Alkaline Phosphatase Gene Expression Research Articles

Using Integrated Multi-Omics to Explore the Differences in the Three Developmental Stages of Thelephora ganbajun Zang

Thelephora ganbajun Zang, a rare wild macrofungus, has significant culinary and medicinal value. However, it also has a high cost attributed to its inability to achieve artificial cultivation and its strict environmental requirements. To reveal the intricacies of its development, we conducted a comprehensive analysis of the proteome and metabolome in three pivotal developmental stages: the mycelium, the primordium, and the fruiting body. In our investigation, genes exhibiting various expression levels across multi-omics analyses were identified as potential candidates implicated in growth, development, or metabolic regulation. The aim of this study was to provide a clearer direction for understanding the fundamental metabolic activities and growth stages of this species. Label-free proteomic sequencing revealed a critical juncture in ectomycorrhiza formation, particularly during the transition from the mycelium to the primordium. Secreted proteins, signaling proteins, membrane proteins, and proteins with unidentified functions were rapidly synthesized, with certain amino acids contributing to the synthesis of proteins involved in signaling pathways or hormone precursor substances. In the metabolomics analysis, the classification of secondary metabolites revealed a noteworthy increase in lipid substances and organic acids, contributing to cell activity. The early mycelial development stage exhibited vigorous cell metabolism, contrasting with a decline in cell division activity during fruiting body formation. In our findings, the integration of metabolomic and transcriptomic data highlighted the potential key role of folate biosynthesis in regulating early ectomycorrhiza development. Notably, the expression of alkaline phosphatase and dihydrofolate synthase genes within this pathway was significantly up-regulated in the mycelium and fruiting body stages but down-regulated in the primordium stage. This regulation primarily influences dihydrofolate reductase activity and B vitamin synthesis.

Magnetic colloidal nanoformulations to remotely trigger mechanotransduction for osteogenic differentiation

Nowadays, diseases associated with an ageing population, such as osteoporosis, require the development of new biomedical approaches to bone regeneration. In this regard, mechanotransduction has emerged as a discipline within the field of bone tissue engineering. Herein, we have tested the efficacy of superparamagnetic iron oxide nanoparticles (SPIONs), obtained by the thermal decomposition method, with an average size of 13 nm, when exposed to the application of an external magnetic field for mechanotransduction in human bone marrow-derived mesenchymal stem cells (hBM-MSCs). The SPIONs were functionalized with an Arg-Gly-Asp (RGD) peptide as ligand to target integrin receptors on cell membrane and used in colloidal state. Then, a comprehensive and comparative bioanalytical characterization of non-targeted versus targeted SPIONs was performed in terms of biocompatibility, cell uptake pathways and mechanotransduction effect, demonstrating the osteogenic differentiation of hBM-MSCs. A key conclusion derived from this research is that when the magnetic stimulus is applied in the first 30 min of the in vitro assay, i.e., when the nanoparticles come into contact with the cell membrane surface to initiate endocytic pathways, a successful mechanotransduction effect is observed. Thus, under the application of a magnetic field, there was a significant increase in runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) gene expression as well as ALP activity, when cells were exposed to RGD-functionalized SPIONs, demonstrating osteogenic differentiation. These findings open new expectations for the use of remotely activated mechanotransduction using targeted magnetic colloidal nanoformulations for osteogenic differentiation by drug-free cell therapy using minimally invasive techniques in cases of bone loss.

P035 3D bioprinting of gelatin derivatives: towards novel small intestinal in vitro models

