Alkaline Phosphatase Gene Expression Research Articles

Osteoblasts Regulate the Expression of ADAMTS and MMPs in Chondrocytes through ERK Signaling Pathway.

Degradative enzymes such as matrix metalloproteinase (MMP) and disintegrin metalloproteinase with platelet thrombin-sensitive protein-like motifs (ADAMTS) play a key role in the development of osteoarthritis (OA). We aimed to investigate the effects of OA subchondral osteoblasts on the expression of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 in chondrocytes and the regulation of mitogen-activated protein kinase (MAPK) signaling pathway. A rat knee OA model was constructed by cutting the anterior cruciate ligament of the knee joints, and normal rat articular cartilage chondrocytes (N-ACC), OA rat articular cartilage chondrocytes (O-ACC), normal subchondral bone osteoblasts (N-SBO), and OA subchondral bone osteoblasts (O-SBO) were isolated and extracted. The expressions of O-ACC and O-SBO COL1 and COL2 were detected respectively. Chondrocytes were identified by immunofluorescence of COL2 and toluidine blue staining, and osteoblasts were identified by COL1 immunofluorescence, alkaline phosphatase (ALP), and Alizarin Red staining. Gene expression of COL1, COL2, and aggrecan in normal chondrocytes and OA chondrocytes, and gene expression of osteoblast ALP and osteocalcin (OCN) were detected by RT-PCR to identify the two chondrocytes and the two osteoblast phenotypes. The constructing N-ACC group, O-ACC group, N-ACC + N-SBO group, N-ACC + O-SBO group, O-ACC + N-SBO group, O-ACC + O-SBO group, I+ N-ACC + O-SBO group, and I + O-ACC + O-SBO group cell cultures, and the expression of ERK, ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes in chondrocytes cultured for 0, 24, 48, and 72 h were detected by RT-PCR. The protein expressions of pERK, ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 were detected by Western blot. · The X-ray showed that the knee joint space of the affected limb became narrow.. · The results of RT-PCR of COL2 and aggrecan gene in OA and normal chondrocytes suggest that the relative expression of COL2 in OA articular chondrocytes (0.24 ± 0.07) issignificantly lower than that in normal cartilage (0.61 ± 0.07) (p < 0.05). The relative expression of AGG (0.37 ± 0.16) in OA chondrocytes was significantly lower than that of normal chondrocytes AGG (1.30 ± 0.25) (p < 0.05). The expression of COL1 was very low, and was not statistically significant.. · The results of RT-PCR of the osteoblast ALP and OCN gene indicated that gene expression of ALP (12.30 ± 1.17) and OCN (20.47 ± 4.19)was upregulated when compared with the relative expression of ALP (4.66 ± 0.71) (p < 0.05) and OCN (12.17 ± 2.76) (p < 0.05) in normal osteoblasts, indicating that osteoblasts of OA have greater osteogenic potential than normal osteoblasts.. · The expressions of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes and proteins in OA chondrocytes or normal chondrocytes were basically unchanged when they were cocultured with normal osteoblasts. Indirect coculture of OA osteoblasts and chondrocytes could promote the expression of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes and proteins in chondrocytes. Overexpression of ADAMTS and MMP in coculture systems can be reversed by MAPK-ERK inhibitors.. · OA subchondral bone osteoblasts can promote the overexpression of ADAMTS and MMPs in chondrocytes.. · The ERK signaling pathway may be involved in the regulation of the effect of subchondral bone osteoblasts on chondrocytes..

Biological Characteristics and Odontogenic Differentiation Effects of Calcium Silicate-Based Pulp Capping Materials.

