Primary Human Osteoblasts Research Articles

Fabrication of 3D melt electrowritting multiphasic scaffold with bioactive and osteoconductivite functionalities for periodontal regeneration

This work aimed to design a multifunctional biphasic 3D scaffold for periodontal tissue regeneration. A 3D fibrous scaffold made from medical-grade poly (ε-caprolactone) (PCL) with high porosity (>90%) and well-oriented fibres was fabricated by a custom design melt electrowriting (MEW) device. A biomimetic process was employed to form a bioactive calcium phosphate (CaP) layer with nanostructure (nanoflakes-like) morphology onto the 3D MEW fibrous surface to stimulate rapid bone formation. Primary human osteoblasts (hOBs) were seeded within the coated 3D fibrous scaffolds for 28 days to acquire the bone compartment of the tissue-engineered construct (TEC). The biphasic construct was obtained by placing an established in vitro periodontal ligament (PDL) cell sheet onto the surface of the bone compartment. Subsequently, a decellularized multiphasic TEC by exploiting a lyophilization approach was obtained. Laser scanning confocal microscopy and scanning electron microscopy confirmed the retention of a functional extracellular matrix within the PDL and bone compartments following scaffold decellularization and lyophilization processes. These findings suggest that lyophilized decellularized biphasic 3D constructs with high porosity constitute a viable ‘off the shelf’ strategy for developing an extracellular matrix-based product to facilitate periodontal regeneration.

Bioessential Inorganic Molecular Wire-Reinforced 3D-Printed Hydrogel Scaffold for Enhanced Bone Regeneration.

Materials with physicochemical properties and biological activities similar to those of the natural extracellular matrix are in high demand in tissue engineering. In particular, Mo3 Se3 - inorganic molecular wire (IMW) is a promising material composed of bioessential minerals and possess nanometer-scale diameters, negatively charged surfaces, physical flexibility, and nanotopography characteristics, which are essential for interactions with cell membrane proteins. Here, an implantable 3D Mo3 Se3 - IMW enhanced gelatin-GMA/silk-GMA hydrogel (IMW-GS hydrogel) is developed for osteogenesis and bone formation, followed by biological evaluations. The mechanical properties of the 3D printed IMW-GS hydrogel are improved by noncovalent interactions between the Mo3 Se3 - IMWs and the positively charged residues of the gelatin molecules. Long-term biocompatibility with primary human osteoblast cells (HOBs) is confirmed using the IMW-GS hydrogel. The proliferation, osteogenic gene expression, collagen accumulation, and mineralization of HOBs improve remarkably with the IMW-GS hydrogel. In in vivo evaluations, the IMW-GS hydrogel implantation exhibits a significantly improved new bone regeneration of 87.8 ± 5.9% (p < 0.05) for 8 weeks, which is higher than that from the gelatin-GMA/silk-GMA hydrogel without Mo3 Se3 - IMW. These results support a new improved strategy with in vitro and in vivo performance of 3D IMW enhanced scaffolds in tissue engineering.

Surface Free Energy and Composition Changes and Ob Cellular Response to CHX-, PVPI-, and ClO2-Treated Titanium Implant Materials.

The study evaluated the interaction of a titanium dental implant surface with three different antibacterial solutions: chlorhexidine, povidone-iodine, and chlorine dioxide. Implant surface decontamination is greatly challenging modern implant dentistry. Alongside mechanical cleaning, different antibacterial agents are widely used, though these could alter implant surface properties. Commercially pure (CP) grade 4 titanium (Ti) discs were treated with three different chemical agents (chlorhexidine 0.2% (CHX), povidone-iodine 10% (PVPI), chlorine dioxide 0.12% (ClO2)) for 5 min. Contact angle measurements, X-ray photoelectron spectroscopy (XPS) analysis, and cell culture studies were performed. Attachment and proliferation of primary human osteoblast cells were investigated via MTT (dimethylthiazol-diphenyl tetrazolium bromide), alamarBlue, LDH (lactate dehydrogenase), and fluorescent assays. Contact angle measurements showed that PVPI-treated samples (Θ = 24.9 ± 4.1) gave no difference compared with controls (Θ = 24.6 ± 5.4), while CHX (Θ = 47.2 ± 4.1) and ClO2 (Θ = 39.2 ± 9.8) treatments presented significantly higher Θ values. All samples remained in the hydrophilic region. XPS analysis revealed typical surface elements of CP grade 4 titanium (Ti, O, and C). Both MTT and alamarBlue cell viability assays showed similarity between treated and untreated control groups. The LDH test revealed no significant difference, and fluorescent staining confirmed these results. Although there was a difference in surface wettability, a high proliferation rate was observed in all treated groups. The in vitro study proved that CHX, PVPI, and ClO2 are proper candidates as dental implant decontamination agents.