Abstract Background Despite significant progress achieved in the field of inflammatory bowel disease research, the precise cause of the disease has yet to be elucidated. Intestinal in vitro models can provide a fast and inexpensive alternative for in vivo studies. Nonetheless, many in vitro models lack the crypt or crypt-villus architecture as seen in the large and small intestine respectively and therefore fail to mimic the complexity of the gut. In this study, we aim to improve the physiology of small intestinal in vitro models by the development of hydrogels that mirror the villi and crypts of the digestive tract. Methods This study focused on the development of gelatin-methacryloyl-aminoethyl-methacrylate (gel-MA-AEMA)-, and gelatin-methacryloyl-norbornene (gel-MA-NB)-based biomaterial inks to fabricate 3D constructs, mimicking villi or a combination of villi and crypts, with digital light processing. The constructs were mechanically and morphologically evaluated with parallel plate rheology and cryo-scanning electron microscopy respectively. To assess the biocompatibility of the 3D constructs, a Caco-2/HT29-MTX co-culture in a 9:1 ratio was maintained for 21 days and confluency was confirmed with immunofluorescence. Gene expression, transepithelial electrical resistance (TEER) and paracellular permeability of the cells cultured on the constructs were compared to cells cultured on flat gel-MA-AEMA and gel-MA-NB hydrogels, a collagen type I coating or uncoated tissue culture plastic. Results Both gel-MA-AEMA and gel-MA-NB hydrogels exhibited physiologically relevant stiffness (1.9 ± 0.63 kPa and 1.64 ± 0.63 kPa respectively), but only the gel-MA-AEMA based biomaterial ink could be successfully utilized for printing constructs with villi and crypts. On all construct designs, confluency was reached and paracellular permeability of small sized marker molecules in combination with TEER measurements suggested the formation of a functional barrier over time, which was further confirmed by immunofluorescence and increased gene expression of tight junction proteins, occludin and ZO-1. The gene expression of enterocyte differentiation markers, villin-1, sucrase isomaltase and alkaline phosphatase, suggested the superior differentiation of Caco-2 cells on the ‘only villi’ and ‘villi and crypts’ constructs compared to flat hydrogels, collagen type I coating or uncoated tissue culture plastic. Conclusion Although both hydrogels promoted functional barrier formation and enterocyte differentiation, gel-MA-AEMA was more suited for DLP than gel-MA-NB. In addition, culturing intestinal epithelial cells on the 3D constructs ameliorated cell differentiation compared to conventional 2D setups.

Current Evidence on the Effects of Endocrine-Disrupting Chemicals (EDCs) on Bone Growth and Health

AbstractEndocrine-disrupting chemicals (EDCs) are a heterogeneous group of natural and man-made chemicals from environmental sources that mimic natural hormones. They can have adverse effects on the morphology, physiology, growth, and development of different organs and systems, among these bone health can be affected too. EDCs work as agonists or antagonists on hormonal receptors in hormone-sensitive cells, influence gene expression by epigenetic mechanisms, stimulate or inhibit cell maturation, and affect the synthesis and metabolism of hormones. This review aims to summarize current evidence on the effects of exposure to EDCs on bone from early gestational to birth and long-term adverse effects. Single and mixtures of endocrine-disrupting chemicals can disrupt bone structure by modifying differentiation, increasing osteoclast activity, inhibiting pre-osteoblasts differentiation into mature osteoblasts and osteocytes, inducing changes in signaling pathways downstream of receptors, and ultimately remodeling and modifying the equilibrium between bone resorption and formation leading to increased bone resorption, morphological, and functional changes in bone maturation. EDCs can affect the IGF system, alkaline phosphatase, and osteocalcin gene expression. Findings are relative to both in vitro and in vivo studies. Studies have shown that prenatal exposure to EDCs leads to growth retardation, delayed ossification, and changes in bone length and size and in bone geometry with a lowering of bone mineral density and area-adjusted bone mineral content. Current knowledge on bone health, growth, mineral content, and development from molecular to clinical findings highlights how endocrine-disrupting chemicals can negatively affect these processes. Mechanisms, however, are not fully understood and need further investigation.

Functionalization of poly (lactic-co-glycolic acid) nano‑calcium sulphate and fucoidan 3D scaffold using human bone marrow mesenchymal stromal cells for bone tissue engineering application

This study aimed to functionalize a novel porous PLGA (Poly lactic-co-glycolic acid) composite scaffold in combination with nano‑calcium sulphate (nCS) and/or fucoidan (FU) to induce osteogenic differentiation of human bone marrow stromal cells. The composite scaffolds (PLGA-nCS-FU, PLGA-nCS or PLGA-FU) were fabricated and subjected to characterization using Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), Scanning electron microscopy (SEM) and Energy Dispersive X-Ray (EDX). The biocompatibility and osteogenic induction potential of scaffolds on seeded human bone marrow derived mesenchymal stromal cells (hBMSCs) were studied using cell attachment and alamar blue cell viability and alkaline phosphatase (ALP), osteocalcin and osteogenic gene expression, respectively. The composition of different groups was reflected in FTIR, XRD and EDX. The SEM micrographs revealed a difference in the surface of the scaffold before and after FU addition. The confocal imaging and SEM micrographs confirmed the attachment of cells onto all three composite scaffolds. However, the AB assay indicated a significant increase (p < 0.05) in cell viability/proliferation seeded on PLGA-nCS-FU on day 21 and 28 as compared with other combinations. A 2-fold significant increase (p < 0.05) in ALP and OC secretion of seeded hBMSCs onto PLGA-nCS-FU was observed when compared with other combinations. A significant increase in RUNX2, OPN, COL-I and ALP genes were observed in the cells seeded on PLGA-nCS-FU on day 14 and 28 as compared with day 0. In conclusion, the incorporation of both Fucoidan and Nano‑calcium sulphate with PLGA showed a promising improvement in the osteogenic potential of hBMSCs. Therefore, PLGA-nCS-FU could be the ideal candidate for subsequent pre-clinical studies to develop a successful bone substitute to repair critical bone defects.

