gene markers alkaline phosphatase (ALP) and osteocalcin (BGLAP), are respectively upregulated by 3-fold and 1.6-fold by day 14. These mature osteoblasts then upregulate the receptor activator of nuclear factor-kB ligand (RANKL) which recruits osteoclast cells, as captured by a nearly 2-fold higher osteoclast expression of tartrate-resistance acid-phosphatase (ACP5). This also activates antagonist osteoprotegerin (OPG) expression in osteoblasts, decreasing osteoclast resorption potential and ACP5 expression by day 21. The pro-osteogenic environment with garlic extract release is further quantified by a 4× increase in phosphatase activity and visibly captured in immunofluorescent tagged confocal images. Also corroborated by enhanced collagen formation in a preliminary in vivo rat distal femur model, this work collectively reveals how garlic extract can enhance bioceramic scaffolds for bone tissue regenerative applications.

Bioenergetic profile and redox tone modulate in vitro osteogenesis of human dental pulp stem cells: new perspectives for bone regeneration and repair.

BACKGROUND: Redox signaling and energy metabolism are known to be involved in controlling the balance between self-renewal and proliferation/differentiation of stem cells. In this study we investigated metabolic and redox changes occurring during in vitro human dental pulp stem cells (hDPSCs) osteoblastic (OB) differentiation and tested on them the impact of the reactive oxygen species (ROS) signaling. METHODS: hDPSCs were isolated from dental pulp and subjected to alkaline phosphatase and alizarin red staining, q-RT-PCR, and western blotting analysis of differentiation markers to assess achievement of osteogenic/odontogenic differentiation. Moreover, a combination of metabolic flux analysis and confocal cyto-imaging was used to profile the metabolic phenotype and to evaluate the redox tone of hDPSCs. RESULTS: In differentiating hDPSCs we observed the down-regulation of the mitochondrial respiratory chain complexes expression since the early phase of the process, confirmed by metabolic flux analysis, and a reduction of the basal intracellular peroxide level in its later phase. In addition, dampened glycolysis was observed, thereby indicating a lower energy-generating phenotype in differentiating hDPSCs. Treatment with the ROS scavenger Trolox, applied in the early-middle phases of the process, markedly delayed OB differentiation of hDPSCs assessed as ALP activity, Runx2 expression, mineralization capacity, expression of stemness and osteoblast marker genes (Nanog, Lin28, Dspp, Ocn) and activation of ERK1/2. In addition, the antioxidant partly prevented the inhibitory effect on cell metabolism observed following osteogenic induction. CONCLUSIONS: Altogether these results provided evidence that redox signaling, likely mediated by peroxide species, influenced the stepwise osteogenic expansion/differentiation of hDPSCs and contributed to shape its accompanying metabolic phenotype changes thus improving their efficiency in bone regeneration and repair.

Abundance and phylogenetic distribution of eight key enzymes of the phosphorus biogeochemical cycle in grassland soils.

Grassland biomes provide valuable ecosystem services, including nutrient cycling. Organic phosphorus (Po) represents more than half of the total P in soils. Soil microorganisms release organic P through enzymatic processes, with alkaline phosphatases, acid phosphatases and phytases being the key P enzymes involved in the cycling of organic P. This study analysed 74 soil metagenomes from 17 different grassland biomes worldwide to evaluate the distribution and abundance of eight key P enzymes (PhoD, PhoX, PhoA, Nsap-A, Nsap-B, Nsap-C, BPP and CPhy) and their relationship with environmental factors. Our analyses showed that alkaline phosphatase phoD was the dataset's most abundant P-enzyme encoding genes, with a wide phylogenetic distribution. Followed by the acid phosphatases Nsap-A and Nsap-C showed similar abundance but a different distribution in their respective phylogenetic trees. Multivariate analyses revealed that pH, Tmax , SOC and soil moisture were associated with the abundance and diversity of all genes studied. PhoD and phoX genes strongly correlated with SOC and clay, and the phoX gene was more common in soils with low to medium SOC and neutral pH. In particular, P-enzyme genes tended to respond in a positively correlated manner among them, suggesting a complex relationship of abundance and diversity among them.

