Increased Alkaline Phosphatase Activity Research Articles

32736 Characterizing calciphylaxis-like lesions in mice predisposed to vascular calcification

Calciphylaxis is a rare yet fatal form of vascular calcification often associated with end-stage renal disease (uremic calciphylaxis) and nonuremic causes. Calciphylaxis is associated with medial arteriolar vascular calcification and results in ischemic subcutaneous necrosis. We hypothesized that tissue-nonspecific alkaline phosphatase (TNAP), which is known to cause osteoblastic transformation of vascular cells, can contribute to the calcification of small arterioles in the skin. Our study aimed to characterize a mouse model of nonuremic calciphylaxis secondary to alkaline phosphatase overexpression in the vasculature. Eleven wild-type (WT) and 11 TNAP mice were evaluated at 23-week-old age on a regular diet. Alizarin and hematoxylin and eosin (H&E) staining of trunk skin cryosections revealed calcium deposits in TNAP mice. As visualized by vascular contrast micro-CT, calcifications appeared primarily vascular in nature and localized to the dermis and subcutaneous adipose tissue. Noncontrast micro-CT (n = 4 per group) revealed that calcification was present in all TNAP mice compared with its complete absence in WT mice. The presence of vascular calcification in adipose-rich trunks of the TNAP mice is consistent with the pattern of lesion formation in human calciphylaxis patients. Our mice tissues are most reflective of the earliest histologic manifestations, which lack ulcerations or necrotic lesions, but may be characterized by gross skin changes with intact epithelium. Our findings suggest that increased alkaline phosphatase activity in the vasculature can contribute to the pathophysiology of nonuremic calciphylaxis.

Volatile Constituents and Anti-Osteoporotic Activity of the n-Hexane Extract From Homalomena gigantea Rhizome

This study analyzed the chemical composition and anti-osteoporosis activity of the n-hexane extract of Homalomena gigantea rhizome. Sixty compounds, representing 92.0% of the extract, were identified by gas chromatography-mass spectrometry. Linalool (15.3%), oplopanone (9.8%), ( Ε)-α-atlantone (5.6%), khusinol acetate (5.4%), bullatantriol (4.3%), and β-sitosterol (3.8%) were the main constituents. The anti-osteoporotic activity of the n-hexane extract was determined by measuring alkaline phosphatase (ALP) activity, collagen content, and the mineralization of MC3T3-E1 cells. At concentrations of 4.0 and 20.0 µg/mL, the n-hexane extract increased ALP activity by 8.2% and 23.7%, and increased collagen secretion by MC3T3-E1 cells by 114.9% and 112.4%, respectively. At 4 µg/mL, the extract significantly promoted the mineralization of MC3T3-E1 cells by as much as 133.2% compared to the negative control. These results suggested that H. gigantea rhizome contains a natural anti-osteoporotic compound.

The effect of hypergravity, hyperbaric pressure, and hypoxia on osteogenic differentiation of adipose stem cells

Human adipose stem cells (ASCs) hold great potential for regenerative medicine approaches, including osteogenic regeneration of bone defects, that fail to heal autonomously. Osteogenic differentiation of stem cells is dependent on the stimulation of biophysical factors. In the present study, the effects of hypergravity, hypoxia, and hyperbaric treatment were investigated on adipose stem cell (ASC) metabolic activity, quantified by PrestoBlue conversion, and cell numbers, evaluated by crystal violet staining. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity and cresolphthalein staining of calcium deposition. Differentiation was performed for 12 days, which was accompanied by periodical stimulation. Increasing gravity forces up to 50x g did not affect ASC viability, but it enhanced osteogenic markers with a strongest effect between 20 and 30x g. Hyperbaric stimulation at 3 bar decreased ASC cell numbers but increased ALP activity and calcium deposition. Hypoxia at 8 % atmospheric oxygen did not affect ASC proliferation, while cell numbers were reduced at 3 % oxygen. Furthermore, hypoxic conditions produced opposing results on osteogenic markers, as ALP activity increased whereas cresolphthalein staining decreased upon stimulation. These data demonstrated that intermittent short duration of basal physical or chemical impulses interfere with the osteogenic differentiation of ASCs. Our findings could be of specific relevance in ASC based therapies for regenerative medicine and bone tissue engineering approaches.