Superoxide dismutase 2 scavenges ROS to promote osteogenic differentiation of human periodontal ligament stem cells by regulating Smad3 in alveolar bone-defective rats.

Osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is an essential event in alveolar bone regeneration. Oxidative stress may be the main inhibiting factor of hPDLSC osteogenesis. Superoxide dismutase 2 (SOD2) is a key antioxidant enzyme, but its effect on hPDLSC osteogenic differentiation is unclear. Several surface markers were detected by flow cytometry, and the differentiation potential of hPDLSCs was validated by alkaline phosphatase (ALP), Alizarin Red S, and Oil Red O staining. Osteogenic indicators of hPDLSCs were detected by real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and ALP staining. Furthermore, alveolar bone defect rat models were analyzed through micro-CT, hematoxylin and eosin, and Masson staining. The intracellular reactive oxygen species (ROS) level was evaluated by a ROS assay kit. Finally, the expression of SOD2, Smad3, and p-Smad3 in hPDLSCs was detected by RT-qPCR and Western blotting (WB). SOD2 positively regulated the gene and protein expressions of ALP, BMP6, and RUNX2 in hPDLSCs (p<0.05). Ideal bone formation and continuous cortical bone were obtained by transplanting LV-SOD2 hPDLSCs (lentivirus vector for overexpressing SOD2 in hPDLSCs) in vivo. Exogenous H2 O2 downregulated osteogenic indicators (ALP, BMP6, RUNX2) in hPDLSCs (p<0.05); this was reversed by overexpression of SOD2. WB results showed that the Smad3 and p-Smad3 signaling pathways participated in the osteogenic process of SOD2 in hPDLSCs. SOD2 positively regulated hPDLSC osteogenic differentiation in vitro and in vivo. Mechanistically, SOD2 promotes hPDLSC osteogenic differentiation by regulating the phosphorylation of Smad3 to scavenge ROS. This work provides a theoretical basis for the treatment of alveolar bone regeneration.

Synergistic effect of FGF-2 and TGF-β1 on the mineralization of human umbilical cord perivascular cells

ObjectiveHuman umbilical cord perivascular cells (HUCPVCs) are derived from the human umbilical cord perivascular tissue and are expected to replace mesenchymal stromal cells in the future. We investigated the synergistic effects of fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 1 (TGF-β1) on HUCPVC mineralization. DesignWe prepared HUCPVCs with (FGF(+)HUCPVCs) or without FGF-2 (FGF(-)HUCPVCs) in the presence of activated vitamin D3, a bone morphogenic protein inhibitor, and TGF-β1. We examined the cell proliferative capacity, expression of various hard tissue-forming cell gene markers, and mineralization induction ability and identified the crystalline phases of the mineralized nodules. ResultsFGF(+)HUCPVCs exhibited higher intracellular alkaline phosphatase (ALP) gene expression and ALP activity, and their cell proliferation rate was higher than that of FGF(-)HUCPVCs. The expression levels of osteoblast marker genes increased in FGF(+)HUCPVCs, whereas those of elastic fiber and muscle cell markers increased in FGF(-)HUCPVCs. The expression of genes related to matrix vesicle-mediated mineralization was increased in FGF(+)HUCPVCs. While FGF(-)HUCPVCs displayed myofibroblast-like properties and could not induce mineralization, FGF(+)HUCPVCs demonstrated the ability to produce mineralized nodules. The resulting mineralized nodules consisted of hydroxyapatite as the major phase and minor amounts of octacalcium phosphate. The mineralized nodules exhibited the morphological characteristics of bone hydroxyapatite, composed of fibrous hydroxyapatite nanorods and polycrystalline sheets. ConclusionWe found that FGF-2 synergizes with TGF-β1 and is a key factor in the differentiation of HUCPVCs into osteoblast-like cells. Thus, HUCPVCs can potentially serve as a new stem cell source for future bone regeneration and dental treatments.

Mesoporous Bioactive Glass-Incorporated Injectable Strontium-Containing Calcium Phosphate Cement Enhanced Osteoconductivity in a Critical-Sized Metaphyseal Defect in Osteoporotic Rats.