The spatio-temporal distribution of alkaline phosphatase activity and phoD gene abundance and diversity in sediment of Sancha Lake.

The bacterial phoD gene encoding alkaline phosphatase (ALP) plays an important role in the release of soluble reactive phosphorus (SRP) from organic phosphorus in ecosystems. However, phoD gene diversity and abundance in ecosystems is poorly understood. In the present study, we sampled the surface sediments and the overlying water of Sancha Lake at 9 different sampling sites, a typical eutrophic sub-deep freshwater lake in China, in April 15 (spring) and November 3 (autumn), 2017. High-throughput sequencing and qPCR were performed to analyze the diversity and abundance of the bacterial phoD gene in the sediments. We further discussed the relationships between the diversity and abundance of the phoD gene and environmental factors and ALP activity. A total of 881,717 valid sequences were obtained from 18 samples and were classified into 41 genera, 31 families, 23 orders, 12 classes, and 9 phyla and grouped into 477 OTUs. The dominant phyla were Proteobacteria and Actinobacteria. The phylogenetic tree based on the sequences of the phoD gene was plotted and composed of three branches. The genetic sequences were aligned predominantly with genera Pseudomonas, Streptomyces, Cupriavidus, and Paludisphaer. The phoD-harboring bacterial community structure showed a significant difference in spring and autumn, but no apparent spatial heterogeneity. The phoD gene abundances at different sampling points were significantly higher in autumn than in spring. In autumn and spring, the phoD gene abundance was significantly higher in the tail of lake and where cage culture used to be intensive. pH value, dissolved oxygen (DO), total organic carbon (TOC), ALP, and phosphorus were important environmental factors affecting the diversity of the phoD gene and the phoD-harboring bacterial community structure. Changes in phoD-harboring bacterial community structure, phoD gene abundance, and ALP activity were negatively correlated with SRP in overlying water. Our study indicated phoD-harboring bacteria in the sediments of Sancha Lake with the characteristics of high diversity and significant spatial and temporal heterogeneity in abundance and community structure, which played a important role in the release of SRP.

Modulatory activity of a bovine hydrolyzed collagen-hydroxyapatite food complex on human primary osteoblasts after simulating its gastrointestinal digestion and absorption.

Introduction: Introduction: osteoporosis is the most prevalent bone disease and one of the main causes of chronic disability in middle and advanced ages. Conventional pharmacological treatments are still limited, and their prolonged use can cause adverse effects that motivate poor adherence to treatment. Nutritional strategies are traditionally based on supplementing the diet with calcium and vitamin D. Recent studies confirm that the results of this supplementation are significantly improved if it is accompanied by the intake of oral hydrolyzed collagen. Objective: to evaluate the possible in vitro osteogenic activity of a peptide-mineral complex formed by bovine hydrolyzed collagen and bovine hydroxyapatite (Phoscollagen®, PHC®). Methods: the digestion and absorption of PHC® were simulated using the dynamic gastrointestinal digester of AINIA and Caco-2 cell model, respectively. Primary cultures of human osteoblasts were treated with the resulting fraction of PHC® and changes were evaluated in the proliferation of preosteoblasts and in the mRNA expression of osteogenic biomarkers at different stages of osteoblast maturation: Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OC) and type I collagen (ColA1). Results: an increase in preosteoblastic proliferation was observed (p ≤ 0,05). No changes were detected in the biomarkers of osteoblasts with 5 days of differentiation, but were with 14 days, registering an increase in Runx2 (p = 0.0008), ColA1 (p = 0.035), OC (p = 0.027) and ALP (without significance). Conclusion: these results show that the PHC® peptide-mineral complex stimulates the activity of mature osteoblasts, being capable of promoting bone formation.