Effects of long‐term phosphorus fertilizer inputs and seasonal conditions on organic soil phosphorus cycling under grazed pasture

AbstractSoil microbes and phosphatase enzymes play a critical role in organic soil phosphorus (P) cycling. However, how long‐term P inputs influence microbial P transformations and phosphatase enzyme activity under grazed pastures remains unclear. We collected top‐soil (0–75 mm) from a grazed pasture receiving contrasting P inputs (control, 188 kg ha−1 year−1 of single super phosphate [SSP], and 376 kg ha−1 year−1 of SSP) for more than 65 years. Olsen P, microbial biomass P, and acid and alkaline phosphatase enzyme activities were measured regularly over a 2‐year period. Pasture dry matter and soil chemical properties were also investigated. Results showed that long‐term P inputs significantly increased pasture dry matter, total N, and the concentrations of –N but significantly decreased soil pH and the concentrations of –N. Total C was not affected by P fertilization. Although Olsen P significantly increased with increasing long‐term P inputs, microbial biomass P was similar under P fertilized treatments. Long‐term P inputs decreased acid phosphatase activity but increased alkaline phosphatase activity. Microbial biomass P was similar across seasons in the control but decreased in spring and autumn while increased in summer and winter under P fertilized treatments. Acid and alkaline phosphatase activities were significantly affected by season and followed similar seasonal trends being maximum in summer and minimum in winter regardless of P treatment. Correlation and principal component analysis revealed that acid and alkaline phosphatase activities were significantly positively correlated with soil temperature and significantly negatively correlated with soil moisture. In contrast, Olsen P and microbial biomass P were weakly correlated with environmental conditions. The findings of this study highlight the intertwined relationship between organic P cycling and the availability of C and N in soil systems and the need to integrate both soil moisture and temperature in models predicting organic P mineralization, especially in the context of global climate change.

Melatonin protects gingival mesenchymal stem cells and promotes differentiation into osteoblasts

Melatonin (MEL) has antioxidant properties and participates in osteogenic differentiation. In periodontitis, in which increased oxidative stress and bone resorption are involved, mesenchymal stem cells derived from the gingiva (GMSCs) combined with MEL could be relevant for osteogenic regeneration. In this study, we studied the antioxidant and differentiating effect of MEL on an in vitro system of GMSCs. Primary culture of GMSCs from Wistar rats was developed to evaluate differentiation into osteoblasts with an appropriate medium with or without MEL. Marker genes of mesenchymal stem cells by real time-polymerase chain reaction, clonogenic capacity, and cell migration after wound assay were used to characterize GMSCs as mesenchymal stem cells. Alkaline phosphatase activity and the alizarin red technique were used to evaluate osteogenic activity and differentiation. MEL increased alkaline phosphatase activity and alizarin red values, promoting osteogenic differentiation. Besides this, MEL protected GMSCs in a model of cellular damage related to oxidative stress, returning viability to baseline. MEL was more effective in promoting and protecting GMSCs by the production of osteogenic cells when oxidative stress is present. This evidence supports the use of MEL as a novel bone-regenerative therapy in periodontal diseases.

An In Vitro Model to Test the Influence of Immune Cell Secretome on Mesenchymal Stromal Cell Osteogenic Differentiation.

