ALP Expression Research Articles

Cytotoxic and Osteoblast Differentiation Induction Properties of Crude Polar Extract of Piper sarmentosum leaves

Piper sarmentosum or ‘kaduk’ is a well-known herb plant in Malaysia. Its extracts were found to exhibit a bone-protective effect against osteoporotic rats. Our study aim is to morphologically observe the effect of P. sarmentosum ethyl acetate extract on the differentiation of human peripheral blood stem cells (PBSCs) into osteoblasts. P. sarmentosum extracts (1-900 µg/mL) prepared using 1% dimethyl sulfoxide (DMSO) were used in the cytotoxicity assay. Then, the differentiation assay was performed using concentrations of 1-50 µg/mL. The untreated cells acted as the negative control, while the cells cultured in 50 µg/mL ascorbic acid and 10 mM β-glycerophosphate was a positive control. The cytotoxicity effect and proliferation capacity of the cells were analyzed using Trypan Blue exclusion method, while the differentiation of PBSCs was observed using von Kossa staining and ALP gene expression analysis. The result showed a decrease in cells’ viability in a dose-dependent manner during cytotoxicity assay. After 14 days of the differentiation, a constant rate of proliferation could be observed in the treated cells and positive control, while the untreated cells showed an increase in proliferation. The mineralization of extract-treated cells showed significant differences (p<0.05) as compared to the negative control. The expression of ALP was also upregulated on day 14 of differentiation compared to day 0. In conclusion, the extract was capable to induce osteoblast differentiation of human peripheral blood stem cell and potential to be used in tissue regeneration

22. The Role of Orai1 in Osteogenic Differentiation Induced by Nanoparticulate Mineralized Collagen Glycosaminoglycan Materials

PURPOSE: Development of synthetic regenerative materials inspired by the extracellular matrix (ECM) has been a topic of significant interest due to the known properties of the ECM to direct cell fate determination via tunable material composition and biomechanical properties. Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) is a low density, open-cell foam that has been evaluated in our laboratory to induce osteogenic differentiation of primary osteoprogenitors in vitro as well as regeneration of rabbit skull defects in vivo without pre-seeding with progenitor cells or addition of exogenous growth factors, which was not observed in non-mineralized collagen glycosaminoglycan (Col-GAG) materials. Given that the critical difference between MC-GAG and Col-GAG is the mineral content, the spatial concentration of inorganic ions on MC-GAG may be critical to its osteogenic properties. This is supported by prior research demonstrating that high calcium concentrations in the bone microenvironment stimulate osteoblastic differentiation. How these biochemical cues are transmitted to cells and activate downstream osteogenic signaling pathways should be further elucidated. Orai1 is a critical subunit of store-operated calcium release-activated calcium channels (CRAC) that has been implicated to be involved in bone homeostasis. In this work, we evaluated the contribution of Orai1 to MC-GAG-mediated osteogenesis. METHODS: To examine the role of extracellular calcium signaling through store-operated calcium channels, such as Orai1, on osteogenic differentiation of primary bone marrow-derived human mesenchymal stem cells (hMSCs), we treated 2D cell cultures with a small molecule inhibitor of store-operated calcium entry (SOCE), MRS1845, for 14 days. hMSCs were subsequently seeded on MC-GAG or Col-GAG for 7 days with or without MRS1845. Baseline Orai1 gene and protein expression was evaluated. Next, small interfering RNAs (siRNAs) targeting ORAI1 and a scrambled control were used to specifically knockdown ORAI1 expression in hMSCs cultured on Col-GAG and MC-GAG. Osteogenic gene and protein expression were measured using quantitative RT-PCR and western blot analyses, respectively. Mineralization was evaluated by Alizarin Red staining. RESULTS: To understand whether store-operated calcium entry was essential to osteogenic differentiation, we treated hMSCs in 2D cultures with a small molecule inhibitor of SOCE, MRS1845, and found a reduction in calcium depositions visualized with Alizarin Red staining when compared to untreated hMSCs. In 3D cultures, MRS1845 downregulated the gene expression of early osteogenic marker ALP and protein expression of the intracellular osteogenic mediator Runx2 specifically on MC-GAG compared to controls. As small molecule inhibitors lack channel specificity, we knocked down ORAI1 expression through siRNA transfection. When MC-GAG was compared with Col-GAG, baseline ORAI1 gene expression was 1.8-fold higher (p=0.02) and Orai1 protein expression was upregulated (p=0.003). ORAI1 knockdown reduced the protein expression of Runx2 and gene expression of late osteogenic marker BSP2 specifically on MC-GAG compared to scrambled control siRNA. Mineralization significantly diminished on MC-GAG with ORAI1 knockdown, whereas no effects were evident on Col-GAG as demonstrated by Alizarin Red staining. CONCLUSIONS: ORAI1-mediated calcium ion signaling was required for MC-GAG-induced osteogenic differentiation. Further elucidation of the osteogenic mechanisms and refinement of calcium levels on MC-GAG may facilitate its clinical application in the reconstruction of osseous defects.