We compared calcium silicate-based pulp capping materials to conventional calcium hydroxide in terms of their biological properties and potential effects on odontogenic differentiation in human dental pulp stem cells (hDPSCs). We cultured hDPSCs on disks (7-mm diameter, 4-mm high) of ProRoot MTA (Dentsply Tulsa Dental Specialties), Biodentine (Septodont), TheraCal LC (Bisco), or Dycal (Dentsply Tulsa Dental Specialties). Cell viability was assessed with cell counting (CCK) and scanning electron microscopy (SEM). Odontogenic activity was assessed by measuring alkaline phosphatase (ALP) activity and gene expression (quantitative real-time PCR). CCK assays showed that Dycal reduced cell viability compared to the other materials (p < 0.05). SEM showed low and absent cell attachment on TheraCal LC and Dycal disks, respectively. hDPSCs exposed to TheraCal LC and Dycal showed higher ALP activity on day 6 than the control group (p < 0.05). The day-9 Runx2 expression was higher in the ProRoot MTA and TheraCal LC groups than in the control group (p < 0.05). On day 14, the ProRoot MTA group showed the highest dentin sialophosphoprotein levels (not significant; p > 0.05). In conclusion, various pulp capping materials, except Dycal, exhibited biological properties favorable to hDPSC viability. ProRoot MTA and TheraCal LC promoted higher Runx2 expression than Biodentine. Future studies should explore the odontogenic potential of pulp capping materials.

The Effect of Strontium-Substituted Hydroxyapatite Nanofibrous Matrix on Osteoblast Proliferation and Differentiation.

Natural bone tissue consists primarily of bioapatite and collagen. Synthetic hydroxyapatite (HA) possesses good biocompatibility, bioactivity, and osteoconductivity due to its chemical and biological similarity to bioapatite. Hence, HA has been widely used as a bone graft, cell carrier and drug/gene delivery carrier. Moreover, strontium-substituted hydroxyapatite (SrHA) can enhance osteogenic differentiation and inhibit adipogenic differentiation of mesenchymal stem cells. Hence, SrHA has the potential to be used as a bone graft for bone regeneration. It is widely accepted that cell adhesion and most cellular activities are sensitive to the topography and molecular composition of the matrix. Electrospun polymer or polymer-bioceramic composite nanofibers have been demonstrated to enhance osteoblast differentiation. However, to date, no studies have investigated the effect of nanofibrous bioceramic matrices on osteoblasts. In this study, hydroxyapatite nanofiber (HANF) and strontium-substituted hydroxyapatite nanofiber (SrHANF) matrices were fabricated by electrospinning. The effect of the HANF components on MG63 osteoblast-like cells was evaluated by cell morphology, proliferation, alkaline phosphatase activity (ALP) and gene expression levels of RUNX2, COLI, OCN and BSP. The results showed that MG63 osteoblast-like cells exhibited higher ALP and gene expression levels of RUNX2, COLI, BSP and OCN on the SrHANF matrix than the HANF matrix. Hence, SrHANFs could enhance the differentiation of MG63 osteoblast-like cells.

Surface Epitaxial Nano-Topography Facilitates Biomineralization to Promote Osteogenic Differentiation and Osteogenesis.

Biomimetic modification of hydroxyapatite on a polymer surface is a potent strategy for activating biological functions in bone tissue engineering applications. However, the polymer surface is bioinert, and it is difficult to introduce a uniform calcium phosphate (CaP) layer. To overcome this limitation, we constructed a specific nano-topographical structure onto a poly(ε-caprolactone) substrate via surface-directed epitaxial crystallization. Formation of the CaP layer on the nano-topological surface was enhanced by 2.34-fold compared to that on a smooth surface. This effect was attributed to the abundant crystallization sites for CaP deposition because of the increased surface area and roughness. Bone marrow mesenchymal stromal cells (BMSCs) were used to examine the biological effect of biomineralized surfaces. We clearly demonstrated that BMSCs responded to surface biomineralization. Osteogenic differentiation and proliferation of BMSCs were significantly promoted on the biomineralized nano-topological surface. The expression of alkaline phosphatase and osteogenic-related genes as well as extracellular matrix mineralization was significantly enhanced. The proposed strategy shows potential for designing bone repair scaffolds.

Comparative immunolocalization of tissue nonspecific alkaline phosphatase and ectonucleotide pyrophosphatase/phosphodiesterase 1 in murine bone