Immune cells and their soluble factors have an important role in the bone healing process. Modulation of the immune response, therefore, offers a potential strategy to enhance bone formation. To investigate the influence of the immune system on osteogenesis, we developed and applied an in vitro model that incorporates both innate and adaptive immune cells. Human peripheral blood mononuclear cells (PBMCs) were isolated and cultured for 24 h and subsequently stimulated with immune-modulatory agents; C-class CpG oligodeoxynucleotide (CpG ODN C), polyinosinic acid-polycytidylic acid [Poly(I:C)], and lipopolysaccharide (LPS); all pathogen recognition receptor agonists, that target Toll-like receptors (TLRs) 9, 3, and 4, respectively. The conditioned medium (CM) obtained from PBMCs after 24 h was used to investigate its effects on the metabolic activity and osteogenic differentiation capacity of human bone marrow-derived mesenchymal stromal cells (MSCs). Conditioned media from unstimulated PBMCs did not affect the metabolic activity and osteogenic differentiation capacity of MSCs. The CM from CpG ODN C and LPS-stimulated PBMCs increased alkaline phosphatase activity (ALP) of MSCs by approximately threefold as compared with the unstimulated control, whereas Poly(I:C) CM did not enhance ALP activity of MSCs. Moreover, direct stimulation of MSCs with the immune-modulatory stimuli did not result in increased ALP. These results demonstrate that soluble factors present in CM from PBMCs stimulated with immune-modulatory factors enhance osteogenesis of MSCs. This in vitro model can serve as a tool in screening immune-modulatory stimulants from a broad variety of immune cells for (indirect) effects on osteogenesis and also to identify soluble factors from multiple immune cell types that may modulate bone healing.

PAP Polypeptide Promotes Osteogenesis in Jaw Bone Defect Repair by Inhibiting Inflammatory Reactions.

Jaw defects are common in oral and maxillofacial diseases and require surgical repair in extreme cases. Given the limitations in availability and efficacy of autologous bone grafts or allografts, great effort has been made in finding suitable, biocompatible, and effective artificial bone materials. Considering the key role of inflammation in bone resorption, we sought to identify a polypeptide with anti-inflammatory and bone-promoting effects. Rat bone marrow-derived mesenchymal cells (BMSCs) were treated with lipopolysaccharide (LPS) to induce an inflammatory environment, and 1,538 differentially abundant polypeptides were identified using mass spectrometry. Based on mass spectrometry signal intensity, multiple of difference, and structural stability, PAP was screened out as the polypeptide with the lowest abundance in the inflammatory condition. PAP showed no cytotoxicity to BMSCs with increasing concentrations. PAP (10 μM) also increased alkaline phosphatase activity and mRNA expression of Ocn, Bmp2, and Runx2 in a concentration-dependent manner, which confirmed that it can promote osteogenic induction of rat BMSCs. Moreover, PAP reduced LPS-induced expression of inflammatory cytokines (TNF-α, IL-1β, IL-6) and reactive oxygen species and inhibited polarization of RAW 264.7 macrophages to the inflammatory type. Finally, a skull defect mouse model was established, and mice were injected with LPS and/or PAP. Micro-CT, histological analysis, and immunohistochemical staining showed that PAP significantly reduced the number of LPS-induced bone resorption pits and maintained bone integrity. Overall, the polypeptide PAP screened using LPS stimulation of BMSCs is not cytotoxic and can inhibit the inflammatory reaction process to promote osteogenesis. This study thus provides a basis for development of PAP as a new osteogenic material in the repair of jaw defects.

Local Wisdom and Diversity of Medicinal Plants in Cha Miang Forest in Mae Kampong Village, Chiang Mai, Thailand, and Their Potential for Use as Osteoprotective Products.

“People-Forest-Miang” communities are villages located in the cultivated area of Camellia sinensis var. assamica, or Cha Miang, in northern Thailand. Cha Miang forests are a form of agriculture relying on forest-rich bioresources. This study focuses on a survey of the diversity of medicinal plants used by “People-Forest-Miang” communities in Mae Kampong Village, Chiang Mai, Thailand. The results demonstrated that 73 species of medicinal plants were used to prevent and treat various ailments. The highest number of species (30.14%) was used for musculoskeletal system disorders, followed by digestive system disorders (21.92%) and unspecified medicinal disorders (15.07%). The alkaline phosphatase (ALP) is the most widely recognized biochemical marker for osteoblast activity. The ALP activity of ethanol and deionized water extracts of the nine selected medicinal plants used for musculoskeletal system disorders were examined in the MG63 cell line. The results showed that the numerous water extracts, including MKP1, MKP2, MKP5, MKP6, MKP7, MKP8, and MKP9, and the ethanolic extracts—namely, MKP2, MKP3, MKP7, and MKP9—significantly increased ALP activity in the MG-63 cell line. The findings indicate that some medicinal plants may be further studied for active chemicals and developed as natural active pharmaceutical ingredients for osteoprotective products.