Cell-cell interactions and fluctuations in the direction of motility promote directed migration of osteoblasts in direct current electrotaxis.

Under both physiological (development, regeneration) and pathological conditions (cancer metastasis), cells migrate while sensing environmental cues in the form of mechanical, chemical or electrical stimuli. In the case of bone tissue, osteoblast migration is essential in bone regeneration. Although it is known that osteoblasts respond to exogenous electric fields, the underlying mechanism of electrotactic collective movement of human osteoblasts is unclear. Here, we present a computational model that describes the osteoblast cell migration in a direct current electric field as the motion of a collection of active self-propelled particles and takes into account fluctuations in the direction of single-cell migration, finite-range cell-cell interactions, and the interaction of a cell with the external electric field. By comparing this model with in vitro experiments in which human primary osteoblasts are exposed to a direct current electric field of different field strengths, we show that cell-cell interactions and fluctuations in the migration direction promote anode-directed collective migration of osteoblasts.

Effect of a 5-aminolevulinic acid gel and 660 nm red LED light on human oral osteoblasts: a preliminary in vitro study

This study aimed to evaluate the effects of a new photodynamic protocol (ALAD-PDT) on primary human osteoblasts (hOBs). The ALAD-PDT protocol consists of a heat-sensitive gel with 5% 5-delta aminolevulinic acid commercialized as Aladent (ALAD), combined with 630 nm LED. For this purpose, the hOBs, explanted from human mandible bone fragments, were used and treated with different ALAD concentrations (10%, 50%, 100% v/v) incubated for 45 min and immediately afterwards irradiated with a 630 nm LED device for 7 min. The untreated and unirradiated cells were considered control (CTRL). The cellular accumulation of the photosensitizer protoporphyrin IX (PpIX), the proliferation, the alkaline phosphatase (ALP) activity, and the calcium deposition were assessed. All concentrations (10, 50, 100%) determined a significant increment of PpIX immediately after 45 min of incubation (0 h) with the highest peak by ALAD (100%). The consequent 7 min of light irradiation caused a slight decrease in PpIX. At 48 h and 72 h, any increment of PpIX was observed. The concentration 100% associated with LED significantly increased hOB proliferation at 48 h (+ 46.83%) and 72 h (+ 127.75%). The 50% and 100% concentrations in combination to the red light also stimulated the ALP activity, + 12.910% and + 14.014% respectively. The concentration 100% with and without LED was selected for the assessment of calcium deposition. After LED irradiation, a significant increase in calcium deposition was observed and quantified (+ 72.33%). In conclusion, the ALAD-PDT enhanced proliferation, the ALP activity, and mineralized deposition of human oral osteoblasts, highlighting a promising potential for bone tissue regeneration.

In Vitro Cytotoxicity and Mineralization Potential of an Endodontic Bioceramic Material.

The interest in bioceramic materials has been steadily growing for different applications in endodontics. With the continued introduction of new bioceramic-based materials into the market, it is of great importance to assess the biocompatibility before providing recommendations on their clinical use. This study evaluated the in vitro cytotoxicity and mineralization potential of two consistencies of unset premixed bioceramic material (TotalFill BC RRM putty and TotalFill BC sealer) compared with an epoxy resin-based sealer (AH Plus) on osteoblast cells. Overall, 100% extracts were obtained by weighing 0.1 g of each material in 1 mL of cell culture media. Primary human osteoblast (HOB) cells (n = 4) were treated with different concentrations (100, 50, 25, 12.50, and 6.25%) of each extract. XTT assay and Alizarin Red S staining were used to evaluate the cytotoxic effect and the biomineralization potential, respectively. Data were analyzed by one-way analysis of variance followed by Tukey's post hoc tests. The cytotoxicity assay after 24 h treatment showed that all materials at high concentrations of the extract (100 and 50%) were toxic to HOB (p < 0.001). On the contrary to TotalFill BC RRM Putty, AH Plus and TotalFill BC sealer were toxic at 25% concentration. However, at 12.5% concentration and lower, all materials were nontoxic. The mineralization potential analyzed after 7 and 14 days showed that TotalFill BC material-treated cells could deposit mineralized nodules in the normal and osteogenic medium unlike AH plus-treated cells. At low concentrations, TotalFill BC materials showed higher biocompatibility to HOB cells than AH Plus, enhanced the viability of the cells, maintained their typical morphology, and induced the formation of mineralized nodules. Despite the encouraging data, clinical trials are needed to identify the effect of this material on the long-term outcome of endodontic treatment.

