Expression Of Osteogenic Marker Genes Research Articles

Porous gelatin-based phosphorylated scaffold: Microstructure, cell response and osteogenic differentiation of human adipose-derived mesenchymal stem cells

To repair the highly organized composite bone tissue, the design and development of biomimetic three-dimensional scaffolds is an active strategy. Here, we describe the fabrication route of a bioengineered gelatin-based scaffold for potential application in bone tissue engineering. First, phosphorus-containing polyurethane (PU–P) polymer was synthesized by polymerization starting from phosphorous polyol and methylene diisocyanate (DMI). Second, a gelatin/phosphorus-containing polyurethane (Gelatin/PU-P) scaffold was prepared by solution mixing, ultrasonication, and freeze-drying of gelatin and PU-P polymer. Next, prepared scaffold samples were characterized by FT-IR, SEM microscopy, porosity measurement, swelling, degradability, and mechanical tests. The scaffold with a porosity of about 78 % was achieved, and at the same time, it had good dimensional stability so that it only lost 43.61 % of its initial weight after 35 days immersing in PBS. Moreover, Gelatin/PU-P showed flexibility, large elongations at break (420 %) and high tensile strength (14 MPa). Finally, biological assessments revealed that the scaffold significantly supported the viability of Adipose-derived mesenchymal stem cells (ADMS). High ALP activity, enhancement in mineralization, and greater expression of osteogenic gene markers in cells grown on the Gelatin/PU-P confirmed the osteoinductivity of the scaffold. These findings suggested that Gelatin/PU-P can be used as a bioactive scaffold for bone tissue engineering.

Jaboticaba Peel Extract Attenuates Ovariectomy-Induced Bone Loss by Preserving Osteoblast Activity.

Therapies to prevent osteoporosis are relevant since it is one of the most common non-communicable human diseases in the world and the most prevalent bone disorder in adults. Since jaboticaba peel extract (JPE) added to the culture medium enhanced the osteogenic potential of mesenchymal stem cells (MSCs) derived from osteoporotic rats, we hypothesized that JPE prevents the development of ovariectomy-induced osteoporosis. Ovariectomized rats were treated with either JPE (30 mg/kg of body weight) or its vehicle for 90 days, starting 7 days after the ovariectomy. Then, the femurs were subjected to microcomputed tomography and histological analyses, and the osteoblast and adipocyte differentiation of MSCs was evaluated. JPE attenuated ovariectomy-induced bone loss, as evidenced by higher bone volume/total volume and trabecular number, along with lower trabecular separation and bone marrow adiposity. These protective effects of JPE on bone tissue are due to its ability to prevent the imbalance between osteoblast and adipocyte differentiation of MSCs, since, compared with MSCs derived from ovariectomized rats treated with vehicle, MSCs treated with JPE exhibited higher gene and protein expression of osteogenic markers and extracellular matrix mineralization, as well as lower gene expression of adipogenic markers. These data highlight the potential therapeutic use of JPE to prevent osteoporosis.

Managing Oxidative Stress Using Vitamin C to Improve Biocompatibility of Polycaprolactone for Bone Regeneration In Vitro.

Increased oxidative stress in bone cells is known to negatively alter favorable bone regeneration. This study aimed to develop a porous polycaprolactone (PCL) membrane incorporated with 25 wt % Vitamin C (PCL-Vit C) and compared it to the PCL membrane to control oxidative stress and enhance biomineralization in vitro. Both membranes were characterized using SEM-EDS, FTIR spectroscopy, and surface hydrophilicity. Vitamin C release was quantified colorimetrically. Assessments of the viability and attachment of human fetal osteoblast (hFOB 1.19) cells were carried out using XTT assay, SEM, and confocal microscopy, respectively. ROS generation and wound healing percentage were measured using flow cytometry and ImageJ software, respectively. Mineralization study using Alizarin Red in the presence or absence of osteogenic media was carried out to measure the calcium content. Alkaline phosphatase assay and gene expression of osteogenic markers (alkaline phosphatase (ALP), collagen Type I (Col1), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN)) were analyzed by real-time PCR. SEM images revealed smooth, fine, bead-free fibers in both membranes. The FTIR spectrum of pure vitamin C was replaced with peaks at 3436.05 and 2322.83 cm-1 in the PCL-Vit C membrane. Vitamin C release was detected at 15 min and 1 h. The PCL-Vit C membrane was hydrophilic, generated lower ROS, and showed significantly higher viability than the PCL membrane. Although both PCL and PCL-Vit C membranes showed similar cellular and cytoskeletal morphology, more cell clusters were evident in the PCL-Vit C membrane. Lower ROS level in the PCL-Vit C membrane displayed improved cell functionality as evidenced by enhanced cellular differentiation with more intense alizarin staining and higher calcium content, supported by upregulation of osteogenic markers ALP, Col1, and OPN even in the absence of osteogenic supplements. The presence of Vitamin C in the PCL-Vit C membrane may have mitigated oxidative stress in hFOB 1.19 cells, resulting in enhanced biomineralization facilitating bone regeneration.