Highly elastic and bioactive bone biomimetic scaffolds based on platelet lysate and biomineralized cellulose nanocrystals

Bone is a vascularized organic-inorganic composite tissue that shows a heavily-mineralized extracellular matrix (ECM) on the nanoscale. Herein, the nucleation of calcium phosphates during the biomineralization process was mimicked using negatively-charged cellulose nanocrystals (CNCs). These mineralized-CNCs were combined with platelet lysate to produce nanocomposite scaffolds through cryogelation to mimic bone ECM protein-mineral composite nature and take advantage of the bioactivity steaming from platelet-derived biomolecules. The nanocomposite scaffolds showed high microporosity (94–95%), high elasticity (recover from 75% strain cycles), injectability, and modulated platelet-derived growth factors sequestration and release. Furthermore, they increased alkaline phosphatase activity (up to 10-fold) and up-regulated the expression of bone-related markers (up to 2-fold), without osteogenic supplementation, demonstrating their osteoinductive properties. Also, the scaffolds promoted the chemotaxis of endothelial cells and enhanced the expression of endothelial markers, showing proangiogenic potential. These results suggest that the mineralized nanocomposite scaffolds can enhance bone regeneration by simultaneously promoting osteogenesis and angiogenesis.

Revealing the mechanisms of Weishi Huogu I capsules used for treating osteonecrosis of the femoral head based on systems pharmacology with one mechanism validated with in vitro experiments

Ethnopharmacological relevanceWeishi Huogu I (WH I) capsules, developed through traditional Chinese medicine, have been used to treat clinical osteonecrosis of the femoral head (ONFH) for decades. However, the mechanisms have not been systematically studied.Aim of the study: In this study, the mechanisms of WH I capsules used in treating ONFH were examined through a systems pharmacology strategy, and one mechanism was validated with in vitro experiments. Materials and methodsWH I capsules compounds were identified by screening databases; then, a database of the potential active compounds was constructed after absorption, distribution, metabolism and excretion (ADME) evaluation. The compounds were identified through a systematic approach in which the probability of an interaction of every candidate compound with each corresponding target in the DrugBank database was calculated. Gene Ontology (GO) and pathway enrichment analyses of the targets was performed with the Metascape and KEGG DISEASE databases. Then, a compound-target network (C-T) and target-pathway network (T-P) of WH I capsule components were constructed, and network characteristics and related information were used for systematically identifying WH I capsule multicomponent-target interactions.Furthermore, the effects of WH I capsule compounds identified through the systematic pharmacology analysis of the osteogenic transformation of human umbilical mesenchymal stem cells (HUMSCs) were validated in vitro. ResultsIn total, 152 potentially important compounds and 176 associated targets were identified. Twenty-two crucial GO biological process (BP) or pathways were related to ONFH, mainly in regulatory modules regulating blood circulation, modulating growth, and affecting pathological processes closely related to ONFH.Furthermore, the GO enrichment analysis showed that corydine, isorhamnetin, and bicuculline were enriched in “RUNX2 regulates osteoblast differentiation”, significantly increased alkaline phosphatase activity and calcium deposition and upregulated runt-related transcription factor 2 mRNA and protein expression and osteocalcin mRNA expression in HUMSCs, suggesting that these compounds promoted the mesenchymal stem cell (MSC) osteogenic transformation. ConclusionsThe study showed that the pharmacological mechanisms of WH I capsule attenuation of ONFH mainly involve three therapeutic modules: blood circulation, modulating growth, and regulating pathological processes. The crosstalk between GOBPs/pathways may constitute the basis of the synergistic effects of the compounds in WH I capsules in attenuating ONFH. One of the pharmacological mechanisms in the WH I capsule effect on ONFH involves enhancement of the osteogenic transformation of MSCs, as validated in experiments performed in vitro; however, more mechanisms should be validated in further studies.