In this study, the in vitro and in vivo bone formation behavior of mesoporous bioactive glass (MBG) particles incorporated in a pasty strontium-containing calcium phosphate bone cement (pS100G10) was studied in a metaphyseal fracture-defect model in ovariectomized rats and compared to a plain pasty strontium-containing calcium phosphate bone cement (pS100) and control (empty defect) group, respectively. In vitro testing showed good cytocompatibility on human preosteoblasts and ongoing dissolution of the MBG component. Neither the released strontium nor the BMG particles from the pS100G10 had a negative influence on cell viability. Forty-five female Sprague-Dawley rats were randomly assigned to three different treatment groups: (1) pS100 (n = 15), (2) pS100G10 (n = 15), and (3) empty defect (n = 15). Twelve weeks after bilateral ovariectomy and multi-deficient diet, a 4 mm wedge-shaped fracture-defect was created at the metaphyseal area of the left femur in all animals. The originated fracture-defect was substituted with pS100 or pS100G10 or left empty. After six weeks, histomorphometrical analysis revealed a statistically significant higher bone volume/tissue volume ratio in the pS100G10 group compared to the pS100 (p = 0.03) and empty defect groups (p = 0.0001), indicating enhanced osteoconductivity with the incorporation of MBG. Immunohistochemistry revealed a significant decrease in the RANKL/OPG ratio for pS100 (p = 0.004) and pS100G10 (p = 0.003) compared to the empty defect group. pS100G10 showed a statistically higher expression of BMP-2. In addition, a statistically significant higher gene expression of alkaline phosphatase, osteoprotegerin, collagen1a1, collagen10a1 with a simultaneous decrease in RANKL, and carbonic anhydrase was seen in the pS100 and pS100G10 groups compared to the empty defect group. Mass spectrometric imaging by time-of-flight secondary ion mass spectrometry (ToF-SIMS) showed the release of Sr2+ ions from both pS100 and pS100G10, with a gradient into the interface region. ToF-SIMS imaging also revealed that resorption of the MBG particles allowed for new bone formation in cement pores. In summary, the current work shows better bone formation of the injectable pasty strontium-containing calcium phosphate bone cement with incorporated mesoporous bioactive glass compared to the bioactive-free bone cement and empty defects and can be considered for clinical application for osteopenic fracture defects in the future.

Effect of doxycycline doped nanoparticles on osteogenic/cementogenic and anti-inflammatory responses of human cells derived from the periodontal ligament

ObjectivesThis work aimed to evaluate if doxycycline-doped polymeric nanoparticles possessed any anti-inflammatory effect and promote osteogenic/cementogenic differentiation of stem cells from human periodontal ligament (PDLSCs). MethodsThe polymeric nanoparticles (NPs) were produced by a polymerization/precipitation process and doped with doxycycline (Dox-NPs). PDLSCs were cultured in the presence or absence of the NPs under osteogenic medium or IL-1β treatment. Cells’ differentiation was assessed by gene expression analysis of osteogenic/cementogenic markers alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2). An anti-inflammatory effect was also ascertained by analyzing IL-1β gene expression. Adipogenic and chondrogenic differentiation was used to confirm the multipotency of PDLSCs. ResultsGene expression of ALP and RUNX2 in PDLSCs was significantly upregulated by the osteogenic medium (ALP: p<0.001; RUNX2: p = 0.005) while Dox-NPs further enhanced ALP gene expression of PDLSCs treated with the osteogenic medium. Furthermore, Dox-NPs suppressed the up-regulation of IL-1β when cells were subjected to an inflammatory challenge. ConclusionsDox-NPs enhanced PDLSCs differentiation into osteoblasts/cementoblasts lineages while providing an anti-inflammatory effect. Clinical significanceDue to their biocompatibility as well as anti-inflammatory and osteogenic/cementogenic effects, Dox-NPs are potential candidates for being used in periodontal regeneration.

Sortilin Is Upregulated in Osteoarthritis-Dependent Cartilage Calcification and Associated with Cellular Senescence.

Osteoarthritis (OA) is a chronic joint disease characterized by articular cartilage calcification, loss of articular cartilage, bone changes, pain, and disability. Cartilage calcification is one hallmark of OA and is predominantly caused by basic calcium crystals formed due to an imbalance of the pyrophosphate pathway. Sortilin is a transmembrane protein that contributes to vascular calcification in atherosclerosis by externalizing alkaline phosphatase (ALP)-containing vesicles. Calcification in atherosclerosis and osteoarthritis has been associated with cellular senescence. The aim of this study was to investigate the potential role of sortilin and senescence in osteoarthritis-dependent cartilage calcification. Osteoarthritic cartilage from human knee joints was collected after joint replacement, and samples were analyzed by immunohistochemistry and quantitative RT-PCR analysis. Human chondrocytes were treated with osteogenic medium for up to 21 days to induce calcification. Western blots for sortilin and ALP, as well as an ALP activity assay, were performed. Human chondrocytes were treated with mitomycin C to induce senescence, and sortilin expression was quantified at the protein and gene levels. Sections of knee joints from a murine model of osteoarthritis were stained for sortilin and p16 and analyzed by immunohistochemistry. Treatment of wild-type chondrocytes using an osteogenic medium similar to human chondrocytes was performed. Osteoarthritic cartilage from mouse and human knee joints showed an increased number of sortilin and p16-positive chondrocytes compared to healthy cartilage. This observation was corroborated by increased gene expression of sortilin and p16 in mild and moderate osteoarthritic cartilage samples. To investigate the mechanism of sortilin regulation, human chondrocytes were treated with osteogenic medium to induce calcification. Sortilin protein levels and expression were increased after 7 days of stimulation, whereas ALP levels and activity were upregulated after 21 days of stimulation. Similar observations were made in a murine osteoarthritis model. Mechanistically, senescent chondrocytes induced by mitomycin C showed an upregulation of sortilin and ALP gene expression compared to non-senescent chondrocytes. Our data indicate that sortilin and ALP are upregulated during cartilage calcification, which is associated with chondrocyte senescence and thus might contribute to the pathogenesis of osteoarthritis. Cellular senescence seems to induce sortilin expression.