The Cell-Penetrating Peptide GV1001 Enhances Bone Formation via Pin1-Mediated Augmentation of Runx2 and Osterix Stability.

Peptide-based drug development is a promising direction due to its excellent biological activity, minimal immunogenicity, high in vivo stability, and efficient tissue penetrability. GV1001, an amphiphilic peptide, has proven effective as an anti-cancer vaccine, but its effect on osteoblast differentiation is unknown. To identify proteins interacting with GV1001, biotin-conjugated GV1001 was constructed and confirmed by mass spectrometry. Proteomic analyses were performed to determine GV1001's interaction with osteogenic proteins. GV1001 was highly associated with peptidyl-prolyl isomerase A and co-immunoprecipitation assays revealed that GV1001 bound to peptidyl-prolyl cis-trans isomerase 1 (Pin1). GV1001 significantly increased alkaline phosphatase (ALP) activity, bone nodule formation, and the expression of osteogenic gene markers. GV1001-induced osteogenic activity was enhanced by Pin1 overexpression and abolished by Pin1 knockdown. GV1001 increased the protein stability and transcriptional activity of Runx2 and Osterix. Importantly, GV1001 administration enhanced bone mass density in the OVX mouse model, as verified by µCT analysis. GV1001 demonstrated protective effects against bone loss in OVX mice by upregulating osteogenic differentiation via the Pin1-mediated protein stabilization of Runx2 and Osterix. GV1001 could be a potential candidate with anabolic effects for the prevention and treatment of osteoporosis.

Beneficial effects of bioactive peptides extracted from Spirulina platensis and Gracilaria gracilis algae on bone regeneration/osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells are used in the treatment of many diseases, particularly in the repair of bone injuries. Algae with various medicinal applications are considered important natural resources. There is limited research on the effects of bioactive peptides from algae extraction on mesenchymal stem cells. In this study the impact of bioactive proteins, protein lysates and peptide fractions (<3, <30 and <50 kDa) isolated from two algae species, Spirulina platensis and Gracilaria gracilis on the proliferation and osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs) was investigated. The proteins were extracted ant hydrolyzed with trypsin enzyme to create peptides, which were then separated by ultrafiltration. hAMSCs were exposed to different concentrations of bioactive compounds (100, 300, 500 and 700 µg/ml) for varying time periods. Cell proliferation was assessed using the with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and to evaluate differentiation into bone tissue, the amount of mineral deposition was measured with alizarin red staining, and alkaline phosphatase enzyme activity was determined using a colorimetric method. The expression of Runx2, Osteocalcin, and β-Catenin genes expression was analyzed using RT-qPCR on days 7, 14 and 21 post-treatment. The results indicated that the <3 kDa peptide fraction of S. platensis and G. gracilis had no cytotoxic effects, increased cell proliferation at a concentration of 300 μg/ml, and enhanced the expression of osteogenic marker genes, alkaline phosphatase enzyme a activity, and calcium deposition in the extracellular matrix. In general, fractions that show positive effects on hAMSC differentiation have the potential to treat bone defects and promote osteoregeneration.

Silk Fibroin-Laponite Porous Microspheres as Cell Microcarriers for Osteogenic Differentiation.