MO555: Exosomes From Endothelial Cells to Smooth Muscle Cells Induce Vascular Calcification in Early Chronic Kidney Disease

Abstract BACKGROUND AND AIMS Vascular calcification can occur in the early stages of chronic kidney disease (CKD) and result in a poor prognosis. How the calcification signal in the blood is transduced from endothelial cells in the tunica intima to vascular smooth muscle cells in the tunica media in the early stages of CKD is still unknown. The aim of this study was to explore the role of exosomes in signal transduction in vascular calcification. METHOD Exosomes were isolated from normal phosphate- and high phosphate-stimulated human umbilical vein endothelial cells (HUVECs) by superspeed centrifugation. We used transmission electron microscopy, molecular size analysis and Western blot analysis of exosome markers to identify exosomes (Figure 1). VSMC calcification was assessed by Alizarin Red Staining and Western blot. Proteomics analysis and miRNA microarray analysis were carried out to detect the differences in exosomal proteins and miRNAs. The results were analysed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. RESULTS Fluorescence microscopy verified that VSMCs took up exosomes from HUVECs. Exosomes isolated from high phosphate-stimulated HUVECs (HP-HUVEC-exos) induced VSMC calcification (assessed by Alizarin Red Staining), increased osteopontin expression and increased alkaline phosphatase activity (Figure 2). Proteomics analysis showed different protein expressions including STAT1, STAT3, HDAC1, PARP1, WDR5 et al. MiRNA microarray analysis showed different miRNA levels including miR-223–3p, miR-6776–5p, miR-6851–5p, miR-2116–3p, miR-6849–5p et al. (Figure 3). Target GO analysis showed that both miRNA and protein difference focused on some biological processes (RNA splicing, mRNA processing, RNA targeting), molecular function (cadherin binding, cell adhesion molecule binding, ubiquitin-like protein ligase binding) and cellular component (focal adhesion, cell-substrate junction, spliceosomal complex). KEGG analysis revealed that both miRNA and protein differences were focused on pathways associated with the cell cycle, virus infection and protein processing in the endoplasmic reticulum. CONCLUSION Exosome-mediated miRNA and protein transfer from endothelial cells to VSMCs may induce vascular calcification via RNA processing, protein processing and cell adhesion.

Circ_0058792 regulates osteogenic differentiation through miR-181a-5p/Smad7 axis in steroid-induced osteonecrosis of the femoral head

ABSTRACT Osteonecrosis of the femoral head (ONFH) caused by steroids is a severe orthopedic disorder resulting from the use of high-dose steroid drugs, characterized by structural changes in the bone, joint dysfunction, and femoral head collapse. CircRNAs and miRNAs have increasingly been suggested to play pivotal roles in osteogenic differentiation and osteogenesis. Significant upregulation of circ_0058792 was observed in patients with steroid-induced ONFH. Bioinformatic analysis showed that circ_0058792 might act as a sponge for miR-181a-5p. This study further investigated the mechanisms underlying the role of circ_0058792 and miR-181a-5p in osteogenic differentiation in methylprednisolone-induced ONFH rats and MC3T3-E1 cells. The results showed a notable decrease in the serum of miR-181a-5p in methylprednisolone-induced ONFH rats. Silencing of circ_0058792 using siRNAs and overexpression of miR-181a-5p significantly increased alkaline phosphatase activity and matrix mineralization capacity. Additionally, markers for osteogenic differentiation were significantly upregulated in miR-181a-5p-transfected cells. However, overexpression of circ_0058792 and the addition of the miR-181a-5p inhibitor reversed this increase. Smad7 was identified to be miR-181a-5p’s direct target and circ_0058792 was confirmed to be miR-181a-5p’s competing endogenous RNA (ceRNA). Upregulation of miR-181a-5p promotes phosphorylation of Smad2 and Smad3. Furthermore, circ_0058792 and miR-181a-5p had opposing effects on Smad7 expression. Collectively, these findings indicate that circ_0058792 regulates osteogenic differentiation by sponging miR-181a-5p via the TGF-β/Smad7 pathway. These findings elucidated the functions of circ_0058792 and miR-181a-5p in the regulation of steroid-induced ONFH. Our findings also indicated that circ_0058792 and miR-181a-5p are possible diagnostic markers and therapeutic targets for treating steroid-induced ONFH.