Pro-Osteogenic and Anti-Inflammatory Synergistic Effect of Orthosilicic Acid, Vitamin K2, Curcumin, Polydatin and Quercetin Combination in Young and Senescent Bone Marrow-Derived Mesenchymal Stromal Cells.

During aging, bone marrow mesenchymal stromal cells (MSCs)-the precursors of osteoblasts-undergo cellular senescence, losing their osteogenic potential and acquiring a pro-inflammatory secretory phenotype. These dysfunctions cause bone loss and lead to osteoporosis. Prevention and intervention at an early stage of bone loss are important, and naturally active compounds could represent a valid help in addition to diet. Here, we tested the hypothesis that the combination of two pro-osteogenic factors, namely orthosilicic acid (OA) and vitamin K2 (VK2), and three other anti-inflammatory compounds, namely curcumin (CUR), polydatin (PD) and quercetin (QCT)-that mirror the nutraceutical BlastiMin Complex® (Mivell, Italy)-would be effective in promoting MSC osteogenesis, even of replicative senescent cells (sMSCs), and inhibiting their pro-inflammatory phenotype in vitro. Results showed that when used at non-cytotoxic doses, (i) the association of OA and VK2 promoted MSC differentiation into osteoblasts, even when cultured without other pro-differentiating factors; and (ii) CUR, PD and QCT exerted an anti-inflammatory effect on sMSCs, and also synergized with OA and VK2 in promoting the expression of the pivotal osteogenic marker ALP in these cells. Overall, these data suggest a potential role of using a combination of all of these natural compounds as a supplement to prevent or control the progression of age-related osteoporosis.

Expression of Stro-1, Runx-2, Osterix, and Alp in Alveolar Bone Regeneration Process Following the Administration of Hydroxyapatite Gypsum Puger (HAGP) Scaffold

Introduction: Tooth extraction before denture placement could result in trauma and damage to up to 50% of the alveolar bone, inducing bone resorption, and affecting the patient’s quality of life. Hydroxyapatite Gypsum Puger (HAGP) can be used as an alternative to bone graft material which degrades slowly, affecting the proliferation and activity of cells that are responsible for bone tissue engineering. This study aimed to analyze the regeneration mechanism of alveolar bone by administering the HAGP scaffold and observing the Stro-1, Runx-2, Osterix, and ALP expression. Methods: Laboratory experimental research was conducted and we used 150-355µm HAGP scaffold particles, applied in vivo inside alveolar sockets of the rats for 7, 14, and 28 days, followed by immunohistochemical examination of Stro-1, Runx-2, Osterix, and ALP expressions. Results: The HAGP scaffold group showed that the Stro-1 expression was significantly higher than the K(-) group, and the Runx-2 expression increased on day 7 and decreased on day 28 in the HAGP and K(-) groups. Osterix expression increased from day 7, 14, to day 28. The high expression of Osterix on day 28 means it took over the Runx-2 function. In ALP there was a significant increase on day 7. ALP expression was a sign of early osteoblast differentiation and production by cells, this extracellular matrix mineralization is an indicator of the osteogenic process. Conclusion: Alveolar bone regeneration mechanism in rats revealed that the expression of Stro-1, Runx-2, Osterix, and ALP was higher in the HAGP scaffold group compared to the control group on days 7,14, and 28.

Osteogenic differentiation of bone marrow stromal stem cells on a novel β titanium alloy-based micro-nano composite

The incorporation of nanoparticles into metallic biomaterials has been recognized as a significant approach to improve their bioactivity for biomedical applications. In this study, friction stir processing (FSP) was employed to incorporate TiO2 nanoparticles as bioactive reinforcement into the β titanium alloy Ti–35Nb–2Ta–3Zr (TNTZ) matrix to construct an integrated micro-nano composite layer. The effect of different TiO2 contents on the osteogenic capacity of rat bone marrow stromal stem cells (BMSCs) was studied. The TiO2/TNTZ composite layer exhibited a refined grain structure with a mean grain size of approximately 10 μm and a characteristic surface morphology of pronounced peaks and valleys with enhanced RSkw and RKur values. Moreover, TiO2/TNTZ significantly promoted the adhesion, proliferation, and spreading of BMSCs, upregulated the expression of genes related to osteogenesis (ALP and Runx2) up to nearly 1.5 times, and enhanced the expression of ALP and OCN (P < 0.05). The novel FSPed TiO2/TNTZ micro-nano composite possesses excellent surface characteristics and favorable biological activities, manifesting vast potential for upcoming clinical applications.