Silk fibroin (SF) has garnered significant attention as a natural polymer for fabricating porous scaffolds in various engineering applications. However, the limited osteoinductive property of SF has hindered its efficacy in bone repair applications. In this study, we constructed an SF-based injectable porous microcarrier that is doped with laponite (LAP), containing magnesium ions (Mg2+). The influence of freezing temperatures and concentrations of SF and LAP on the structural parameters of SF-LAP microcarriers was investigated. The SF-LAP microcarrier exhibited a porosity of 76.7 ± 1.2% and a controlled pore size of 24.6 ± 4.0 μm. At the 6 weeks of in vitro degradation test, a mild alkaline level in culture medium containing SF-LAP microcarriers was detected. The release of Mg2+ from the SF-LAP microcarrier was maintained at a concentration within the range of 1.2-2.3 mM during the 6 weeks. The seeded human adipose-derived stem cells in the SF-LAP microcarrier demonstrated a significant enhancement in osteogenic differentiation compared with cells seeded in the pure SF microcarrier, as evidenced by quantitative alkaline phosphatase activity and the expression of osteogenic marker genes. These findings underscore the potential of the SF-LAP microcarrier as an ideal cell carrier in the treatment of bone defects.

CA1 Modulates the Osteogenic Differentiation of Dental Follicle Stem Cells by Activating the BMP Signaling Pathway In Vitro.

Carbonic anhydrase 1 (CA1) has been found to be involved in osteogenesis and osteoclast in various human diseases, but the molecular mechanisms are not completely understood. In this study, we aim to use siRNA and lentivirus to reduce or increase the expression of CA1 in Dental follicle stem cells (DFSCs), in order to further elucidate the role and mechanism of CA1 in osteogenesis, and provide better osteogenic growth factors and stem cell selection for the application of bone tissue engineering in alveolar bone fracture transplantation. The study used RNA interference and lentiviral vectors to manipulate the expression of the CA1 gene in DFSCs during in vitro osteogenic induction. The expression of osteogenic marker genes was evaluated and changes in CA1, alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and Bone morphogenetic proteins (BMP2) were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB). The osteogenic effect was assessed through Alizarin Red staining. The mRNA and protein expression levels of CA1, ALP, RUNX2, and BMP2 decreased distinctly in the si-CA1 group than other groups (p < 0.05). In the Lentivirus-CA1 (LV-CA1) group, the mRNA and protein expressions of CA1, ALP, RUNX2, and BMP2 were amplified to varying degrees than other groups (p < 0.05). Apart from CA1, BMP2 (43.01%) and ALP (36.69%) showed significant upregulation (p < 0.05). Alizarin red staining indicated that the LV-CA1 group produced more calcified nodules than other groups, with a higher optical density (p < 0.05), and the osteogenic effect was superior. CA1 can impact osteogenic differentiation via BMP related signaling pathways, positioning itself upstream in osteogenic signaling pathways, and closely linked to osteoblast calcification and ossification processes.

Direct coupled electrical stimulation towards improved osteogenic differentiation of human mesenchymal stem/stromal cells: a comparative study of different protocols

Electrical stimulation (ES) has been described as a promising tool for bone tissue engineering, being known to promote vital cellular processes such as cell proliferation, migration, and differentiation. Despite the high variability of applied protocol parameters, direct coupled electric fields have been successfully applied to promote osteogenic and osteoinductive processes in vitro and in vivo. Our work aims to study the viability, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells when subjected to five different ES protocols. The protocols were specifically selected to understand the biological effects of different parts of the generated waveform for typical direct-coupled stimuli. In vitro culture studies evidenced variations in cell responses with different electric field magnitudes (numerically predicted) and exposure protocols, mainly regarding tissue mineralization (calcium contents) and osteogenic marker gene expression while maintaining high cell viability and regular morphology. Overall, our results highlight the importance of numerical guided experiments to optimize ES parameters towards improved in vitro osteogenesis protocols.

Lanthanum Hydroxide and Chronic Kidney Disease Mineral and Bone Disorder: A Rat Model.