Introducción: Introducción: la osteoporosis es la enfermedad ósea más prevalente y una de las principales causas de discapacidad crónica en las edades medias y avanzadas. Los tratamientos farmacológicos convencionales aún son limitados y su uso prolongado puede provocar efectos adversos que motiven baja adherencia al tratamiento. Las estrategias nutricionales se basan tradicionalmente en suplementar la dieta con calcio y vitamina D. Estudios recientes confirman que los resultados de esta suplementación mejoran significativamente si se acompaña de la ingesta de colágeno hidrolizado oral. Objetivo: evaluar la posible actividad osteogénica in vitro de un complejo péptido-mineral formado por colágeno hidrolizado e hidroxiapatita bovinos (Phoscollagen®, PHC®). Métodos: se simuló la digestión y absorción de PHC® utilizando el digestor dinámico gastrointestinal de AINIA y el modelo celular Caco-2, respectivamente. Cultivos primarios de osteoblastos humanos se trataron con la fracción resultante de PHC® y se evaluaron los cambios en la proliferación de los preosteoblastos y en la expresión del ARNm de los biomarcadores osteogénicos en diferentes etapas de maduración de los osteoblastos: factor de transcripción 2 relacionado con Runt (Runx2), fosfatasa alcalina (ALP), osteocalcina (OC) y colágeno tipo I (ColA1). Resultados: se observó un incremento de la proliferación preosteoblástica (p ≤ 0,05). No se detectaron cambios en los biomarcadores de osteoblastos con 5 días de diferenciación, pero sí con 14 días, registrándose un aumento de Runx2 (p = 0,0008), ColA1 (p = 0,035), OC (p = 0,027) y ALP (sin significancia). Conclusión: estos resultados muestran que el complejo péptido-mineral PHC® estimula la actividad de los osteoblastos maduros, siendo susceptible de promover la formación ósea.

Functional differentiation and complementation of alkaline phosphatases and choreography of DOP scavenging in a marine diatom.

Facing phosphate deficiency, phytoplankton use alkaline phosphatase (AP) to scavenge dissolved organophosphate (DOP). AP is a multitype (e.g., PhoA, PhoD) family of hydrolases and is known as a promiscuous enzyme with broad DOP substrate compatibility. Yet, whether the multiple types differentiate on substrates and collaborate to provide physiological flexibility remain elusive. Here we identify PhoA and PhoDs and document the functional differentiation between PhoA and a PhoD (PhoD_45757) in Phaeodactylum tricornutum. CRISPR/Cas9-based mutations and physiological analyses reveal that (1) PhoA is a secreted enzyme and contributes the majority of total AP activity whereas PhoD_45757 is intracellular and contributes a minor fraction of the total AP activity, (2) AP gene expression compensates for each other after one is disrupted, (3) the DOP→PhoA→phosphate_uptake and the DOP_uptake→PhoD→phosphate pathways function interchangeably for some DOP substrates. These findings shed light on the underpinning of AP's multiformity and have important implications in phytoplankton phosphorus-nutrient niche differentiation, physiological plasticity, and competitive strategy.

How dam construction affects the activity of alkaline phosphatases in reservoir sediments: A study of two highly regulated rivers.

Dam construction causes phosphorus (P) accumulation in reservoir sediments and significantly affects the generation of available P. However, the effect of dam construction on the activity of sediment alkaline phosphatase (ALP), which is encoded by the bacterial phoD gene and participates in P mineralization, in river sediments remains unclear. Here, we investigated the ALP activities in 78 sediment samples collected from the cascade reservoir regions located in the Lancang River and the Jinsha River, two highly regulated rivers in southwestern China. The abundance and community composition of phoD-harboring bacteria were determined based on the phoD gene using quantitative real-time PCR and MiSeq sequencing, respectively. Comparison of control and affected sites indicated that dam construction significantly increased sediment ALP activity in both rivers. The abundances of phoD-harboring bacteria increased and their community compositions varied in response to dam construction; the relative abundances of the dominant genera Methylobacterium and Bradyrhizobium were particularly higher in affected site than control site. Co-occurrence network analyses revealed much higher network connectivity and relative abundances of keystone species in affected sites. Some microbial factors including phoD-harboring bacterial abundances, network clustering coefficients, and relative abundance of keystone species were positively correlated with ALP activity. The relative abundance of keystone species was identified as the most important microbial factor contributing to variation in ALP activity based on structural equation modeling analysis. These findings enhance our understanding of how dam construction affects the functions of phoD-harboring bacteria and their role in the P biogeochemical cycle in highly regulated rivers.