ObjectiveThis study aimed to demonstrate the immunolocalization and gene expression of tissue nonspecific alkaline phosphatase (TNALP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in osteoblasts, preosteoblasts, and osteocytes of murine bone to provide clues for a better understanding of the supply of phosphate ions (Pi) during bone mineralization. MethodsSix-week-old male C57BL/6J mice (n = 6) were fixed with a paraformaldehyde solution, and the right femora were extracted for immunodetection of TNALP and ENPP1, while the left tibiae were used for reverse transcription polymerase chain reaction to evaluate Tnalp and Enpp1 gene expression. ResultsTNALP was intensely localized on the basolateral cell membranes of mature osteoblasts and preosteoblastic cells. There was little immunoreactivity of TNALP on the secretory surface of the osteoblasts and no TNALP reactivity in the osteocytes. In contrast, ENPP1 was observed throughout the cytoplasm of mature osteoblasts and osteocytes embedded in bone but was not observed in preosteoblasts. Together, despite the fact that the osteoid is a site of matrix vesicle-mediated mineralization, ENPP1, which inhibits mineralization by providing pyrophosphates, was localized in close proximity of the osteoid, whereas TNALP, which facilitates mineralization by providing Pi, was relatively distant from the osteoid. ConclusionIt seems likely that the differential localization of TNALP and ENPP1 around the osteoid observed at the microscopic level may provide preferential micro-circumstance for a balanced concentration of Pi and pyrophosphate for bone mineralization.

Interactions between cadmium and zinc on gene expression pattern of differentiation markers in MC3T3-E1 cell line

We evaluated, in vitro, the interactions between cadmium (Cd) and zinc (Zn) during the proliferation and differentiation process using bone MC3T3-E1 cell line. Cells were treated with CdCl2 and/or ZnCl2 for 24 and 48 h and 5 µM CdCl2 was found as low cytotoxic dose and 25 µM ZnCl2 as the best Zn treatment for cell proliferation. Gene expression of some bone markers (Runx2, collagen α1 (Colα1), osteocalcin (Oc), alkaline phosphatase (ALP) and bone sialoprotein (BSP)) was studied at 24, 48 and 72 h. Treatment by CdCl2 depressed Runx2, Colα1, and BSP mRNA levels after 24 h. Oc and ALP gene expression was found to be decreased after 72 h. CdCl2 -exposure decreased ALP activity and Ca deposit in matrix. In concomitant treatment by CdCl2 and ZnCl2, gene expression of osteoblastic markers was found to be up-regulated (p < 0, 05) compared to CdCl2 treated cells, ALP staining and mineralization were increased. Our results show that Zn could prevent Cd-induced toxicity on MC3T3-E1 cells, probably through the restoration of Runx2, col α1, BSP, ALP and Oc and gene expression inhibited by Cd.

Low concentrations of caffeic acid phenethyl ester stimulate osteogenesis in vitro

ObjectiveThe aim of this study was to evaluate the effects of caffeic acid phenethyl ester (CAPE) on osteoblast-like cell cultures (SAOS-2). MethodsSAOS-2 were exposed to CAPE at 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM. Non-exposed cultures were used as control. The following parameters were assayed: 1) cell viability at 1, 3, and 7 days; 2) alkaline phosphatase (ALP) activity at 5 and 10 days; 3) matrix mineralization at 14 days; and 4) Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (SPP1), and osteocalcin (BGLAP) gene expression at 5 and 10 days. The data were analyzed by ANOVA two-way or Kruskal-Wallis (α = 5%). ResultsAt day 1, cell viability was similar among all groups (p > 0.05). At days 3 and 7, cultures exposed to CAPE at 10 μM exhibited a significant reduction in cell viability compared with the others groups (p < 0.05). At day 5, ALP activity was similar among all experimental groups; at day 10, however, the stain intensity was higher in cultures exposed to CAPE at 100 nM and 10 nM in comparison with the other groups (p < 0.05). At days 5 and 10, RUNX2, ALP, SPP1, and BGLAP gene expression was greater in cultures exposed to CAPE in comparison with the control (p < 0.05). At day 14, matrix mineralization was similar in cultures exposed to CAPE at 1 nM and 10 nM (p > 0.05), but superior to those ones observed in the other experimental groups (p < 0.05). ConclusionCAPE at low concentrations can positively module the osteogenesis in vitro.