To investigate the pharmacological effects and molecular mechanisms of lanthanum hydroxide(LH) on ectopic mineralization of soft tissue and abnormal bone in rats with acute kidney injury(AKI). Wistar rats were modeled by 5/6 nephrectomy. After the operation, the rats were divided into different groups, the biochemical indexes of serum collected at different times. LH was administered by intragastric tube at doses of 0.4, 0.2, and 0.1g/kg, respectively. Rats were sacrificed in the 16th week after LH treatment. Observation of pathological changes in tissues were made by specific staining. Western Blot, Real-Time Quantitative PCR, and immunohistochemistry techniques were used to detect the impact on pathway-related proteins. Compared with the control group (no LH administered), the serum phosphate level of the LH group was significantly reduced (p<0.01), calcification of the thoracic aorta was reduced (p<0.05, p<0.01) (Serum biochemical tests before dosing and during drug treatment cycles), renal fibrosis was improved (p<0.01), nuclear entry of nuclear factor kappa-B (NF-κB) was reduced (p<0.01), and the expression of the smooth muscle protein 22α (SM22α) was significantly increased (p<0.01). The expression of osteogenic marker genes was decreased. In addition, compared with the controls, the receptor activator for nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio of the femur in the model group was increased (p<0.05). LH can inhibit the occurrence and development of vascular calcification and bone abnormalities in AKI rats by inhibiting the NF-κB and RANKL/OPG signaling pathways.

Pten knockout in mouse preosteoblasts leads to changes in bone turnover and strength.

Bone development and remodeling are controlled by the phosphoinositide-3-kinase (Pi3k) signaling pathway. We investigated the effects of downregulation of phosphatase and tensin homolog (Pten), a negative regulator of Pi3k signaling, in a mouse model of Pten deficiency in preosteoblasts. We aimed to identify mechanisms that are involved in the regulation of bone turnover and are linked to bone disorders. Femora, tibiae, and bone marrow stromal cells (BMSCs) isolated from mice with a conditional deletion of Pten (Pten cKO) in Osterix/Sp7-expressing osteoprogenitor cells were compared to Cre-negative controls. Bone phenotyping was performed by μCT measurements, bone histomorphometry, quantification of bone turnover markers CTX and procollagen type 1N propeptide (P1NP), and three-point bending test. Proliferation of BMSCs was measured by counting nuclei and Ki-67-stained cells. In vitro, osteogenic differentiation capacity was determined by ALP staining, as well as by detecting gene expression of osteogenic markers. BMSCs from Pten cKO mice were functionally different from control BMSCs. Osteogenic markers were increased in BMSCs derived from Pten cKO mice, while Pten protein expression was lower and Akt phosphorylation was increased. We detected a higher trabecular bone volume and an altered cortical bone morphology in Pten cKO bones with a progressive decrease in bone and tissue mineral density. Pten cKO bones displayed fewer osteoclasts and more osteoblasts (P = .00095) per trabecular bone surface and a higher trabecular bone formation rate. Biomechanical analysis revealed a significantly higher bone strength (P = .00012 for males) and elasticity of Pten cKO femora. On the cellular level, both proliferation and osteogenic differentiation capacity of Pten cKO BMSCs were significantly increased compared to controls. Our findings suggest that Pten knockout in osteoprogenitor cells increases bone stability and elasticity by increasing trabecular bone mass and leads to increased proliferation and osteogenic differentiation of BMSCs.

DOT1L decelerates the development of osteoporosis by inhibiting SRSF1 transcriptional activity via microRNA-181-mediated KAT2B inhibition

ObjectiveOur study explored the function of DOT1L in osteoporosis (OP) via the microRNA (miR)-181/KAT2B/SRSF1 axis. MethodsOsteoclast (OC) number was evaluated via TRAP staining, and serum CTXI, PINP, and ALP contents were tested by ELISA. Following identification of bone marrow mesenchymal stem cells (BMSCs), OC differentiation was induced by M-CSF and RANKL, followed by the detection of OC differentiation and the expression of bone resorption-related genes, DOT1L, miR-181, KAT2B, and SRSF1. ResultsOverexpressed DOT1L or miR-181 stimulated calcified nodule formation and increased alkaline phosphatase activity and osteogenic marker gene expression. KAT2B knockdown enhanced the osteogenic differentiation of BMSCs by reducing SRSF1 acetylation. The enhancement of OC differentiation induced by overexpressed SRSF1 was inhibited by simultaneous DOT1L or miR-181 overexpression. DOT1L suppressed OP development in vivo via the miR-181/KAT2B/SRSF1 axis. ConclusionDOT1L overexpression slowed down bone loss and promoted bone formation via the miR-181/KAT2B/SRSF1 axis, thereby alleviating OP development.