Preparation of co-electrospinning membrane loaded with simvastatin and substance P to accelerate bone regeneration by promoting cell homing, angiogenesis and osteogenesis.

Bone regeneration is a complex process that requires the coordination of various biological events. Developing a tissue regeneration membrane that can regulate this cascade of events is challenging. In this study, we aimed to fabricate a membrane that can enrich the damaged area with mesenchymal stem cells, improve angiogenesis, and continuously induce osteogenesis. Our approach involved creating a hierarchical polycaprolactone/gelatin (PCL/GEL) co-electrospinning membrane that incorporated substance P (SP)-loaded GEL fibers and simvastatin (SIM)-loaded PCL fibers. The membrane could initiate a burst release of SP and a slow/sustained release of SIM for over a month. In vitro experiments, including those related to angiogenesis and osteogenesis (e.g., migration, endothelial network formation, alkaline phosphatase activity, mineralization, and gene expression), clearly demonstrated the membrane's superior ability to improve cell homing, revascularization, and osteogenic differentiation. Furthermore, a series of in vivo studies, including immunofluorescence of CD29+/CD90+ double-positive cells and immunohistochemical staining for CD34 and vWF, confirmed the co-electrospinning membrane's ability to enhance MSC migration and revascularization response after five days of implantation. After one month, the Micro-CT and histological (Masson and H&E) results showed accelerated bone regeneration. Our findings suggest that a co-electrospinning membrane with time-tunable drug delivery could advance the development of tissue engineering therapeutic strategies and potentially improve patient outcomes.

Sema4D is diminished in leukocyte platelet-rich fibrin and impairs pre-osteoblastic MC3T3-E1 cells' functionality.

ObjectiveSemaphorin 4D (Sema4D) is a coupling factor expressed on osteoclasts that may hinder osteoblast differentiation. Since the leukocyte platelet-rich fibrin (L-PRF) membrane promotes growth factor concentration, this study aims to quantify the amount of Sema4D in L-PRF membranes, and analyze the impact of Sema4D on osteoblast cell function in vitro. DesignEnzyme-linked immunosorbent assay (ELISA) was used to quantify the levels of Sema4D in both L-PRF and whole blood (serum). To analyze the impairment of Sema4D on osteoblasts, MC3T3-E1 cells were induced to osteogenic differentiation and exposed to Sema4D ranging from 10 to 500 ng/ml concentrations. The following parameters were assayed: 1) cell viability by MTT assay after 24, 48, and 72 h; 2) matrix mineralization by Alizarin Red staining after 14 days, 3) Runt-related transcription factor 2 (RUNX-2), osteocalcin (OCN), osteonectin (ONC), bone sialoprotein (BSP) and alkaline phosphatase (ALP) gene expression by qPCR. For all data, the significance level was set at 5%. ResultsThe amount of Sema4D in the whole blood (serum) was higher than in L-PRF. Osteoblasts exposed to Sema4D at all tested concentrations exhibited a decrease in matrix mineralization formation as well in RUNX-2, OCN, ONC, BSP, and ALP gene expression (p < 0.05). ConclusionThe presence of Sema4D, a molecule known for suppressing osteoblast activity, diminishes within L-PRF, enhancing its ability to facilitate bone regeneration.