GelMA Hydrogel Reinforced with 3D Printed PEGT/PBT Scaffolds for Supporting Epigenetically-Activated Human Bone Marrow Stromal Cells for Bone Repair.

Epigenetic approaches using the histone deacetylase 2 and 3 inhibitor-MI192 have been reported to accelerate stem cells to form mineralised tissues. Gelatine methacryloyl (GelMA) hydrogels provide a favourable microenvironment to facilitate cell delivery and support tissue formation. However, their application for bone repair is limited due to their low mechanical strength. This study aimed to investigate a GelMA hydrogel reinforced with a 3D printed scaffold to support MI192-induced human bone marrow stromal cells (hBMSCs) for bone formation. Cell culture: The GelMA (5 wt%) hydrogel supported the proliferation of MI192-pre-treated hBMSCs. MI192-pre-treated hBMSCs within the GelMA in osteogenic culture significantly increased alkaline phosphatase activity (p ≤ 0.001) compared to control. Histology: The MI192-pre-treated group enhanced osteoblast-related extracellular matrix deposition and mineralisation (p ≤ 0.001) compared to control. Mechanical testing: GelMA hydrogels reinforced with 3D printed poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) scaffolds exhibited a 1000-fold increase in the compressive modulus compared to the GelMA alone. MI192-pre-treated hBMSCs within the GelMA–PEGT/PBT constructs significantly enhanced extracellular matrix collagen production and mineralisation compared to control (p ≤ 0.001). These findings demonstrate that the GelMA–PEGT/PBT construct provides enhanced mechanical strength and facilitates the delivery of epigenetically-activated MSCs for bone augmentation strategies.

Facilitative role of circPVT1 in osteogenic differentiation potentials of bone marrow mesenchymal stem cells from patients with osteoporosis through the miR-30d-5p/ITGB3 axis

ObjectiveThe critical role of circular RNAs (circRNAs) in osteoporosis (OP) has been highlighted. We tried to explore the role of circPVT1 in OP in relation to microRNA-30d-5p (miR-30d-5p) and ITGB3. MethodsAfter bone marrow collection, bone marrow mesenchymal stem cells (BMSCs) were isolated and identified. Then, Pearson coefficient was used to analyze the correlation among circPVT1, miR-30d-5p and ITGB3, and the binding sites were predicted and verified. Gain- and loss-of function assays in circPVT1, miR-30d-5p and ITGB3 were performed to analyze their effect on osteogenic differentiation of BMSCs. ResultsThe osteogenic differentiation of BMSCs from OP patients was significantly decreased, and reduced circPVT1 expression was found in the BMSCs from OP patients. Overexpression of circPVT1 stimulated the formation of calcified nodules, increased alkaline phosphatase activity, and enhanced the expression of osteogenic marker genes in the BMSCs from OP patients. Additionally, circPVT1 expression was negatively correlated with miR-30d-5p, and miR-30d-5p was negatively correlated with ITGB3 in OP patients. Mechanically, circPVT1 regulated the osteogenic differentiation potential of BMSCs by relieving the inhibition of miR-30d-5p on ITGB3 through the competitive endogenous RNA mechanism. ConclusionOur study highlighted a circPVT1/miR-30d-5p/ITGB3 axis in regulating osteogenic differentiation potential of BMSCs from OP patients.