Circular RNA circ-3626 promotes bone formation by modulating the miR-338-3p/Runx2 axis

ObjectiveDisorders of bone homeostasis are the key factors leading to metabolic bone disease, such as senile osteoporosis, which is characterized by age-related bone loss. Bone marrow stromal cells (BMSCs) possess high osteogenic capacity which has been regarded as a practical approach to preventing bone loss. Previous studies have shown that the osteogenic differentiation ability of BMSCs is significantly decreased in senile osteoporosis. Recently, circular RNAs (circRNAs) have been regarded as critical regulators in controlling the osteogenic differentiation of BMSCs by sponging microRNAs (miRNAs). Our study aimed to discover new and critical osteogenesis-related circRNAs that can promote bone formation in senile osteoporosis. MethodsWe detected the dysregulated circRNAs of BMSCs upon osteogenic differentiation induction and identified the critical osteogenic circRNA (circ-3626). The relationship between circ-3626 and osteoporosis was further verified in clinical bone samples and aged mice by qPCR. Moreover, circ-3626 AAV was constructed to examine the osteogenic effect of circ-3626 on bone formation via using Micro-CT, double calcein labeling, and the three-point bending tests. Bioinformatics analysis, Luciferase report gene assays, FISH, RNA pull-down, qPCR, Western Blots, and alizarin red staining assay explore the effects and mechanisms of circ-3626 on osteogenic differentiation of BMSCs. ResultsCirc-3626 was identified as a pivotal osteogenesis-related circRNA via RNA sequencing. The results of alizarin red staining, Western blots, and qPCR assays suggest that overexpressing circ-3626 dramatically accelerates the osteogenic capability of BMSCs. Furthermore, the bone repair capability of aging mice could be significantly improved by circ-3626 AAV treatment. Micro RNA miR-338-3p was identified as the downstream target of circ-3626. Overexpression of circ-3626 increases the expression of Runx2 by sponging miR-338-3p, thereby promoting the osteogenic differentiation of BMSCs by upregulating the expression of osteogenic genes. In addition, Western blots, and qPCR assays suggest circ-3626 AAV treatment promote the expression of Runx2 and osteogenic marker genes. ConclusionThus, we demonstrate that circ-3626 plays a pivotal role in promoting bone formation through the miR-338-3p/Runx2 axis and may provide new strategies for preventing and treating the bone loss of senile osteoporosis.

Chitin scaffolds derived from the marine demosponge Aplysina fistularis stimulate the differentiation of dental pulp stem cells.

The use of stem cells for tissue regeneration is a prominent trend in regenerative medicine and tissue engineering. In particular, dental pulp stem cells (DPSCs) have garnered considerable attention. When exposed to specific conditions, DPSCs have the ability to differentiate into osteoblasts and odontoblasts. Scaffolds are critical for cell differentiation because they replicate the 3D microenvironment of the niche and enhance cell adhesion, migration, and differentiation. The purpose of this study is to present the biological responses of human DPSCs to a purified 3D chitin scaffold derived from the marine demosponge Aplysina fistularis and modified with hydroxyapatite (HAp). Responses examined included proliferation, adhesion, and differentiation. The control culture consisted of the human osteoblast cell line, hFOB 1.19. Electron microscopy was used to examine the ultrastructure of the cells (transmission electron microscopy) and the surface of the scaffold (scanning electron microscopy). Cell adhesion to the scaffolds was determined by neutral red and crystal violet staining methods. An alkaline phosphatase (ALP) assay was used for assessing osteoblast/odontoblast differentiation. We evaluated the expression of osteogenic marker genes by performing ddPCR for ALP, RUNX2, and SPP1 mRNA expression levels. The results show that the chitin biomaterial provides a favorable environment for DPSC and hFOB 1.19 cell adhesion and supports both cell proliferation and differentiation. The chitin scaffold, especially with HAp modification, isolated from A. fistularis can make a significant contribution to tissue engineering and regenerative medicine.