Chalcone T4 Inhibits RANKL-Induced Osteoclastogenesis and Stimulates Osteogenesis In Vitro.

Chalcones are phenolic compounds produced during the biosynthesis of flavonoids that have numerous biological activities, including anti-inflammatory, antioxidant and anticancer. In this in vitro study, we investigate a newly synthesized chalcone (Chalcone T4) in the context of bone turnover, specifically on the modulation of osteoclast differentiation and activity and osteoblast differentiation. Murine macrophages (RAW 264.7) and pre-osteoblasts (MC3T3-E1) were used as models of osteoclasts and osteoblasts, respectively. Differentiation and activity osteoclasts were induced by RANKL in the presence and absence of non-cytotoxic concentrations of Chalcone T4, added in different periods during osteoclastogenesis. Osteoclast differentiation and activity were assessed by actin ring formation and resorption pit assay, respectively. Expression of osteoclast-specific markers (Nfatc1, Oscar, Acp5, Mmp-9 and Ctsk) was determined by RT-qPCR, and the activation status of relevant intracellular signaling pathways (MAPK, AKT and NF-kB) by Western blot. Osteoblast differentiation and activity was induced by osteogenic culture medium in the presence and absence of the same concentrations of Chalcone T4. Outcomes assessed were the formation of mineralization nodules via alizarin red staining and the expression of osteoblast-related genes (Alp e Runx2) by RT-qPCR. Chalcone T4 reduced RANKL-induced osteoclast differentiation and activity, suppressed Oscar, Acp5 and Mmp-9 expression, and decreased ERK and AKT activation in a dose-dependent manner. Nfact1 expression and NF-kB phosphorylation were not modulated by the compound. Mineralized matrix formation and the expression of Alp and Runx2 by MC3T3-E1 cells were markedly stimulated by Chalcone T4. Collectively, these results demonstrate that Chalcone T4 inhibits in osteoclast differentiation and activity and stimulates osteogenesis, which indicates a promising therapeutic potential in osteolytic diseases.

Metabolic shift and the effect of mitochondrial respiration on the osteogenic differentiation of dental pulp stem cells.

Metabolism shifts from glycolysis to mitochondrial oxidative phosphorylation are vital during the differentiation of stem cells. Mitochondria have a direct function in differentiation. However, the metabolic shift and the effect of mitochondria in regulating the osteogenic differentiation of human dental pulp stem cells (hDPSCs) remain unclear. Human dental pulp stem cells were collected from five healthy donors. Osteogenic differentiation was induced by osteogenic induction medium. The activities of alkaline phosphatase, hexokinase, pyruvate kinase, and lactate dehydrogenase were analyzed by enzymatic activity kits. The extracellular acidification rate and the mitochondrial oxygen consumption rate were measured. The mRNA levels of COL-1, ALP, TFAM, and NRF1 were analyzed. The protein levels of p-AMPK and AMPK were detected by western blotting. Glycolysis decreased after a slight increase, while mitochondrial oxidative phosphorylation continued to increase when cells grew in osteogenic induction medium. Therefore, the metabolism of differentiating cells switched to mitochondrial respiration. Next, inhibiting mitochondrial respiration with carbonyl cyanide-chlorophenylhydrazone, a mitochondrial uncoupler inhibited hDPSCs differentiation with less ALP activity and decreased ALP and COL-1 mRNA expression. Furthermore, mitochondrial uncoupling led to AMPK activation. 5-Aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, simulated the effect of mitochondrial uncoupling by inhibiting osteogenic differentiation, mitochondrial biogenesis, and mitochondrial morphology. Mitochondrial uncoupling and activation of AMPK depressed mitochondrial oxidative phosphorylation and inhibited differentiation, suggesting that they may serve as regulators to halt osteogenic differentiation from impaired mitochondrial oxidative phosphorylation.