In vitro facilitating role of polygonatum sibiricum polysaccharide in osteogenic differentiation of bone marrow mesenchymal stem cells from patients with multiple myeloma.

BACKGROUND: Bone marrow mesenchymal stem cells (BMMSCs) were proved to play a vital role in multiple myeloma (MM). Polygonatum sibiricum polysaccharide (PSP) was found to have anti-tumor pharmacological effects, yet its interaction with BMMSCs remained poorly understood. Therefore, we explore the effect of PSP on osteogenic differentiation of BMMSCs. METHODS: BMMSCs were isolated by density gradient centrifugation. CD90 and CD34 were detected by flow cytometry (FCM). Osteogenic marks were detected by quantitative real-time PCR (qRT-PCR) and Western blotting (WB). The vitality of cells treated with different concentrations of PSP was observed by Cell Counting Kit-8 (CCK-8). ALP staining kit was used to detect the activity of alkaline phosphatase (ALP). Alizarin red staining detected the formation of mineralized nodules. Osteoblast-associated genes were evaluated by qRT-PCR and WB. The phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) signaling pathways were tested by WB. RESULTS: The BMMSCs showed good growth under an inverted microscope. FCM showed that CD34 and CD45 was low-expressed, whereas CD44, CD90 and CD105 was highly expressed. Compared with the Control group, the expressions of Runx2 and ALP in cells were significantly increased. CCK-8 showed that different concentrations of PSP had no significant effect on the viability of BMMSCs. BMMSCs treated with 25 mg/l PSP were stained the most deeply by ALP. Mineralized nodules in PSP groups dramatically increased, and hit a peak under the action of 25 mg/l PSP. PSP up-regulated p-PI3K, p-AKT, and p-mTOR, but had no significant effect on PI3K, AKT, and mTOR. CONCLUSION: PSP induced osteogenic differentiation of BMMSCs from MM patients.

Pre-conditioning of Mesenchymal Stem Cells with Piper longum L. augments osteogenic differentiation.

ETHNOPHARMACOLOGICAL RELEVANCE: The Indian Traditional Medicine, Ayurveda prescribes Piper longum L. popularly known as Long Pepper (Pippali) for the treatment of inflammatory and degenerative diseases. Therapeutic benefits of Piper longum L. are mainly attributed to the anti-inflammatory and arthritic potential. AIM OF THE STUDY: This study was aimed to explore the activity of Piper longum L. fruit extract on proliferation and osteogenic differentiation of human Wharton's Jelly Mesenchymal Stem Cells (WJMSCs) to find out it's possible role as anti-osteoporotic agent. MATERIALS AND METHODS: Proliferation of WJMSCs treated with Piper longum L. fruit extract was assessed by MTT assay and Cell Cycle Analysis. Effect of Piper longum L. preconditioning on osteogenic differentiation was performed. Ca2+ accumulation and matrix mineralization (Von Kossa and Alizarin Red Staining), alkaline phosphatase (ALP) activity and gene expression of key mRNA (RT PCR) was analyzed. RESULTS: Significant increase in the proliferation of WJMSCs was observed upon treatment of Piper longum L. at 5 µg/mL (P < 0.001) which can be attributed to the significant decrease in apoptotic cells (P < 0.05) as evidenced by cell cycle analysis. Preconditioning of Piper longum L. (10-100 µg/mL) enhanced Ca2+ accumulation and matrix mineralization as observed by Von Kossa and Alizarin Red staining where ALP activity was elevated 3.6 folds as compared to untreated WJMSCs (P < 0.001). RT-PCR analysis exhibited up regulation of Runx2, Osterix, ALP and OPN mRNAs. CONCLUSIONS: We demonstrate for the first time that Piper longum L. fruit extract enhanced osteogenic differentiation of WJMSCs. This finding can be clinically translated into development of an anti-osteoporotic agent.