Effect of amoxicillin and clindamycin on the gene expression of markers involved in osteoblast physiology

Background/purposeAmoxicillin and clindamycin are the most effective decontaminants for intraoral bone grafts before their application in bone regeneration without cytotoxic effects on osteoblasts, but their effects on the gene expression of markers involved in osteoblast growth and differentiation remain unclear. The study objective was to determine the effects of amoxicillin and clindamycin on the gene expression of markers involved in osteoblast growth and differentiation. Materials and methodsReal-time polymerase chain reaction (RT-PCR) was performed to explore the effect of 150 μg/mL clindamycin or 400 μg/mL amoxicillin on the gene expression by primary human osteoblasts (HOBs) of runt-related transcription factor 2 (Runx-2), osterix (OSX), alkaline phosphatase (ALP), osteocalcin (OSC), osteoprotegerin (OPG), receptor activator for nuclear factor κ B ligand (RANKL), type I collagen (Col-I), bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), TGF-β1 and TGF-β receptors (TGF-βR1, TGF-βR2, and TGF-βR3), and vascular endothelial growth factor (VEGF). ResultsTreatment with 150 μg/mL clindamycin significantly increased the gene expression of TFG-β1, TGF-βR1, TGF-βR2, TGF-βR3, RUNX-2, Col-1, OSX, OSC, BMP-2, BMP-7, ALP, VEGF, and RANKL by HOBs. Treatment with 400 μg/mL amoxicillin significantly increased the gene expression of TGF-β R1, Col-I, OSC, RANKL, and OPG alone. ConclusionThese findings suggest that 150 μg/mL clindamycin is the decontaminant of choice to treat intraoral bone grafts before their application in bone regeneration. The osteogenic and antibacterial properties of clindamycin can favor and accelerate the integration of bone grafts in the oral cavity.

Effect of insulin on bone formation ability of rat alveolar bone marrow mesenchymal stem cells.

The alveolar bone marrow mesenchymal stem cells (ABM-MSCs) play an important role in oral bone healing and regeneration. Insulin is considered to improve impaired oral bones due to local factors, systemic factors and pathological conditions. However, the effect of insulin on bone formation ability of ABM-MSCs still needs to be elucidated. The aim of this study was to determine the responsiveness of rat ABM-MSCs to insulin and to explore the underlying mechanism. We found that insulin promoted ABM-MSCs proliferation in a concentration-dependent manner, in which 10-6 M insulin exerted the most significant effect. 10-6 M insulin significantly promoted the type I collagen (COL-1) synthesis, alkaline phosphatase (ALP) activity, osteocalcin (OCN) expression and mineralized matrix formation in ABM-MSCs, significantly enhanced the gene and protein expressions of intracellular COL-1, ALP and OCN. Acute insulin stimulation significantly promoted insulin receptor (IR) phosphorylation, insulin receptor substrate-1 (IRS-1) protein expression and mammalian target of rapamycin (mTOR) phosphorylation, but chronic insulin stimulation decreased these values, while inhibitor NT219 could attenuate these responses. When seeded on β-tricalcium phosphate (β-TCP), ABM-MSCs adhered and grew well, during the 28-day culture period, ABM-MSCs+β-TCP+10-6 M Insulin group showed significantly higher extracellular type I collagen amino-terminus prolongation peptide (TPINP) content, ALP activity, OCN secretion, Ca and P concentration. When implanted subcutaneously in severe combined immunodeficient mice for 1 month, the ABM-MSCs+β-TCP+10-6 M Insulin group obtained the most bone formation and blood vessels. These results showed that insulin promoted the proliferation and osteogenic differentiation of ABM-MSCs in vitro, and enhance osteogenesis and angiogenesis of ABM-MSCs in vivo. Inhibition studies demonstrated that the insulin-induced osteogenic differentiation of ABM-MSCs was dependent of insulin/mTOR signaling. It suggests that insulin has a direct anabolic effect on ABM-MSCs.

Preparation, characterization and biocompatibility of calcium peroxide-loaded polycaprolactone microparticles.