TGF-BETA STIMULATES PERIOSTEAL OSTEOGENIC ACTIVITY

Osteoprogenitors on the inner layer of periosteum are the major cellular contributors to appositional bone growth and bone repair by callus formation. Previous work showed that periosteal-derived cells have little or no osteogenic activity under standard in vitro osteogenic culture conditions. This study was conducted to determine what growth factor(s) can activate periosteal osteogenic capacity.This study was conducted with IACUC approval. Periosteum from five equine donors was digested in collagenase for 3-4 hours at 37C. Isolated periosteal cells were maintained in DMEM/10% FBS medium and exposed to PDGF, Prostaglandin E2, BMP-2 and TGF-b3 at a range of concentrations for 72 hours. Changes in osteogenic gene expression (Runx2, OSX and ALP) were measured by qPCR. Periosteal cells were pre-treated with TGF-b3 or maintained in control medium were transferred into basal or osteogenic medium. Osteogenic status was assessed by Alizarin Red staining for mineralized matrix, ALP enzymatic activity and induction of osteogenic genes.PDGF, PgE2 and BMP-2 had little impact on expression of osteogenic markers by periosteal cells. In contrast, TGF-b3 stimulated significant increases in Osterix (over 100-fold) ALP expression (over 70-fold). Pre-treating periosteal cells with TGF-b3 for 72 hours stimulated rapid cell aggregation and aggregate mineralization once cells were transferred to osteogenic medium, while cells not exposed to TGF-b3 exhibited minimal evidence of osteogenic activity.This study indicate that TGF-b signaling is vital for periosteal osteogenic activity. Transient ‘priming’ of periosteal cells through TGF-b exposure was sufficient to activate subsequent osteogenesis without requiring ongoing growth factor stimulation. TGF beta ligands are secreted by many cell types, including periosteal progenitors and osteocytes, providing opportunities for both autocrine and paracrine pathways to regulate periosteal bone formation under homeostatic and reparative conditions.

Repurposing sarecycline for osteoinductive therapies: an in vitro and ex vivo assessment.

Tetracyclines (TCs) embrace a class of broad-spectrum antibiotics with unrelated effects at sub-antimicrobial levels, including an effective anti-inflammatory activity and stimulation of osteogenesis, allowing their repurposing for different clinical applications. Recently, sarecycline (SA)-a new-generation molecule with a narrower antimicrobial spectrum-was clinically approved due to its anti-inflammatory profile and reduced adverse effects verified with prolonged use. Notwithstanding, little is known about its osteogenic potential, previously verified for early generation TCs. Accordingly, the present study is focused on the assessment of the response of human bone marrow-derived mesenchymal stromal cells (hBMSCs) to a concentration range of SA, addressing the metabolic activity, morphology and osteoblastic differentiation capability, further detailing the modulation of Wnt, Hedgehog, and Notch signaling pathways. In addition, an ex vivo organotypic bone development system was established in the presence of SA and characterized by microtomographic and histochemical analysis. hBMSCs cultured with SA presented a significantly increased metabolic activity compared to control, with an indistinguishable cell morphology. Moreover, RUNX2 expression was upregulated 2.5-fold, and ALP expression was increased around sevenfold in the presence of SA. Further, GLI2 expression was significantly upregulated, while HEY1 and HNF1A were downregulated, substantiating Hedgehog and Notch signaling pathways' modulation. The ex vivo model developed in the presence of SA presented a significantly enhanced collagen deposition, extended migration areas of osteogenesis, and an increased bone mineral content, substantiating an increased osteogenic development. Summarizing, SA is a promising candidate for drug repurposing within therapies envisaging the enhancement of bone healing/regeneration.

Resveratrol prevents ovariectomy-induced bone quality deterioration byimprovingthemicroarchitectural and biophysicochemical properties of bone.

Osteoporosis is a major health problem that is very common worldwide and is characterized by both low bone density and deterioration in bone quality. New treatment options without side effects have become an active area of research in recent years. This study was designed to investigate the preventive effects of resveratrol on bone quality deterioration caused by ovariectomy. Sixty rats were randomly divided into five groups (12 animals per group): Control, Sham-operated (SHAM), ovariectomized (OVX), OVX + Resveratrol-40mg/kg/day (OVX + Res40), OVX + Resveratrol-80mg/kg/day (OVX + Res80). Resveratrol was administered by oral gavage (40 and 80mg/kg/day) for ten weeks. Micro-CT measurements, biomechanical testing, Raman spectroscopyanalysis, and RT-PCR analysis were performed. ALP, OCN, TAS, and TOS levels were also measured from blood serum. Bone strength, bone volume/total volume, trabecular volume, and trabecular thickness were higher in the OVX + RES-80 group than inthe OVX group. Resveratrol increased osteogenic differentiation, as the expression of osteogenic markers ALP, Col1A1, Runx2, OPG, OCN increased in both OVX + RES-80 and OVX + RES-40 groups compared to theOVX group. 80mg/kg/day resveratrol administration decreasedthe levels of ALP, OCN and TOS in ovariectomized rats. Raman spectroscopy findings showed a preventive effect of resveratrol administration against ovariectomy-induced deterioration in biophysiochemical properties of bone tissue. This study revealed that administration of different doses of 80 mg/kg/day and 40 mg/kg/day of resveratrol had protective effects on bone quality deterioration caused by ovariectomy.

Exendin-4 regulates the MAPK and WNT signaling pathways to alleviate the osteogenic inhibition of periodontal ligament stem cells in a high glucose environment.