Circular RNA-FK501 binding protein 51 boosts bone marrow mesenchymal stem cell proliferation and osteogenic differentiation via modulating microRNA-205-5p/Runt-associated transcription factor 2 axis

ObjectiveOsteogenesis is the key process of bone homeostasis differentiation. Numerous studies have manifested that circular RNA (circRNA) is a critical regulator of osteogenesis. The research was to explore circRNA-mediated mechanisms in osteogenesis.MethodsBone marrow mesenchymal stem cells (BMSCs) were cultured and induced to osteogenic differentiation (OD). Then, oe-circ-FKBP5, oe-NC, si-circ-FKBP5, si-NC, miR-205-5p mimic, mimic NC, miR-205-5p inhibitor, inhibitor NC, sh-RUNX2, or sh-NC were transfected into BMSCs. Alkaline phosphatase (ALP) activity was detected by ALP staining, cell mineralization was detected by alizarin red staining, cell proliferation was detected by CCK-8, and cell apoptosis was detected by flow cytometry. Then, the expression of circ-FKBP5, miR-205-5p, RUNX2 and osteogenic marker genes was detected by RT-qPCR, and the expression of RUNX2 protein was detected by Western blot. Finally, the targeting relationship between miR-205-5p and circ-FKBP5 or RUNX2 was verified by bioinformation website analysis and dual luciferase reporter gene detection.ResultsCirc-FK501 binding protein 51 (FKBP5) was distinctly elevated during OD of BMSCs. Elevated circ-FKBP5 boosted the proliferation and OD, as well as expression of osteogenic marker genes while reduced apoptosis of BMSCs. Down-regulation of circ-FKBP5 inhibited BMSCs proliferation, OD and osteogenic marker gene expression, and promoted apoptosis of BMSCs. Subsequently, circ-FKBP5 combined with miR-205-5p and constrained miR-205-5p expression. Silenced miR-205-5p boosted proliferation, OD, and expression of osteogenic marker genes and suppressed apoptosis of BMSCs. However, up-regulation of miR-205-5p inhibited BMSC proliferation, OD and osteogenic marker gene expression, and promoted apoptosis. Additionally, miR-205-5p targeted Runt-associated transcription factor 2 (RUNX2). Repression of RUNX2 turned around the effect of circ-FKBP5 overexpression on BMSCs.ConclusionIn brief, circ-FKBP5 boosted BMSC proliferation and OD by mediating the miR-205-5p/RUNX2 axis.

Effect of icariin surface treatment on the resorption of denuded roots after replantation in rat.

Limiting the incidence of resorption associated with delayed replantation of avulsed teeth is critical for long-term tooth survival. In this study, we assessed whether icariin, a natural product with anti-osteoclastic properties, could reduce root resorption in a rat model of tooth replantation. Cytocompatibility of icariin (10, 20, 40 and 80 μM) was evaluated by CCK-8 proliferation assay in vitro, and an osteoclastogenesis assay was performed to evaluate the effect of icariin on the differentiation of rat bone marrow macrophages and human peripheral blood monocytes into tartrate-resistant acid phosphatase-stained (TRAP+ ) multinucleated giant cells (MNGCs). Differentiation of human periodontal ligament stem cells (hPDLSCs) treated with icariin (10 μM) was also evaluated at 5, 10 and 21 days of osteogenic induction. The first maxillary molars of five-week-old male Sprague-Dawley rats were extracted, denuded of PDL, then treated either with neutralized collagen solution (Carrier control) or icariin in collagen (3 μg/μL) before replantation into their sockets. The animals were euthanized 2 weeks post-surgery for micro-computed tomography (micro-CT) imaging and histological analyses. Icariin was cytocompatible and significantly reduced the differentiation of TRAP+ MNGCs in a dose-dependent manner compared to the control. Moreover, icariin enhanced alkaline phosphatase activity, expression of osteogenic marker genes and proteins, and calcium deposition in hPDLSCs. Micro-CT imaging of the replanted samples demonstrated a significantly higher volume of remaining roots in the icariin-treated group than in the control group. Histological analysis revealed a marked number of resorptive lacunae with TRAP activity in the control group, whereas icariin-treated samples showed signs of functional healing and reduced osteoclastic activity. Icariin was biocompatible and demonstrated potent anti-osteoclastic and pro-osteogenic properties that reduced resorption and promoted functional healing of denuded roots in a rat maxillary first molar model of replantation. These findings indicate that root surface treatment with icariin may be a clinically relevant and practical method for improving the retention and survival of teeth with compromised PDL after delayed replantation following traumatic avulsion.