To explore the physicochemical characteristics and biocompatibility of calcium peroxide (CPO)-loaded polycaprolactone (PCL) microparticle. The CPO/PCL particles were prepared. The morphology and elemental distribution of CPO, PCL and CPO/PCL particles were observed with scanning electron microscopy and energy dispersive spectroscopy, respectively. Rat adipose mesenchymal stem cells were isolated and treated with different concentrations (0.10%, 0.25%, 0.50%, 1.00%) of CPO or CPO/PCL particles. The mesenchymal stem cells were cultured in normal media or osteogenic differentiation media under the hypoxia/normoxia conditions, and the amount of released O2 and H2O2 after CPO/PCL treatment were detected. The gene expressions of alkaline phosphatase (ALP), Runt-associated transcription factor 2 (RUNX2), osteopontin (OPN) and osteocalcin (OCN) were detected by realtime RT-PCR. SD rats were subcutaneously injected with 1.00% CPO/PCL particles and the pathological changes and infiltration of immune cells were observed with HE staining and immunohistochemistry at day 7 and day 14 after injection. Scanning electron microscope showed that CPO particles had a polygonal structure, PCL particles were in a small spherical plastic particle state, and CPO/PCL particles had a block-like crystal structure. Energy dispersive spectroscopy revealed that PCL particles showed no calcium mapping, while CPO/PCL particles showed obvious and uniform calcium mapping. The concentrations of O2 and H2O2 released by CPO/PCL particles were lower than those of CPO group, and the oxygen release time was longer. The expressions of Alp, Runx2, Ocn and Opn increased with the higher content of CPO/PCL particles under hypoxia in osteogenic differentiation culture and normal culture, and the induction was more obvious under osteogenic differentiation conditions (all P<0.05). HE staining results showed that the muscle tissue fibers around the injection site were scattered and disorderly distributed, with varying sizes and thicknesses at day 7 after particle injection. Significant vascular congestion, widened gaps, mild interstitial congestion, local edema, inflammatory cell infiltration, and large area vacuolization were observed in some tissues of rats. At day 14 after microparticle injection, the muscle tissue around the injection site and the tissue fibers at the microparticle implantation site were arranged neatly, and the gap size was not thickened, the vascular congestion, local inflammatory cell infiltration, and vacuolization were significantly improved compared with those at day 7. The immunohistochemical staining results showed that the expressions of CD3 and CD68 positive cells significantly increased in the surrounding muscle tissue, and were densely distributed in a large area at day 7 after particle injection. At day 14 of microparticle injection, the numbers of CD3 and CD68 positive cells in peripheral muscle tissue and tissue at the site of particle implantation were lower than those at day 7 (all P<0.01). CPO/PCL particles have good oxygen release activity, low damage to tissue, and excellent biocompatibility.

Relaxin treatment stimulates the differentiation of mesenchymal stem cells into osteoblasts

Background/purposeSeveral studies have determined that relaxin stimulates differentiation and regulates the activity of mature osteoclasts, but little is known about its effect on the differentiation of mesenchymal cells towards the osteogenic lineage. Therefore, this study aimed to determine the effect of relaxin on the proliferation and differentiation of the osteoblastic lineage of mesenchymal cells derived from human dental pulp (hDPSC). Materials and methodsIn this in vitro study, hDPSC were characterized and treated with relaxin at different doses (10–80 ng/ml) and times (1–21 days). Morphology was assessed by microscopy, and proliferation was assessed using a resazurin assay. Osteoblastic differentiation was evaluated by Alizarin Red staining, alkaline phosphatase (ALP) labeling, and changes in the expression of the osteoblastic differentiation genes RUNX2 and BMP2. ResultsRelaxin treatment did not induce changes in the proliferation or viability of hDPSCs; however, larger cells and increased cytoplasmic prolongation were observed. Relaxin treatment (20 and 80 ng/ml) significantly increased calcified nodule formation on days 14 and 21. The cytochemical signals for ALP, RUNX2, and BMP2 gene expression were significantly (P < 0.05) increased by the relaxin treatment. ConclusionRelaxin treatment does not induce changes in hDPSC proliferation but induces morphological changes, increases ALP detection, calcified nodule formation, and increases expression of RUNX2 and BMP2, suggesting the induction of osteoblastic differentiation of hDPSC.

Enzyme-Responsive Zr-Based Metal-Organic Frameworks for Controlled Drug Delivery: Taking Advantage of Clickable PEG-Phosphate Ligands.

We report for the first time the controlled drug release from a nanoscale Zr-based metal-organic framework (MOF), UiO-66, in the presence of the enzyme alkaline phosphatase (ALP). This unprecedented reactivity was possible thanks to the prior functionalization of the MOF with N3-PEG-PO3 ligands, which were designed for three specific aims: (1) to impart colloidal stability in phosphate-containing media; (2) to endow the MOF with multifunctionality thanks to azide groups for the covalent attachment of an imaging agent by click-chemistry; and (3) to confer stimuli-responsive properties, specifically the selective release of doxorubicin triggered by the enzymatic activity of ALP. Cell studies revealed that the functionalization of the MOF with N3-(PEG)20-PO3 ligands improved their intracellular stability and led to a sustained drug release compared to the bare MOF. More importantly, an enhanced drug release was observed in cells with higher expression of ALP genes (HeLa versus MDA-MB-231 and MCF7), confirming the ALP-responsiveness of the system inside living cells.

In vitro effects of photobiomodulation on cell migration and gene expression of ALP, COL-1, RUNX-2, and osterix in cementoblasts.