Diabetes mellitus (DM) increases the destruction of periodontal tissue and impairs osteogenesis differentiation. Exendin-4 (Ex-4), a glucagon-like peptide-1 (GLP-1) analogue, can be used for treating DM and promotes bone regeneration. The aim of this study was to explore the effect and mechanism of Ex-4 on improving the osteogenesis of periodontal ligament stem cells (PDLSCs) in a high glucose environment. Alkaline phosphatase staining and alizarin red staining were used to detect the osteogenic differentiation of PDLSCs. The results showed that 10 nM Ex-4 could reduce the osteogenesis inhibition of PDLSCs induced by high glucose. RT-PCR and western blot results showed that Ex-4 increased the osteogenesis-related gene expression of ALP, Runx2, and Osx, and upregulated the phosphorylation of P38, JNK, and ERK1/2; the peak effect was observed in the range 0.5-1.0 h. Mitogen-activated protein kinase (MAPK) inhibitors PD98059, SB203580, and SP600125 blocked the effects of Ex-4 on MAPK activation and decreased the expression of ALP, Runx2, and Osx in PDLSCs. Moreover, after Ex-4 treatment, the total β-catenin, p-GSK3β, LEF, and Runx2 protein levels increased under normal or high glucose environments. In conclusion, our results indicated that Ex-4 regulates the MAPK and WNT signaling pathways to alleviate the osteogenic inhibition of PDLSCs in a high glucose environment.

Preparation, Characterization and Osteoblastic Compatibility Evaluation of Bifunctional Polyvinyl Alcohol Hydrogels Containing RGD and Hyaluronic Acid

In recent years, polyvinyl alcohol hydrogel has been repeatedly confronted with urgent needs for more enhanced bio-activity in the field of bone tissue engineering. We used sulfydryl-alkyne click chemical reaction to prepare a bifunctional polyvinyl alcohol hydrogel containing RGD peptide and hyaluronic acid. Polyvinyl alcohol grafted with hyaluronic acid can reduce degradation rate and improve mechanical properties. RGD peptide can improve cell adhesion and migration on material surface. The bifunctional polyvinyl alcohol hydrogel is prepared by click chemistry reaction and characterized by scanning electron microscopy, 1H NMR, Ellman’s test and Kjeldahl nitrogen determination. The physical properties of bifunctional polyvinyl alcohol hydrogel are evaluated in terms of tensile properties and water contact angle test. According to varying hydrogel composition, five groups are designed to conduct the experiment as follows: Control group, PVA group, PVA-HA group, PVAPA-RGD group, and PVA-HA-SH/PVAPA-RGD group, each of which is to be co-cultured with MC3T3-E1 cells to observe cell morphology by laser scanning confocal microscope. Then, CCK-8 method and cell counting method were used to detect the proliferation activity and cell adhesion rate, respectively. ALP activity and qRT-PCR were used to detect osteogenic differentiation and expression of osteogenic marker genes ALP, OCN, Runx2, Col I and OPN. The experimental results show that the tensile strength of the bifunctional polyvinyl alcohol hydrogel is 1.42±0.61 MPa, with tensile modulus as 0.58±0.03 MPa, and elongation at break as 297.53±42.36%. The water contact angle is 18.40±2.2°. After three days of culture, the proliferation activity ranks in order as PVA-HA-SH/PVAPA-RGD group &gt; PVA-RGD group &gt; PVA-HA group &gt; PVA group &gt; Control group. After 4 hours of culture, cell adhesion is sequenced as PVA-HA-SH/PVAPA-RGD group &gt; bicomponent group &gt; PVA group &gt; Control group. After 48 hours of culture, immunofluorescence staining demonstrates more sufficient cellular spreading and adherence, clearer actin fibers, more vinculin numbers in PVA-HA-SH/PVAPA-RGD group. In addition, significantly higher ALP activity (after 3 days’ culturing) and osteogenic gene expression level (after 7 days’ culturing) have been witnessed in PVA-HA-SH/PVAPA-RGD group than those of other groups. The overall experimental results have confirmed its excellent hydrophilicity and fine mechanical properties of the bifunctional polyvinyl alcohol hydrogel system, which would exert a remarkable effect on proliferation, adhesion and osteogenic differentiation of MC3T3-E1 cells.