Roles of Alkaline Phosphatase PhoA in Algal Metabolic Regulation under Phosphorus-replete Conditions.

Alkaline phosphatase (AP) in plants and algae is known to hydrolyze dissolved organophosphate (DOP) in order to obtain phosphorus when the preferred dissolved inorganic phosphorus (DIP) is present in limited supply. By conducting comparative analyses of physiologies and transcriptomes on a mutant of PhoA type AP (mPhoA) and wild type (WT) of the marine diatom Phaeodactylum tricornutum CCAP 1055/1 under P-replete and P-depleted conditions, we document other roles of this gene than DOP scavenging. PhoA mutation created by CRISPR/Cas9 diminished its DOP hydrolase activity but led to significant increases in cellular contents of pigment, carbon, and lipids, photosynthetic rate, growth rate, and the transcriptional levels of their corresponding metabolic pathways. All the results in concert indicate that besides P-nutrient scavenging under DIP deficiency, AP also functions, under the P-replete condition, to constrain pigment biosynthesis, photosynthesis, fatty acid biosynthesis, and cell division. These functions have important implications in maintaining metabolic homeostasis and preventing premature cell division.

Effects of Cirsium setidens (Dunn) Nakai on the osteogenic differentiation of stem cells.

Cirsium setidens (Dunn) Nakai, commonly known as gondre, is a perennial herb that grows predominantly in South Korea. It contains several bioactive phytochemicals with antioxidant, anti­cancer, anti­tumor and anti­inflammatory properties. The present study aimed to investigate the effects of methanolic extracts of gondre on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). As characterized by nuclear magnetic resonance spectroscopy and matrix­assisted laser deposition/ionization (time­of­flight) mass spectrometry, the methanol extract of gondre was found to be enriched with pectolinarin. After 48 h, enhanced viability of hPDLSCs was observed in the presence of gondre compared with under control conditions, suggesting the biocompatibility of gondre. Notably, biocompatibility was markedly affected by gondre concentration in cultured media. Relatively high cell viability was observed in medium containing 0.05% gondre. Furthermore, mineralization was significantly higher in hPDLSCs in the presence of gondre compared with that in control cells, indicating their mineralization potential. Increased expression of various transcription markers, such as collagen 1, runt­related transcription factor 2, bone sialoprotein and alkaline phosphatase, was also detected when hPDLSCs were stimulated with gondre compared with in the control groups, further confirming the superior osteogenic potential of gondre extract for tissue engineering applications, particularly in bone tissues.

Sol-Gel Synthesis, in vitro Behavior, and Human Bone Marrow-Derived Mesenchymal Stem Cell Differentiation and Proliferation of Bioactive Glass 58S.

Background: Bioactive glasses 58S, are silicate-based materials containing calcium and phosphate, which dissolved in body fluid and bond to the bone tissue. This type of bioactive glass is highly biocompatible and has a wide range of clinical applications. Methods: The 58S glass powders were synthesized via sol-gel methods, using tetraethyl orthosilicate, triethyl phosphate, and calcium nitrate, as precursors. Upon the analyses of phase and chemical structures of bioactive glass in different gelation times (12, 48, and 100 h), the appropriate heat treatment (at 525, 575, and 625 °C) was performed to eliminate nitrate compounds and stabilize the glass powder samples. The in vitro assay in SBF solution revealed the bioactivity of the synthesized 58S glass through the morphological (SEM), chemical structure (FTIR), release of calcium, phosphorous and silicon elements, pH variations, and weight loss measurements. The behavior of MSCs in the presence of bioactive glass powders was studied by MTT cytotoxicity, cell staining, ALP activity and biomineralization tests, as well as by the evaluation of ALP, osteocalcin, osteonectin, collagen I, and RUNX2 gene expression. Results: The results confirmed a gelation time of 100 h and a calcination temperature of 575 °C at optimal conditions for the synthesis of nitrate-free bioactive glass powders. Conclusion: The glass spherical nanoparticles in the range of 20-30 nm possess the improved bioactivity and osteogenic properties as demanded for bone tissue engineering.