Improved Osteogenesis by Noggin siRNA Collagen-Bioactive Glass Composites

Collagen-bioactive glass (COL–BG) composites have significant bone regeneration ability due to the considerable osteogenic performance and desirable biological properties of collagen. In the current research, the influence of COL–BG composite scaffolds loaded with noggin siRNA on osteogenesis in MC3T3 cells have been comprehensively studied. We prepared three types of COL–BG composites scaffolds, namely, unloaded, loaded with noggin siRNA, and loaded with negative control siRNA. The effect of the aqueous extracts achieved from these scaffolds on MC3T3 cell proliferation was monitored using a CCK8 test, whilst the effect on osteogenesis was assessed with ALP (alkaline phosphatase) activity assays, quantitative real-time PCR, and Alizarin Red staining (ARS). After cells were cultured for three and five days, proliferation in the presence of the extracts of composite scaffolds was remarkably greater than in the no-scaffold blank (P &lt; 0.05). After 14 days of cultivation, MC3T3 cells exposed to COL–BG composites scaffold loaded with noggin siRNA exhibited greater ALP performance (P &lt; 0.05) as well as higher mRNA expression (P &lt; 0.01) of Runx2, BSP, and ALP genes in comparison with two other composite scaffolds, whereas ARS exhibited a larger number of mineralized nodules. Overall, these outcomes illustrate that COL–BG composite scaffold loaded with noggin siRNA is effective in enhancing osteogenesis. Thus, we suggest that this material has a promising potential for bone tissue engineering and is able to locally improve bone formation in bone defects.

Endocannabinoids Regulate Stem Cells of the Apical Papilla via a Cannabinoid Receptor and TRPV1-Independent Mechanism

IntroductionEndogenous cannabinoids (endocannabinoids [eCBs]) have been shown to have a multitude of functions including neurotransmission and immune modulatory effects. This study aimed to evaluate if stem cells of the apical papilla (SCAP) express the receptors and enzymes of the endocannabinoid system (ECS) and whether eCBs regulate their proliferation and mineralization potential. MethodsGene expression of the main components of the ECS and transient receptor potential vanilloid 1 (TRPV1) was evaluated in SCAP cultures. SCAP were treated with 2 concentrations of eCBs and/or capsazepine, a TRPV1 antagonist. SCAP viability was evaluated after 1, 4, and 7 days. Osteogenic differentiation was assessed after 14 days, and the gene expression of mineralization markers was assessed after 7 days. ResultsThe enzymes of ECS and TRPV1 but not the cannabinoid receptors (cannabinoid receptors 1 and 2) were expressed in SCAP. Anandamide, 2-arachidonoylglycerol, and N-arachidonoylphenolamine (AM-404) reduced SCAP viability in all experimental periods at the highest concentration compared with the group with no treatment. Anandamide and AM-404 did not inhibit SCAP differentiation potential, but 2-arachidonoylglycerol at the highest concentration did. SCAP treated with AM-404 presented a down-regulation in gene expression of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSPP) compared with the proliferation medium group but not with control group. ConclusionsSCAP expressed the genes of the main components of ECS and TRPV1, and eCBs can affect SCAP viability, mineralization, and gene expression.

The biocompatibility and mineralization potential of mineral trioxide aggregate containing calcium fluoride–An in vitro study

Background/purposeMTA is used to induce hard tissue regeneration in various procedures. This study evaluated the biocompatibility and mineralization potential of mineral trioxide aggregate (MTA) containing calcium fluoride (CaF2). To verify if the change of components affected physical properties, the setting time, solubility, and surface roughness were measured. Materials and methodsHuman dental pulp cells (HDPCs) were treated with powder and set MTA containing CaF2 (0, 1, 5, and 10 wt %). The proliferation of HDPCs was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mineralization potential of HDPCs was investigated with the relative gene expression of alkaline phosphatase (ALP), collagen type I (ColI), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2) using real-time reverse transcription polymerase chain reaction (RT-PCR). For investigating the physical properties, setting time and solubility were tested. Surface profiles of material were analyzed by a non-contact surface profiler and a scanning electron microscope (SEM). ResultsMTA-5% CaF2 mixtures increased the proliferation and the mineralization-related gene expression of HDPCs to a greater degree than pure MTA. The addition of CaF2 to MTA delayed the setting, but the difference was only significant in the MTA-10% CaF2. Solubility and surface roughness was not altered. ConclusionThe addition of more than 5% CaF2 can be considered to increase the regeneration potential of pulp cells without adverse effects on physical property.