Biological Role of miRNA-302a-3p/DKK1 in Osteogenesis of Bone-Marrow Derived Mesenchymal Stem Cells

To elucidate the involvement of miRNA-302a-3p in osteogenesis of BMSCs through targeting DKK1, thus influencing the progression of osteoporosis (OP). MiRNA-302a-3p levels in serum of 40 OP patients and 40 healthy controls were detected by qRT-PCR. BMD was determined in OP patients expressing high or low level of miRNA-302a-3p. In BMSCs undergoing osteogenesis at the different time points, dynamic expressions of ALP, RUNX2 and Bglap were determined. Influences of miRNA-302a-3p on ALP activity and osteogenic capacity in BMSCs were assessed by ALP activity determination, ALP staining and ARS. The interaction between miRNA-302a-3p and DKK1 was verified by dual-luciferase reporter gene assay and Spearman correlation test. In addition, DKK1 involvement in osteogenesis of BMSCs influenced by miRNA-302a-3p was evaluated. MiRNA-302a-3p level was reduced in serum of OP patients. BMD was higher in OP patients expressing high level of miRNA-302a-3p. During the process of osteogenesis, overexpression of miRNA-302a-3p upregulated osteogenesis-associated genes, ALP activity and capacity of mineralization in BMSCs. Knockdown of miRNA-302a-3p achieved the opposite trends. DKK1 was identified to be the target gene binding miRNA-302a-3p, and its level was negatively regulated by miRNA-302a-3p. Notably, DKK1 was responsible for accelerating osteogenesis in BMSCs regulated by miRNA-302a-3p. Downregulation of MiRNA-302a-3p inhibits osteogenesis in BMSCs via downregulating DKK1, thereafter accelerating the progression of OP.

Incorporation of strontium-containing bioactive particles into PEOT/PBT electrospun scaffolds for bone tissue regeneration

The combination of biomaterials and bioactive particles has shown to be a successful strategy to fabricate electrospun scaffolds for bone tissue engineering. Among the range of bioactive particles, hydroxyapatite and mesoporous bioactive glasses (MBGs) have been widely used for their osteoconductive and osteoinductive properties. Yet, the comparison between the chemical and mechanical characteristics as well as the biological performances of these particle-containing scaffolds have been characterized to a limited extent. In this work, we fabricated PEOT/PBT-based composite scaffolds incorporating either nanohydroxyapatite (nHA), strontium-containing nanohydroxyapatite (nHA_Sr) or MBGs doped with strontium ions up to 15 wt./vol% and 12,5 wt./vol% for nHA and MBG, respectively. The composite scaffolds presented a homogeneous particle distribution. Morphological, chemical and mechanical analysis revealed that the introduction of particles into the electrospun meshes caused a decrease in the fiber diameter and mechanical properties, yet maintaining the hydrophilic nature of the scaffolds. The Sr2+ release profile differed according to the considered system, observing a 35-day slowly decreasing release from strontium-containing nHA scaffolds, whereas MBG-based scaffolds showed a strong burst release in the first week. In vitro, culture of human bone marrow-derived mesenchymal stromal cells (hMSCs) on composite scaffolds demonstrated excellent cell adhesion and proliferation. In maintenance and osteogenic media, all composite scaffolds showed high mineralization as well as expression of Col I and OCN compared to PEOT/PBT scaffolds, suggesting their ability to boost bone formation even without osteogenic factors. The presence of strontium led to an increase in collagen secretion and matrix mineralization in osteogenic medium, while gene expression analysis showed that hMSCs cultured on nHA-based scaffolds had a higher expression of OCN, ALP and RUNX2 compared to cells cultured on nHA_Sr scaffolds in osteogenic medium. Yet, cells cultured on MBGs-based scaffolds showed a higher gene expression of COL1, ALP, RUNX2 and BMP2 in osteogenic medium compared to nHA-based scaffolds, which is hypothesized to lead to high osteoinductivity in long term cultures.

Analysis of the Potency of Robusta Coffee (Coffea canephora) to Increase the Expression of FGF2, Collagen 1 and ALP in the Periodontal Ligament During Orthodontic Tooth Movement

Orthodontic treatment is relatively long and various efforts have been made to speed up the movement of orthodontic teeth. Coffee contains substances called caffeine (1,3,7 trimethylxanthine), chlorogenic acid and caffeic acid, as well as flavonoids which have antioxidant effects. Chlorogenic acid and caffeic acid are antioxidants that can reduce oxidative stress in osteoblasts and promote osteogenesis. The study aimed to analyze the expression of FGF2, Collagen 1 and ALP, on the movement of orthodontic teeth after administration of Robusta coffee extract. 24 wistar rats divided into 4 groups, namely groups C1 and C2: Rats were given orthodontic mechanical force (OMF) for 2 and 3 weeks and groups T1 and T2: Mice were given OMF and Robusta coffee extract for 2 and 3 weeks. OMF in mice (rats) was carried out by means of the right maxillary first molars teeth and on both maxillary incisors were given ligature wires. Then the right maxillary first molars moved to the mesial with a Niti closed coil spring. Observations were made on the day 15 and day 22 with immunohistochemical examinations to determine the expression of FGF2, Collagen 1 and ALP. The results indicated that administration of Robusta coffee extract for 2 and 3 weeks significantly increased the [expression of FGF2, Collagen 1 and ALP. The expression of FGF2, Collagen 1 and ALP (expression of GFß1, IGF1 and PDGF). The expression of TGFß1, IGF1 and PDGF during 3 weeks was greater than that at 2 weeks (p &lt; 0.05. Robusta coffee extract can be used as an alternative ingredient in accelerating orthodontic treatment. HIGHLIGHTS Orthodontic treatment requires quite a long time and various efforts have been made to speed uporthodontic tooth movement, for example with drugs Robusta coffee extract contains ingredients that have antioxidant effects that can reduce oxidativestress on osteoblasts and promote osteogenesis Robusta coffee extract effectively increases the expression of FGF2, Collagen-1 and ALP which are bone formation markers GRAPHICAL ABSTRACT