Osteogenic differentiation of human amniotic mesenchymal stem cells by phycocyanin and phycoerythrin pigments isolated from Spirulina platensis and Gracilaria gracilis algae

Bone regeneration is a multistep and regular physiological process that occurs normally in fracture repair and bone defects. However, some factors such as aging, particular diseases and some drugs prevent or slowdown bone natural healing. Cell therapy using stem cells and differentiation activating factors is an effective treatment method for bone regeneration triggering in unusual conditions. Therefore, in the present study the effect of phycocyanin and phycoerythrin pigments which isolated from Spirulina platensis and Gracilaria gracilis algae was investigate on osteogenic differentiation potency of human Amniotic Mesenchymal Stem Cells (hAMSCs). For this purpose, hAMSCs were exposed to 300, 500, and 700 µg/ml concentrations of phycocyanin and phycoerythrin pigments and then the cells viability was measured with MTT assay in 48 and 72 h after treatment. The osteo-differentiation level of cells was studied by measuring ALP activity using calorimetric method and Alizarin red staining for calcium deposition in 7 and 21 days after treatment. Also, total RNA of cells was extracted in different time periods and then cDNA synthesized with specific primers, and relative expression of Runx2, β-catenin and Osteocalcin genes were investigated using SYBR Green RT-qPCR technique. Osteogenic differentiation of hAMSCs that treated with pigments was confirmed by mineral deposits staining and increased level of ALP activity. Furthermore, these pigments elevated significantly the expression of osteogenic marker genes compared to control samples and caused hAMSCs to differentiate into osteoblast cells. According to these results, phycocyanin and phycoerythrin may suggest as suitable osteogenic supplements with low toxicity, low cost and high efficiency, although the molecular mechanism of its efficacy is not available yet.

Surgical suction filter-derived bone graft displays osteogenic miRNA and mRNA patterns

PurposeRecently, a surgical suction filter device was introduced which aims at generating a suction filter-derived bone grafting substitute (SF-BGS). The osteogenic capacity of this grafting material, however, is unclear. MicroRNAs (miRNAs) and osteogenic mRNAs may influence these processes. The aim of this study was therefore to investigate the quality of the SF-BGS by determining the expression of miRNAs and osteogenic mRNAs.MethodsSamples were collected during non-union surgery. Upon exposure of the intramedullary canal, the surgical vacuum system was fitted with the suction filter device containing collagen complex and synthetic β-TCP: (Ca3(PO4)2, granule size 5–8 mm, total volume 10 mL (Cerasorb Foam®, Curasan AG, Kleinostheim, Germany). As a control, venous blood was used as in current clinical practice. Samples were snap-frozen and mechanically disrupted. MiRNAs and mRNAs were isolated, transcribed, and pooled for qPCR analysis. Lastly, mRNA targets were determined through in silico target analyses.ResultsThe study population consisted of seven patients with a posttraumatic long bone non-union (4♀; mean age 54 ± 16 years). From the array data, distinct differences in miRNA expression were found between the SF-BGS and control samples. Osteogenic marker genes were overall upregulated in the SF-BGS. Qiagen IPA software identified 1168 mRNA targets for 43 of the overall deregulated miRNAs.ConclusionThis study revealed distinctly deregulated and exclusively expressed osteogenic miRNAs in SF-BGS, as well as overall enhanced osteogenic marker gene expression, as compared to the venous blood control group. These expression profiles were not seen in control samples, indicating that the derived material displays an osteogenic profile. It may therefore be a promising tool to generate a BGS or graft extender when needed.

Pretreatment of Mesenchymal Stem Cells with Electrical Stimulation as a Strategy to Improve Bone Tissue Engineering Outcomes.

Electrical stimulation (EStim), whether used alone or in combination with bone tissue engineering (BTE) approaches, has been shown to promote bone healing. In our previous in vitro studies, mesenchymal stem cells (MSCs) were exposed to EStim and a sustained, long-lasting increase in osteogenic activity was observed. Based on these findings, we hypothesized that pretreating MSC with EStim, in 2D or 3D cultures, before using them to treat large bone defects would improve BTE treatments. Critical size femur defects were created in 120 Sprague-Dawley rats and treated with scaffold granules seeded with MSCs that were pre-exposed or not (control group) to EStim 1 h/day for 7 days in 2D (MSCs alone) or 3D culture (MSCs + scaffolds). Bone healing was assessed at 1, 4, and 8 weeks post-surgery. In all groups, the percentage of new bone increased, while fibrous tissue and CD68+ cell count decreased over time. However, these and other healing features, like mineral density, bending stiffness, the amount of new bone and cartilage, and the gene expression of osteogenic markers, did not significantly differ between groups. Based on these findings, it appears that the bone healing environment could counteract the long-term, pro-osteogenic effects of EStim seen in our in vitro studies. Thus, EStim seems to be more effective when administered directly and continuously at the defect site during bone healing, as indicated by our previous studies.