Protective effect of mitophagy against aluminum-induced MC3T3-E1 cells dysfunction

Aluminum (Al) is a ubiquitous environmental metal toxicant that causes osteoblast (OB) damage which leads to Al-related bone diseases. Mitochondrial damage plays a key role in Al-related bone diseases, and while mitophagy can clear damaged mitochondria and improve OB function, the relationship between mitophagy and Al-induced OB dysfunction is unknown. To explore the role of mitophagy in Al-induced OB dysfunction in vitro, we used 2 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 0.4 μM Cyclosporin A (CsA) to activate and inhibit mitophagy, respectively. MC3T3-E1 cells were treated with 0 mM AlCl3 (control group); 2 mM AlCl3 (Al group); 2 μM CCCP (CCCP group); 2 μM CCCP and 2 mM AlCl3 (CCCP + Al group); 0.4 μM CsA (CsA group); 0.4 μM CsA and 2 mM AlCl3 (CsA + Al group). The results showed that Al induced ultrastructural and functional impairment of MC3T3-E1 cells. Compared to the Al group, mitophagy activation caused mitochondrial membrane potentials to collapse, up-regulated PINK1, Parkin, and LC3 expression, down-regulated p62 expression, and increased mitophagosome numbers. Mitophagy activation also reduced Al-induced oxidative stress and MC3T3-E1 cell functional damage, as seen in improvement in cell viability, cellular calcium and phosphorus contents, and collagen I, osteocalcin, and bone alkaline phosphatase gene expression. Mitophagy inhibition had the opposite effects on activation. Overall, these results show that mitophagy can protect against Al-induced OB dysfunction.

Green Tea Catechin, Epigallocatechin Gallate, Prevents Bone Loss in Hindlimb-Unloading Mice

Abstract Objectives Osteoporosis is a major health problem in the elderly characterized by bone loss and micro-architectural deterioration of bone tissue and associated with an increased risk of fracture. Prolonged bed rest, or physical inactivity during space flight causes rapid and marked bone loss. The effects of catechin, the main ingredient of Japanese green tea, on the bone are currently under study. It has been shown that green tea catechin modulates bone resorption in osteoclasts. However, there is no evidence supporting its inhibitory effect on bone loss during physical inactivity. In the present study, we investigated whether green tea catechin prevented bone loss through skeletal hindlimb-unloading in mice. Methods Female 8-week-old ddY mice were divided into five groups (n = 6–8 each) and subjected to: (1) normal housing fed a control diet, (2) sham unloading fed a control diet, (3) hind limb-unloading fed a control diet, (4) hind limb-unloading fed a 0.05% epigallocatechin gallate (EGCG)-containing diet, and (5) hind limb-unloading fed a 0.25% EGCG-containing diet for three weeks. Purified EGCG (97%) was used for green tea catechin. Results Bone mineral density of the tibia significantly decreased in hind limb-unloading mice. Treatment with 0.25% EGCG prevented bone loss and maintained trabecular bone mineral density more significantly than in cortical bones. The 0.25% EGCG diet inhibited decrease in the gene expression of alkaline phosphatase, a marker of bone formation, in the bone marrow in hind limb-unloading mice. Conclusions These results suggest that EGCG has ability to prevent bone loss induced by hindlimb-unloading in mice. These osteoprotective effects of EGCG may result from the inhibition of unloading-induced decrease in bone formation. Funding Sources This work was supported by the Honjo International Scholarship Foundation of Japan.

Cold Atmospheric Plasma Promotes Regeneration-Associated Cell Functions of Murine Cementoblasts In Vitro.

The aim of the study was to examine the efficacy of cold atmospheric plasma (CAP) on the mineralization and cell proliferation of murine dental cementoblasts. Cells were treated with CAP and enamel matrix derivates (EMD). Gene expression of alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (BGLAP), periostin (POSTN), osteopontin (OPN), osterix (OSX), collagen type I alpha 1 chain (COL1A1), dentin matrix acidic phosphoprotein (DMP)1, RUNX family transcription factor (RUNX)2, and marker of proliferation Ki-67 (KI67) was quantified by real-time PCR. Protein expression was analyzed by immunocytochemistry and ELISA. ALP activity was determined by ALP assay. Von Kossa and alizarin red staining were used to display mineralization. Cell viability was analyzed by XTT assay, and morphological characterization was performed by DAPI/phalloidin staining. Cell migration was quantified with an established scratch assay. CAP and EMD upregulated both mRNA and protein synthesis of ALP, POSTN, and OPN. Additionally, DMP1 and COL1A1 were upregulated at both gene and protein levels. In addition to upregulated RUNX2 mRNA levels, treated cells mineralized more intensively. Moreover, CAP treatment resulted in an upregulation of KI67, higher cell viability, and improved cell migration. Our study shows that CAP appears to have stimulatory effects on regeneration-associated cell functions in cementoblasts.