ZIF-8 modified multifunctional injectable photopolymerizable GelMA hydrogel for the treatment of periodontitis

Periodontitis is a chronic inflammatory disease caused by plaque that leads to alveolar bone resorption. In the treatment of periodontitis, it is necessary to reduce the bacterial load and promote alveolar bone regeneration. In this study, zeolitic imidazolate framework-8 (ZIF-8) is used in the treatment of periodontitis, and an injectable photopolymerizable ZIF-8/gelatin methacryloyl (GelMA) composite hydrogel (GelMA-Z) is constructed. We confirm that ZIF-8 nanoparticles are successfully loaded into GelMA, which demonstrates fluidity and photopolymerizability. GelMA-Z continuously releases Zn2+ and shows good cytocompatibility. In vitro, GelMA-Z can effectively upregulate the expression of osteogenesis-related genes and proteins, increase alkaline phosphatase activity, promote extracellular matrix mineralization by rat bone mesenchymal stem cells, and exert an obvious antibacterial effect against Porphyromonas gingivalis. In vivo, GelMA-Z reduces the bacterial load, relieves inflammation and promotes alveolar bone regeneration in a rat model. The above results show that GelMA-Z has potential prospects in the treatment of periodontitis. Statement of significanceVarious methods have been explored for the treatment of periodontitis. However, current regiments have difficulty achieving ideal alveolar bone regeneration. In this study, we constructed a zeolitic imidazolate framework-8 (ZIF-8)/gelatin methacryloyl (GelMA) composite hydrogel (GelMA-Z). (1) The injectable and photopolymerizable GelMA-Z showed biocompatibility in vitro and in vivo. (2) GelMA-Z continually released zinc ions to promote the osteogenic differentiation of bone mesenchymal stem cells and kill bacteria in vitro. (3) In a rat model, the GelMA-Z pregel solution was used to fill the periodontal pocket and then crosslinked by UV exposure. GelMA-Z can stably remain in the periodontal pocket to reduce the bacterial load, relieve inflammation and promote alveolar bone regeneration. In conclusion, GelMA-Z has great potential for use in the treatment of periodontitis, especially in promoting alveolar bone regeneration.

Effect of polycystin2 on differentiation and maturation of osteoblasts promoted by low-frequency pulsed electromagnetic fields

It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.

Melatonin increases bone mass in normal, perimenopausal, and postmenopausal osteoporotic rats via the promotion of osteogenesis

BackgroundOsteoporosis is a disease threatening the health of millions of individuals. Melatonin is found to be a potential anti-osteoporosis drug. However, whether melatonin plays a role against osteoporosis at different stages of the menopause and the underlying mechanisms are unknown.MethodsOvariectomy was utilized as a model of perimenopausal and postmenopausal osteoporosis. A total of 100 mg/kg melatonin, or solvent alone, was added to the drinking water of the rats over 8 weeks. Perimenopausal rats immediately received intervention following ovariectomy while postmenopausal rats received intervention 8 weeks after ovariectomy. All rats underwent overdose anesthesia following intervention after which blood samples and femurs were collected for further analysis. Rat femurs were scanned using micro-CT and examined histologically. The serum levels of melatonin and osteogenic biochemical markers were measured and the expression of osteogenesis-associated genes (Runx2, Sp7) were quantified by real-time quantitative PCR. Alkaline phosphatase (ALP) activity and the gene expression (Col1a1, Runx2, Alpl, and Bglap) were measured after bone marrow mesenchymal stem cells (BMSCs) were osteogenically induced, both with and without melatonin in vitro. ALP staining and Alizarin Red S staining were used to identify osteogenesis.ResultsAnalysis by micro-CT and histological staining demonstrated that bone mass decreased and bone microarchitecture deteriorated over time after ovariectomy. Intervention with melatonin increased bone mass in normal, perimenopausal, and postmenopausal osteoporotic rats. Serum levels of ALP continuously increased after ovariectomy while osteocalcin levels initially rose, then decreased. Melatonin increased the serum levels of ALP and osteocalcin and mRNA expression levels of Runx2 and Sp7 in normal and postmenopausal rats, the opposite of the markers in perimenopausal rats. In vitro study demonstrated that 100 μmol/L melatonin increased the mRNA expression of Col1a1, Runx2, and Alpl three and/or seven days after intervention, and Alpl and Bglap 14 d after intervention. Melatonin increased ALP activity and the extent of ALP and matrix mineralization in the late stage of osteogenesis.ConclusionsBone mass continuously decreased after ovariectomy, while melatonin increased bone mass and ameliorated bone metabolism in normal, perimenopausal, and postmenopausal osteoporotic rats due to the induction of osteogenic differentiation in BMSCs.