Recombinant Irisin Protects Against Alveolar Bone Destruction During Orthodontic Tooth Movement

Patients with periodontitis have higher risk of alveolar bone loss when seek for orthodontic therapy. Inflammation and osteogenesis are key factors in alveolar bone destruction in periodontitis and orthodontic tooth movement (OTM). Here we evaluated the effects of irisin on alveolar bone destruction in rats with periodontitis and OTM. We isolated and cultured human periodontal ligament stem cells (PDLSCs). Irisin was administrated to PDLSCs. Cell proliferations, osteogenic differentiation, expression of RUNX2 and ALP, and the expression of OPG and RANKL were measured. We induced periodontitis and OTM in rats and treated rats with irisin. The alveolar bone loss, inflammatory cytokine levels, and expression of OPG and RANKL in gingival tissues were measured. Irisin promoted the cell proliferation and osteogenic differentiation of PDLSCs. Irisin elevated the expression of RUNX2, ALP, and OPG while decreased the expression of RANKL in PDLSCs. Irisin ameliorated the alveolar bone loss, suppressed cytokine levels, and increased OPG/RANKL expression ratio in rat with periodontitis and orthodontic tooth movement. Irisin prevented alveolar bone destruction during OTM in rats with periodontitis.

Effect of inoculation density of bone marrow mesenchymal stem cells cultured on calcium phosphate cement scaffold on osteogenic differentiation.

Calcium phosphate cements (CPCs) are biocompatible materials that have been evaluated as scaffolds in bone tissue engineering. At present, the stem cell density of inoculation on CPC scaffold varies. The aim of this study is to analyze the effect of seeding densities on cell growth and osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) on a calcium phosphate cements (CPCs) scaffold. BMMSCs derived from minipigs were seeded onto a CPC scaffold at three densities [1million/mL (1M), 5 million/mL (5M) and 25million/mL 25M)], and cultured for osteogenic induction for 1, 4 and 8 days. Well adhered and extended BMMSCs on the CPC scaffold showed significantly different proliferation rates within each seeding density group at different time points (P <0.05). The number of live cells per unit area in 1M, 5M and 25M increased by 3.5, 3.9 and 2.5 folds respectively. The expression of ALP peaked at 4 days post inoculation with the fold-change being 2.6 and 2.8 times higher in 5M and 25M respectively as compared to 1M. The expression levels of OC, Coll-1 and Runx-2 peaked at 8 days post inoculation. An optimal seeding density may be more conducive for cell proliferation, differentiation, and extracellular matrix synthesis on scaffolds. We suggest the optimal seeding density should be 5million/mL.

Comparison of the effect of NaOCL, curcumin, and EDTA on differentiation, proliferation, and adhesion of dental pulp stem cells.

This study examined the effect of 1.5% NaOCl, 17% EDTA, and curcumin on the proliferation, attachment, and differentiation of dental pulp stem cells (DPSCs) placed on the dentin specimens. MTT assay was performed to evaluate the proliferation of DPSCs on the dentin specimens treated with different concentrations of NaOCl, 17% EDTA, and curcumin (0.97-250μM). Cell-adhering ability of DPSCs was tested via the LDH assay to calculate the attached DPSCs. In addition, the western blotting assay was performed to investigate the expression levels of fibronectin as a cell-adhesion marker and analyze the expressions level of differentiation markers, including DMP-1, OCN, ALP, and DSPP, to detect the odontogenic potential of hDPCs. NaOCl had lower toxicity on DPSCs at lower concentrations (P<0.001). The cytotoxicity of irrigants increased with increased dosage. The difference between the cell-adhesion ability of NaOCl and curcumin was not significant (∼4.4 MU/mL), whereas EDTA (∼3.8 MU/mL) exhibited the lowest release of LDH and less damage to hDPSCs. Regarding fibronectin expression, the pattern differed between irrigants in inducing cell adhesion. NaOCl increased fibronectin expression more than EDTA and curcumin. All the treated groups upregulated the expression of DSPP, DMP-1, OCN, and ALP compared to the control group, in which NaOCl showed a higher effect on the overexpression of differentiation markers. The results showed that all the tested irrigants could be used in regenerative endodontic treatment. However, as an herbal-based and biocompatible irrigant, curcumin exhibited fewer adverse effects than NaOCl and EDTA.