Behavior of rat bone marrow stem cells on titanium surfaces modified by laser-beam and deposition of calcium phosphate

ObjectivesThe aim of this study was to evaluate the behavior of rat bone marrow stem cells seeded on a Ti-15Mo alloy surface modified by laser-beam irradiation followed by calcium phosphate deposition.Materials and methodsA total of four groups were evaluated: polished commercially pure titanium (cpTi): Ti-P; laser irradiation + calcium phosphate deposition on cpTi: Ti-LCP; polished Ti-15Mo alloy: Ti15Mo-P; and laser irradiation + calcium phosphate deposition on Ti-15Mo alloy: Ti15Mo-LCP. Before and after laser irradiation and calcium phosphate deposition on the surfaces, physicochemical and morphological analyses were performed: Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDX). The wettability of the samples was evaluated by contact angle measurement. In addition, the behavior of osteoblast-like cells to these surfaces was evaluated for cell morphology, adhesion, proliferation and viability, evaluation of alkaline phosphatase formation and gene expression of osteogenesis markers.ResultsSurfaces wet-abrade with grit paper (P) showed oriented groves, while the laser irradiation and calcium phosphate deposition (LCP) produced porosity on both cpTi and Ti15Mo alloy groups with deposits of hydroxyapatite (HA) crystals (SEM). EDX showed no contamination after surface modification in both metal samples. A complete wetting was observed for both LCP groups, whereas P surfaces exhibited high degree of hydrophobicity. There was a statistical difference in the intragroup comparison of proliferation and viability (p < 0.05). The ALP activity showed higher values in the Ti15Mo alloy at 10 days of culture. The gene expression of bone related molecules did not present significant differences at 7 and 14 days among different metals and surface treatments.ConclusionTi15-Mo seems to be an alternative alloy to cpTi for dental implants. Surface treatment by laser irradiation followed by phosphate deposition seems to positively interact with bone cells.Clinical relevanceTi-15Mo alloy surface modified by laser-beam irradiation followed by calcium phosphate deposition may improve and accelerate the osseointegration process of dental implants.

Effects of maternal consumption of morphine on rat skeletal system development

BackgroundOpioid abuse is among the most ubiquitous issues world-wide, and when it happens in mothers, it puts them at risk of diseases that can be transferred to the next generation. Previous studies have indicated that morphine addiction during pregnancy could inhibit development in rat embryos and infants.The present study focused on the effects of maternal consumption of morphine on rat skeletal system development and also investigate the molecular pathway of chondrogenesis and osteogenesis of infants from control and addicted rat groups.MethodsThirty-two female rats were randomly assigned to four groups. The groups consisted of one- and seven-day-old female infants which were born of morphine-dependent mothers and a control group for each of them. Experimental groups received oral morphine at the final dose of 0.4 mg/ml/day. Withdrawal signs were confirmation of morphine dependency. Female rats were crossed with male rats and coupling time was recorded. Fixed bones of all groups were processed and then stained by hematoxyline-eosin method. Thickness and cell number of proximal and distal growth plate of bones were measured. The cartilage and bone cells were stained by alcian blue/alizarin red method. Additionally, the gene expression of alkaline phosphatase, osteocalcin, and COLL2 and SOX9 gene expression were studied immuno-histochemically.ResultsUnfavorable effects of morphine on histological measurements were observed in one-day and seven-day infants, with more effects on seven-day infants. The thickness and cell number of the proximal and distal growth plate of morphine-dependent rat offsprings were reduced significantly. Furthermore, morphine reduced growth of primary and secondary ossification centers, and thus, longitudinal bone growth was reduced. Moreover, a decrease in the alkaline phosphatase, osteocalcin, COLL2 and SOX9 gene expression, and the number of stained cells was observed. More adverse effects of morphine in seven-day infants compared to one-day infants which showed the time dependent of morphine to the time length of administration.ConclusionHistochemistry and immunohistochemistry findings on cartilage and bone matrix formation, as well as protein expression of chondrogenic and osteogenic markers suggest that morphine dependence in pregnant mothers may impair intra-cartilaginous osteogenesis in post-natal rats.