A Non-thermal Biocompatible Plasma-Modified Chitosan Scaffold Enhances Osteogenic Differentiation in Bone Marrow Stem Cells.

Non-thermal biocompatible plasma (NBP) was considered as an efficient tool in tissue engineering to modify the surface of biomaterials. Three-dimensional chitosan scaffolds have been extensively used in different ways because it holds some remarkable properties, including biodegradability and biocompatibility. In this study, we evaluated the osteogenic potential of NBP-treated chitosan scaffolds using two different plasma sources: a dielectric barrier discharge (NBP-DBD) and a soft jet (NBP-J). The surface modification of the scaffold was evaluated using scanning electron microscopy. For osteogenic differentiation of cells, proliferation and differentiation were tested by using bone marrow-derived stem cells (BMSCs). We observed that cell viability using NBP-DBD and NBP-J treated chitosan scaffolds yielded significant improvements in cell viability and differentiation. The results obtained with MTT and live/dead assays showed that NBP-modified scaffold increases cell metabolic by MTT assay and live/dead assay. It also observed that the NBP treatment is more effective at 5 min with DBD and was selected for further investigations. Enhanced osteogenic differentiation was observed using NBP-treated scaffolds, as reflected by increased alkaline phosphatase activity. Our findings showed that NBP is an innovative and beneficial tool for modifying chitosan scaffolds to increase their activity, making them suitable as biocompatible materials and for bone tissue engineering.

Protease-activated receptor-2 dependent and independent responses of bone cells to prostate cancer cell secretory products.

Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.

Regulation of gingival fibroblast phenotype by periodontal ligament cells in vitro.

ObjectivesStem cell transplantation has shown modest effects on periodontal tissue regeneration, and it is still unclear how regenerative effects utilizing this modality are mediated. A greater understanding of the basic interactions between implanted and host cells is needed to improve future strategies. The aims of this study were to investigate the effects of periodontal ligament (PDL) cells on expression of periodontal markers and alkaline phosphatase (ALP) activity of gingival fibroblasts (GF).Materials and MethodsPrimary human PDL cells were co‐cultured with primary GF cultures either by direct co‐culture with subsequent FACS sorting or indirect co‐culture using transwell cultures and PDL cell conditioned medium. Expression of periodontal markers, asporin, nestin, and periostin, was assessed by qPCR and immunofluorescence staining. Alkaline phosphatase (ALP) expression was assessed by qPCR, histochemical staining, and activity assessed by para‐nitrophenol enzymatic assay. Single cultures of PDL cells and GF were used as controls. The role of Wnt signaling on ALP activity was assessed via Dkk1‐mediated inhibition.ResultsPDL cells significantly upregulated expression of PDL markers in GF with both direct and indirect co‐culture methods when compared to controls (6.05 vs. 0.73 and 59.48 vs. 17.55 fold change of asporin expression). PDL/GF cell co‐cultures significantly increased ALP activity in GF when compared with single GF cultures. Similar results were obtained when using conditioned medium isolated from PDL cell cultures. Dkk1 caused dose‐dependent reduction in ALP activity of GF cultured in PDL cell conditioned medium.ConclusionsPDL cells stimulate expression of periodontal markers and osteogenic capacity of gingival fibroblasts via paracrine signaling which can be partially inhibited with addition of the Wnt antagonist, Dkk1.Further studies are required to identify specific secreted factors responsible for this activity.