Baicalein inhibits inflammatory response and promotes osteogenic activity in periodontal ligament cells challenged with lipopolysaccharides.

BACKGROUND: Periodontitis is a chronic infection initiated by oral bacterial and their virulence factors, yet the severity of periodontitis is largely determined by the dysregulated host immuno-inflammatory response. Baicalein is a flavonoid extracted from Scutellaria baicalensis with promising anti-inflammatory properties. This study aims to clarify the anti-inflammatory and osteogenic effects of baicalein in periodontal ligament cells (PDLCs) treated with lipopolysaccharides (LPS). METHODS: Human PDLCs were incubated with baicalein (0-100 µM) for 2 h prior to LPS challenge for 24 h. MTT analysis was adopted to assess the cytoxicity of baicalein. The mRNA and protein expression of inflammatory and osteogenic markers were measured by real-time polymerase chain reaction (PCR), western blot and enzyme-linked immunosorbent assay (ELISA) as appropriate. Alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of PDLCs. The expression of Wnt/ß-catenin and mitogen-activated protein kinase (MAPK) signaling related proteins was assessed by western blot. RESULTS: MTT results showed that baicalein up to 100 µM had no cytotoxicity on PDLCs. Baicalein significantly attenuated the inflammatory factors induced by LPS, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), matrix metalloprotein-1 (MMP-1), MMP-2 and monocyte chemoattractant protein 1 (MCP-1) at both mRNA and protein level. Moreover, MAPK signaling (ERK, JNK and p38) was significantly inhibited by baicalein, which may account for the mitigated inflammatory response. Next, we found that baicalein effectively restored the osteogenic differentiation of LPS-treated PDLCs, as shown by the increased ALP and ARS staining. Accordingly, the protein and gene expression of osteogenic markers, namely runt-related transcription factor 2 (RUNX2), collagen-I, and osterix were markedly upregulated. Importantly, baicalein could function as the Wnt/ß-catenin signaling activator, which may lead to the increased osteoblastic differentiation of PDLCs. CONCLUSIONS: With the limitation of the study, we provide in vitro evidence that baicalein ameliorates inflammatory response and restores osteogenesis in PDLCs challenged with LPS, indicating its potential use as the host response modulator for the management of periodontitis.

Natural compounds from Leea macrophylla enhance phagocytosis and promote osteoblasts differentiation by alkaline phosphatase, type 1 collagen, and osteocalcin gene expression.

The current study investigated the immunomodulating and osteoblast differentiation potential of the natural compounds from Leea macrophylla (LMN). Immunomodulatory effects have been investigated by the phagocytosis of Candida albicans using polymorphonuclear neutrophil cells in the in vitro slide method. A bioactivity-guided fractionation technique was used to evaluate the stimulating effect of L. macrophylla methanol extract on osteoblast differentiation using mouse osteoblastic cells. A low dose of LMN was found to stimulate the phagocytic effect better than a higher dose. The natural compounds from L. macrophylla have significantly increased alkaline phosphatase (ALP) and osteocalcin activities. The LMN promoted the osteoblast differentiation through upregulation of ALP, osteocalcin, and type 1 collagen in a dose-dependent manner. These natural compounds also upregulated ALP, osteocalcin, and type 1 collagen gene expressions. The data suggest that LMN has potential anabolic sequel on bone formation and osteoblast differentiation.

Effects of PIN on Osteoblast Differentiation and Matrix Mineralization through Runt-Related Transcription Factor.