The additive effect of iloprost on the biological properties of Mineral trioxide aggregate on mesenchymal stem cells

Background/purposeIloprost has been proposed as a potential biomaterial owing to angiogenic and odontogenic properties. However, the liquid form can limit its use during clinical applications. Mineral trioxide aggregate (MTA) has been used for various dental applications in which cell–material interaction is essential. This study aimed to investigate additive effects of iloprost on the biological properties of MTA on the viability, attachment, migration and differentiation of human mesenchymal stem cells (hMSCs). Materials and methodsStandardized human dentin disks were prepared. MTA was prepared by mixing distilled water or iloprost solution, and the lumen of the disks was filled with MTA or MTA-iloprost. hMSCs on disk alone and hMSCs on culture plates were used as controls. Cell viability and attachment were measured after 1, 7 and 14 days using AlamarBlue assay and scanning electron microscopy (SEM). Cell migration in MTA or MTA-iloprost extracts was determined using a wound-healing model.Osteogenic differentiation was evaluated by real-time reverse transcriptase polymerase chain reaction for alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), and osteopontin (OSP) gene expressions after 7 and 14 days of osteogenic induction. ResultsCells on MTA-iloprost surface showed similar viability with MTA at 1 and 14 days but enhanced cellular viability and cell spreading compared to MTA at 7 days (p < 0.05). Cell migration was similar by MTA-iloprost and MTA extracts (p > 0.05). MTAiloprost significantly upregulated BSP, OCN and OSP expressions compared to MTA (p < 0.05). ConclusionThe addition of iloprost to MTA improved the initial cell viability and osteogenic potential of hMSCs.

Tetracalcium Phosphate/Monetite/Calcium Sulfate Hemihdrate Biocement Powder Mixtures Prepared by the One-Step Synthesis for Preparation of Nanocrystalline Hydroxyapatite Biocement-Properties and In Vitro Evaluation.

A modified one-step process was used to prepare tetracalcium phosphate/monetite/calcium sulfate hemihydrate powder cement mixtures (CAS). The procedure allowed the formation of monetite and calcium sulfate hemihydrate (CSH) in the form of nanoparticles. It was hypothesized that the presence of nanoCSH in small amounts enhances the in vitro bioactivity of CAS cement in relation to osteogenic gene markers in mesenchymal stem cells (MSCs). The CAS powder mixtures with 15 and 5 wt.% CSH were prepared by milling powder tetracalcium phosphate in an ethanolic solution of both orthophosphoric and sulfuric acids. The CAS cements had short setting times (around 5 min). The fast setting of the cement samples after the addition of the liquid component (water solution of NaH2PO4) was due to the partial formation of calcium sulfate dihydrate and hydroxyapatite before soaking in SBF with a small change in the original phase composition in cement powder samples after milling. Nanocrystalline hydroxyapatite biocement was produced by soaking of cement samples after setting in simulated body fluid (SBF). The fast release of calcium ions from CAS5 cement, as well as a small rise in the pH of SBF during soaking, were demonstrated. After soaking in SBF for 7 days, the final product of the cement transformation was nanocrystalline hydroxyapatite. The compressive strength of the cement samples (up to 30 MPa) after soaking in simulated body fluid (SBF) was comparable to that of bone. Real time polymerase chain reaction (RT-PCR) analysis revealed statistically significant higher gene expressions of alkaline phosphatase (ALP), osteonectin (ON) and osteopontin (OP) in cells cultured for 14 days in CAS5 extract compared to CSH-free cement. The addition of a small amount of nanoCSH (5 wt.%) to the tetracalcium phosphate (TTCP)/monetite cement mixture significantly promoted the over expression of osteogenic markers in MSCs. The prepared CAS powder mixture with its enhanced bioactivity can be used for bone defect treatment and has good potential for bone healing.