Alginate hydrogels containing different concentrations of magnesium-containing poly(lactic-co-glycolic acid) microspheres for bone tissue engineering

The easy loss of crosslinking ions in alginate can result in structural collapse and loss of its characteristics as a bone scaffold. A novel injectable tissue engineering scaffold containing poly(lactic-co-glycolic acid) (PLGA) microspheres and alginate was fabricated to improve alginate’s physiochemical and biological properties. MgCO3 and MgO were loaded at a 1:1 ratio into PLGA microspheres to form biodegradable PLGA microspheres containing magnesium (PMg). Subsequently, different concentrations of PMg were mixed into a Ca2+ suspension and employed as crosslinking agents for an alginate hydrogel. A pure Ca2+ suspension was used as the alginate crosslinking agent in the control group. The influence of PMg on the physiochemical properties of the injectable scaffolds, including the surface morphology, degradation rate, Mg2+ precipitation concentration, and the swelling rate, was investigated. MC3T3-E1 cells were seeded onto the hydrogels to evaluate the effect of the resultant alginate on osteoblastic attachment, proliferation, and differentiation. The physicochemical properties of the hydrogels, including morphology, degradation rate, and swelling ratio, were effectively tuned by PMg. Inductively coupled plasma-optical emission spectroscopy results showed that, in contrast to those in pure PMg, the magnesium ions (Mg2+) in alginate hydrogel containing PMg microspheres (Alg-PMg) were released in a dose-dependent and slow-releasing manner. Additionally, Alg-PMg with an appropriate concentration of PMg not only improved cell attachment and proliferation but also upregulated alkaline phosphatase activity, gene expression of osteogenic markers, and related growth factors. These findings indicate that PMg incorporation can regulate the physicochemical properties of alginate hydrogels. The resultant hydrogel promoted cell attachment, matrix mineralization, and bone regeneration. The hydrogel described in this study can be considered a promising injectable scaffold for bone tissue engineering.

Mechanical and Functional Improvement of β-TCP Scaffolds for Use in Bone Tissue Engineering.

Autologous bone transplantation is still considered as the gold standard therapeutic option for bone defect repair. The alternative tissue engineering approaches have to combine good hardiness of biomaterials whilst allowing good stem cell functionality. To become more useful for load-bearing applications, mechanical properties of calcium phosphate materials have to be improved. In the present study, we aimed to reduce the brittleness of β-tricalcium phosphate (β-TCP). For this purpose, we used three polymers (PDL-02, -02a, -04) for coatings and compared resulting mechanical and degradation properties as well as their impact on seeded periosteal stem cells. Mechanical properties of coated and uncoated β-TCP scaffolds were analyzed. In addition, degradation kinetics analyses of the polymers employed and of the polymer-coated scaffolds were performed. For bioactivity assessment, the scaffolds were seeded with jaw periosteal cells (JPCs) and cultured under untreated and osteogenic conditions. JPC adhesion/proliferation, gene and protein expression by immunofluorescent staining of embedded scaffolds were analyzed. Raman spectroscopy measurements gave an insight into material properties and cell mineralization. PDL-coated β-TCP scaffolds showed a significantly higher flexural strength in comparison to that of uncoated scaffolds. Degradation kinetics showed considerable differences in pH and electrical conductivity of the three different polymer types, while the core material β-TCP was able to stabilize pH and conductivity. Material differences seemed to have an impact on JPC proliferation and differentiation potential, as reflected by the expression of osteogenic marker genes. A homogenous cell colonialization of coated and uncoated scaffolds was detected. Most interesting from a bone engineer's point of view, the PDL-04 coating enabled detection of cell matrix mineralization by Raman spectroscopy. This was not feasible with uncoated scaffolds, due to intercalating effects of the β-TCP material and the JPC-formed calcium phosphate. In conclusion, the use of PDL-04 coating improved the mechanical properties of the β-TCP scaffold and promoted cell adhesion and osteogenic differentiation, whilst allowing detection of cell mineralization within the ceramic core material.