Styrax Japonica Sieb. et Zucc. has been used as traditional medicine in inflammatory diseases, and isolated compounds have shown pharmacological activities. Pinoresinol glucoside (PIN) belonging to lignins was isolated from the stem bark of S. Japonica. This study aimed to investigate the biological function and mechanisms of PIN on cell migration, osteoblast differentiation, and matrix mineralization. Herein, we investigated the effects of PIN in MC3T3-E1 pre-osteoblasts, which are widely used for studying osteoblast behavior in in vitro cell systems. At concentrations ranging from 0.1 to 100 µM, PIN had no cell toxicity in pre-osteoblasts. Pre-osteoblasts induced osteoblast differentiation, and the treatment of PIN (10 and 30 µM) promoted the cell migration rate in a dose-dependent manner. At concentrations of 10 and 30 µM, PIN elevated early osteoblast differentiation in a dose-dependent manner, as indicated by increases in alkaline phosphatase (ALP) staining and activity. Subsequently, PIN also increased the formation of mineralized nodules in a dose-dependent manner, as indicated by alizarin red S (ARS) staining, demonstrating positive effects of PIN on late osteoblast differentiation. In addition, PIN induced the mRNA level of BMP2, ALP, and osteocalcin (OCN). PIN also upregulated the protein level of BMP2 and increased canonical BMP2 signaling molecules, the phosphorylation of Smad1/5/8, and the protein level of Runt-related transcription factor 2 (RUNX2). Furthermore, PIN activated non-canonical BMP2 signaling molecules, activated MAP kinases, and increased ß-catenin signaling. The findings of this study indicate that PIN has biological roles in osteoblast differentiation and matrix mineralization, and suggest that PIN might have anabolic effects in bone diseases such as osteoporosis and periodontitis.

Genistein Improves Bone Healing via Triggering Estrogen Receptor Alpha-Mediated Expressions of Osteogenesis-Associated Genes and Consequent Maturation of Osteoblasts.

Osteoporosis-associated fractures may cause higher morbidity and mortality. Our previous study showed the effects of genistein, a phytoestrogen, on the induction of estrogen receptor alpha (ERα) gene expression and stimulation of osteoblast mineralization. In this study, rat calvarial osteoblasts and an animal bone defect model were used to investigate the effects of genistein on bone healing. Treatment with genistein caused a time-dependent increase in alkaline phosphatase (ALP) activity in rat osteoblasts. Levels of cytosolic and nuclear ERα significantly augmented following exposure to genistein. Subsequently, genistein elevated levels of ALP mRNA and protein in rat osteoblasts. Moreover, genistein induced other osteogenesis-associated osteocalcin and Runx2 mRNA and protein expressions. Knocking-down ERα using RNA interference concurrently inhibited genistein-induced Runx2, osteocalcin, and ALP mRNA expression. Attractively, administration of ICR mice suffering bone defects with genistein caused significant increases in the callus width, chondrocyte proliferation, and ALP synthesis. Results of microcomputed tomography revealed that administration of genistein increased trabecular bone numbers and improved the bone thickness and volume. This study showed that genistein can improve bone healing via triggering ERα-mediated osteogenesis-associated gene expressions and subsequent osteoblast maturation.

A Comparative In Vitro Analysis of the Osteogenic Potential of Human Dental Pulp Stem Cells Using Various Differentiation Conditions.

Dental pulp stem cells (DPSCs) have excellent proliferative properties, mineralization potential and can be easily obtained from third molar teeth. Recently, many studies have focused on isolation and differentiation of DPSCs. In our study, we focused on biological properties of non-differentiated DPSCs in comparison with osteogenic differentiated cells from DPSCs. We analyzed morphology as well as mineralization potential using three varied osteogenic differentiation media. After fifteen days of differentiation, calcium deposit production was observed in all three osteogenic differentiation media. However, only one osteogenic medium, without animal serum supplement, showed rapid and strong calcification-OsteoMAX-XF™ Differentiation Medium. Therefore, we examined specific surface markers, and gene and protein expression of cells differentiated in this osteogenic medium, and compared them to non-differentiated DPSCs. We proved a decrease in expression of CD9 and CD90 mesenchymal stem cell surface markers, as well as downregulation in the expression of pluripotency genes (NANOG and OCT-4) and increased levels of expression in osteogenic genes (ALP, BSP, OCN and RUNX2). Moreover, osteogenic proteins, such as BSP and OCN, were only produced in differentiated cells. Our findings confirm that carefully selected differentiation conditions for stem cells are essential for their translation into future clinical applications.