OSTEOGENIC POTENTIAL OF NANOSTRUCTURED CARBONATED HYDROXIAPATITE MICROSPHERES ASSOCIATED WITH MESENCHYMAL STEM CELL IN VITRO AND IN VIVO

<h3>Background</h3> Bone tissue is capable of self-healing in the event of injuries. However, situations such as the patient‘s age or size of the damage can compromise natural healing, bringing to the fore the need for interventions to treat bone injuries. Tissue engineering presents treatment alternatives involving the use of cells and associated biomaterials. In this context, studies using biomaterials claim that carbonated hydroxyapatite (CHA) is more resorbable than hydroxyapatite (HA) and has biological similarity to bone hydroxyapatite. <h3>Methods</h3> Parameters of indirect contact (cytotoxicity, proliferation, and migration) of the extract obtained from spheres of carbonated hydroxyapatite associated with murine bone marrow stem cells (BMSCs) in vitro were evaluated. Then the osteogenic potential of CHA extract in murine BMSCs in vitro was assessed by quantification of calcium matrix, alkaline phosphatase levels and gene expression Col1A1 and Bglap. Non-critical size bone defects were performed in rat femur. After 14 days of healing, bone blocks were obtained for analysis from treated animals and compared to non-treated animals by histological and histomorphometric evaluation. <h3>Results</h3> The CHA extract showed osteoinductive properties in vitro without the need to add osteogenic inducing medium. The ability to induce osteogenic differentiation in undifferentiated cells was confirmed by increased activity of alkaline phosphatase, formation of mineralized nodules and increased expression of the Col1A1 and Bglap genes after 7 and 14 days of differentiation. All groups presented bone neoformation at the injury site 14 days after the operation. Histomorphometric analysis showed no statistical difference between groups in the parameters of bone formation, biomaterial and connective tissue. <h3>Conclusion</h3> The CHA spheres have in vitro osteoinductive properties and, when loaded with murine BMSCs, have the potential for bone repair and regeneration in vivo, presenting biocompatibility and osteoconductive, making it a promising alternative for bone tissue engineering applications.

3D Bioprinting of Mesenchymal Stem Cells and Endothelial Cells in an Alginate-Gelatin-Based Bioink

Aim: Bioink is one of the essential factors in 3D bioprinting that determines the fate of cells, in our case, umbilical cord-derived mesenchymal stem cells (UMSC). The aim was to determine if the presence of the osteoinductive factors in the bioink enhances osteodifferentiation as compared with adding them postprinting and if the UMSC and endothelial cells (EC) coculture result in better osteodifferentiation. Materials &amp; methods: Alginate-gelatin along with UMSC–EC were bioprinted using an extrusion 3D bioprinter. Results &amp; conclusion: The UMSC–EC interaction, as well as intrinsic addition of the differentiation components in the bioink, were observed to play a vital role in increasing the osteogenic differentiation as shown by the histochemical staining, alkaline phosphatase activity and gene expression of osteogenic markers.

Chondrogenic and BMP-4 primings confer osteogenesis potential to human cord blood mesenchymal stromal cells delivered with biphasic calcium phosphate ceramics

Bone marrow mesenchymal stem/stromal cells (BMSCs) show great promise for bone repair, however they are isolated by an invasive bone marrow harvest and their regenerative potential decreases with age. Conversely, cord blood can be collected non-invasively after birth and contains MSCs (CBMSCs) that can be stored for future use. However, whether CBMSCs can replace BMSCs targeting bone repair is unknown. This study evaluates the in vitro osteogenic potential of unprimed, osteogenically primed, or chondrogenically primed CBMSCs and BMSCs and their in vivo bone forming capacity following ectopic implantation on biphasic calcium phosphate ceramics in nude mice. In vitro, alkaline phosphatase (intracellular, extracellular, and gene expression), and secretion of osteogenic cytokines (osteoprotegerin and osteocalcin) was significantly higher in BMSCs compared with CBMSCs, while CBMSCs demonstrated superior chondrogenic differentiation and secretion of interleukins IL-6 and IL-8. BMSCs yielded significantly more cell engraftment and ectopic bone formation compared to CBMSCs. However, priming of CBMSCs with either chondrogenic or BMP-4 supplements led to bone formation by CBMSCs. This study is the first direct quantification of the bone forming abilities of BMSCs and CBMSCs in vivo and, while revealing the innate superiority of BMSCs for bone repair, it provides avenues to induce osteogenesis by CBMSCs.