The aim of this study was to evaluate the effects of photobiomodulation (PBM) on cell migration and alkaline phosphatase (ALP), type I collagen (Col-1), runt-related transcription factor 2 (RUNX-2), and Osterix (OSX) gene expression in a cementoblast culture (OCCM-30), in a microenvironment mimicking an injury on the cementoblast layer, such as it occurs during root resorption. For this, OCCM-30 cells were cultured in 6-well plates and the following parameters were assayed: (1) migration by scratch assay and ALP, Col-1, Runx2, and Osx by real-time PCR. PBM was performed in two protocols using a LED device emitting light at 660 nm (± 30 nm). OCCM-30 cementoblasts were grown and divided into four groups: (1) negative control; (2) positive control (scratch); (3) scratch + PBM with a total energy of 36 J and energy density 1.6 J/cm2; and (4) scratch + PBM with a total energy of 72 J and energy density of 3.2 J/cm2. Data were statistically analyzed, with the level of significance set at 5%. Cementoblasts migrated from the edge of the scratch toward the center, and the wound closed after 24 h, with the PBM3.2J/cm2 group showing the higher cell migration compared with the other groups at 2 h, 6 h, 8 h, and 13 h (p < 0.05). The control and PBM1.6J/cm2 groups showed similar levels of cell migration, with no significant differences (p > 0.05). PBM3.2J/cm2 group exhibited greater ALP, Col-1, OSX, and RUNX2 in comparison with the other experimental groups (p < 0.05). Similar levels of all genes evaluated were observed between the PBM1.6J/cm2 group and the positive control group (p > 0.05). In conclusion, our findings support the effectiveness of photobiomodulation on cementoblast migration and gene expression, which may contribute to the formation of a new cementum layer.

In vitro investigation of canine periodontal ligament-derived mesenchymal stem cells: A possibility of promising tool for periodontal regeneration.

Recent investigations indicate that canine periodontal ligament-derived stem cells (cPDLSCs) may reveal a reliable strategy for repair of periodontal tissues via cell-based tissue engineering approaches. Due to limited research, this study aimed to demonstrate the phenotypic characterization of cPDLSc in comparison with canine bone marrow-derived mesenchymal stem cells (cBMSCs) in vitro. Mesenchymal stem cells (MSCs) were obtained from PDL and BM of five male adult Mongrel dogs. In vitro isolation and expansion as well as biologic characterization including colony unit formation (CFU), osteogenic and adipogenic differentiation, flow cytometric analysis of CD34 and CD44, and RT-PCR of alkaline phosphatase (ALP), osteocalcin (OCN), periostin (POSTN) and S100A4 were performed. Furthermore, electron microscopy analysis was done to complement the comparative research. CFU assay revealed that colonies of cPDLSCs presented 70% confluency with a more finite lifespan than BM-MSCs, showing a significant increase in cPDLSCs. Both types of MSCs showed osteogenic and adipogenic phenotypic characterized with clusters of mineralized depositions and lipid vacuoles, respectively. Both types of MSCs expressed CD44 with limited expression of CD34. RT-PCR of cPDLSCs revealed that expression of ALP, POSTN, OCN and S100A4 genes were significantly higher than those of BMSCs. In addition, comparison of SEM and revealed that cPDLSCs expressed more extracellular collagen fibers. The current study indicated that cPDLSCs show potency as a novel cellular therapy for periodontal regeneration a large animal model.

Hemostatic Collagen Sponge with High Porosity Promotes the Proliferation and Adhesion of Fibroblasts and Osteoblasts.

The use of biomaterial for tissue repair involves the interaction between materials and cells, and the coagulum formation represents the first step of tissue healing. This process is particularly critical in the oral cavity, where the wounds are immediately subjected to the masticatory mechanical stress, saliva invasion, and bacterial attack. Therefore, the present study aimed to explore the structural features and the biological activities of a hemostatic collagen sponge on human gingival fibroblasts (HGFs) and human oral osteoblasts (HOBs). The microstructure of the collagen sponge was characterized by a scanning electron microscope (SEM) and histological analysis. The porosity was also calculated. To investigate biological activities, HGFs and HOBs were cultured on the collagen sponges, and their adhesion was observed at SEM on the third day, while cell viability was investigated at the third and seventh days by Tetrazolium (MTT) assay. For osteoblasts seeded on collagen sponge the mineralization ability was also evaluated by alkaline phosphatase (ALP) assay at the seventh day, and by Alizarin red staining on the 14th. Furthermore, the gene expression of ALP and osteocalcin (OCN) was investigated after 3, 7 and 14 days. SEM images of the sponge without cells showed a highly porous 3D structure, confirmed by the measurement of porosity that was more than 90%. The samples cultured were characterized by cells uniformly distributed and adhered to the sponge surface. Proliferation ended up being promoted, as well as the mineralization ability of the osteoblasts, mainly at the mature stage. In conclusion, this collagen sponge could have a potential use for tissue healing.