The Solanum glaucophyllum Desf. extract reduces mineralized matrix synthesis in osteogenically differentiated rat mesenchymal stem cells in vitro.

The Solanum glaucophyllum Desf. has been used to treat and prevent diseases in human and veterinary medicine. On the other hand, plant poisoning causes several bone diseases, among them osteoporosis, which is characterized by osteoblastic hypoplasia. Because the osteoblast is a cell derived from the differentiation of mesenchymal stem cells (MSCs) from bone marrow, the hypothesis is that the plant reduces the osteogenic differentiation of MSCs. The objective of this study was to evaluate the effects of S. glaucophyllum Desf. extract on MSCs cultured in osteogenic differentiation medium. We determined by liquid chromatography that 1 ml of plant extract contained 3.8 µl of 1,25(OH)2 D3 (calcitriol). Four groups of MSCs cultivated in osteogenic medium were evaluated as follows: (a) treated with 100 µl of extract/L containing 0.4 µg/L of calcitriol; (b) treated with 1 ml of extract/L containing 4 µg/L of calcitriol; (c) treated with 5 ml of extract/L containing 20 µg/L of calcitriol; and (d) a control group without extract. We performed alkaline phosphatase activity assay, analysis of MTT conversion to formazan, and evaluated the percentage of cells, and number and diameter of mineralization nodules. The expression of gene transcripts for osteopontin, bone sialoprotein and BMP-2 was analysed by RT-qPCR. After 21 days, there was a significant reduction in MTT conversion to formazan in treated groups, of the cellularity in the group with 5 ml of extract/L, and in the number and size of mineralization nodules in the groups treated with 1 and 5 ml of extract/L. The 5 ml extract/L concentration also reduced transcript expression of osteopontin. It is concluded that S. glaucophyllum Desf. at concentrations of 1 and 5 ml extract/L reduced mineralized matrix synthesis in MSCs cultivated in osteogenic differentiation medium, which suggests that this is one of the mechanisms by which osteoporosis occurs in intoxicated animals.

Comparison of Proliferation and Osteogenic Differentiation Potential of Rat Mandibular and Femoral Bone Marrow Mesenchymal Stem Cells In Vitro.

This study was conducted to compare the in vitro proliferation and osteogenic differentiation potential of mesenchymal stem cells (MSCs) derived from mandibular (M-MSCs) or femur (F-MSCs) tissues of rats. M-MSC and F-MSC cultures were isolated and established from the same rat. Cultures were observed for morphological changes by microscope and growth characteristics by CCK-8 and cloning assays. Cell adhesion ability on a culture plate and titanium sheet was detected by staining with toluidine blue and Hoechst 33258, respectively. The levels of Ca, P, and ALP (serially) during osteogenic differentiation were evaluated. Cultures were analyzed for mineralization potential with alizarin red and ALP staining methods and for differentiation markers with RT-PCR (ALP, Runx2, and OCN). M-MSCs and F-MSCs were successfully isolated from the same rat with uncontaminated culture, which showed significant differences in morphology. The proliferation rate of M-MSCs was higher than F-MSCs in primary culture, but significantly lower after passage. More colonies are formed from F-MSCs than from M-MSCs. M-MSCs showed a significantly higher mineralization and osteogenic differentiation potential, which might be of significance for use in bone/dental tissue engineering. In vitro, cell passage will decrease the proliferation ability of M-MSCs. The higher mineralization and osteogenic differentiation potential of M-MSCs could make them an approachable stem cell source for further application in stem cell-based clinical therapies.