N-acetyl Cysteine Inhibits Cell Proliferation and Differentiation of LPSInduced MC3T3-E1 Cells Via Regulating Inflammatory Cytokines.

Peri-implantitis is one of the most common complications in oral implantation and could lead to the loss of the function of bone tissues around implants. This study used lipopolysaccharide (LPS) as a stimulant for MC3T3-E1 cells and N-acetyl cysteine (NAC) as an inhibitor to inhibit the effect of LPS to investigate the effect of NAC on the expression of bone formation related factors and inflammatory-related factors of osteoblasts under the action of LPS. In this study, we found that the cell proliferation and cell differentiation were significantly promoted when NAC concentrations were between 0 ~ 0.5 mM, but were inhibited when the concentration exceeded 0.5 mM. LPS had a slightly promoting effect on the cell proliferation before 20 μg/mL but inhibited the cell proliferation after 20 μg/mL. LPS reduced protein and gene expressions of Runx2, ALP and BGP and increased protein and gene expressions of NF-κB and TNF-α. NAC reversibly regulated the LPS's regulation on the expression of MC3T3-E1 cell cytokine gene and protein. The optimal NAC concentration for treating MC3T3-E1 cells is 0.5 mM, and the optimal LPS concentration for stimulating MC3T3-E1 cells is 20 μg/mL. NAC plays an active role in regulating the differentiation of MC3T3-E1 cells, and can inhibit LPS to regulate the differentiation of MC3T3-E1 cells. NAC promotes the expression of an osteogenic factor of MC3T3-E1cells and inhibits the expression of inflammatory cytokines.

MACF1 overexpression in BMSCs alleviates senile osteoporosis in mice through TCF4/miR-335-5p signaling pathway.

The decreased osteogenic differentiation ability of mesenchymal stem cells (MSCs) is one of the important reasons for SOP. Inhibition of Wnt signaling in MSCs is closely related to SOP. Microtubule actin crosslinking factor 1 (MACF1) is an important regulator in Wnt/β-catenin signal transduction. However, whether the specific expression of MACF1 in MSC regulates SOP and its mechanism remains unclear. We established MSC-specific Prrx1 (Prx1) promoter-driven MACF1 conditional knock-in (MACF-KI) mice, naturally aged male mice, and ovariectomized female mice models. Micro-CT, H&E staining, double calcein labeling, and the three-point bending test were used to explore the effects of MACF1 on bone formation and bone microstructure in the SOP mice model. Bioinformatics analysis, ChIP-PCR, qPCR, and ALP staining were used to explore the effects and mechanisms of MACF1 on MSCs' osteogenic differentiation. Microarray analysis revealed that the expression of MACF1 and positive regulators of the Wnt pathway (such as TCF4, β-catenin, Dvl) was decreased in human MSCs (hMSCs) isolated from aged osteoporotic than non-osteoporotic patients. The ALP activity and osteogenesis marker genes (Alp, Runx2, and Bglap) expression in mouse MSCs was downregulated during aging. Furthermore, Micro-CT analysis of the femur from 2-month-old MSC-specific Prrx1 (Prx1) promoter-driven MACF1 conditional knock-in (MACF-cKI) mice showed no significant trabecular bone changes compared to wild-type littermate controls, whereas 18- and 21-month-old MACF1 c-KI animals displayed increased bone mineral densities (BMD), improved bone microstructure, and increased maximum compression stress. In addition, the ovariectomy (OVX)-induced osteoporosis model of MACF1 c-KI mice had significantly higher trabecular volume and number, and increased bone formation rate than that in control mice. Mechanistically, ChIP-PCR showed that TCF4 could bind to the promoter region of the host gene miR-335-5p. Moreover, MACF1 could regulate the expression of miR-335-5p by TCF4 during the osteogenic differentiation of MSCs. These data indicate that MACF1 positively regulates MSCs osteogenesis and bone formation through the TCF4/miR-335-5p signaling pathway in SOP, suggesting that targeting MACF1 may be a novel therapeutic approach against SOP. MACF1, an important switch in the Wnt signaling pathway, can alleviate SOP through the TCF4/miR-335-5p signaling pathway in mice model. It might act as a therapeutic target for the treatment of SOP to improve bone function.