Nanoparticles of naturally occurring PPAR-γ inhibitor betulinic acid ameliorates bone marrow adiposity and pathological bone loss in ovariectomized rats via Wnt/β-catenin pathway

AimsPostmenopausal osteoporosis is one of the world's biggest yet unnoticed health issues. After ovariectomy, declined estrogen level significantly contributes to the elevation of bone marrow adiposity and bone loss leading to osteoporosis. Therapeutics to prevent osteoporosis addressing various aspects are now in short supply. In this study we made an approach to synthesize nanoparticles of naturally occurring PPAR-γ inhibitor, betulinic acid (BA/NPs) and tested the same in altered bone metabolisms developed after ovariectomy. Main methodsThe osteogenic efficacy of BA/NPs was established in human and rat primary osteoblast cells using qRT-PCR and immunoblot analysis. Furthermore, lineage allocations of multipotent bone marrow stromal cells were evaluated. Various aspects of altered bone metabolism after ovariectomy such as bone marrow adiposity and pathological bone loss were evaluated using μCT and histological assessments. Key findingsBA/NPs exert potential osteogenic efficacy by modulating key osteogenic markers such as RUNX2 and BMP2. Mechanistically BA/NPs regulate osteoblastogenesis through Wnt/β-catenin signaling. Further, BA/NPs showed the potential to inhibit the differentiation of multipotent BMSCs towards adipogenesis while favouring the osteogenic lineage via Wnt/β-catenin pathway. In the in vivo study, increased bone marrow adiposity was reduced in ovariectomized rats after BA/NPs treatment as assessed by histology and μCT analysis. Loss of bone mineral density as a hallmark of pathological bone loss was also abrogated by BA/NPs. Furthermore, increased obesity after OVX was also prevented in BA/NPs treated animals. SignificanceOur findings imply that BA/NPs could be used further as a viable drug lead to counteract various pathophysiological challenges after menopause.

Trabecular Titanium for Orthopedic Applications: Balancing Antimicrobial with Osteoconductive Properties by Varying Silver Contents.

Periprosthetic joint infection (PJI) and implant loosening are the most common complications after joint replacement surgery. Due to their increased surface area, additively manufactured porous metallic implants provide optimal osseointegration but they are also highly susceptible to bacterial colonization. Antibacterial surface coatings of porous metals that do not inhibit osseointegration are therefore highly desirable. The potential of silver coatings on arthroplasty implants to inhibit PJI has been demonstrated, but the optimal silver content and release kinetics have not yet been defined. A tight control over the silver deposition coatings can help overcome bacterial infections while reducing cytotoxicity to human cells. In this regard, porous titanium sputtered with silver and titanium nitride with increasing silver contents enabled controlling the antibacterial effect against common PJI pathogens while maintaining the metabolic activity of human primary cells. Electron beam melting additively manufactured titanium alloys, coated with increasing silver contents, were physico-chemically characterized and investigated for effects against common PJI pathogens. Silver contents from 7 at % to 18 at % of silver were effective in reducing bacterial growth and biofilm formation. Staphylococcus epidermidis was more susceptible to silver ions than Staphylococcus aureus. Importantly, all silver-coated titanium scaffolds supported primary human osteoblasts proliferation, differentiation, and mineralization up to 28 days. A slight reduction of cell metabolic activity was observed at earlier time points, but no detrimental effects were found at the end of the culture period. Silver release from the silver-coated scaffolds also had no measurable effects on primary osteoblast gene expression since similar expression of genes related to osteogenesis was observed regardless the presence of silver. The investigated silver-coated porous titanium scaffolds may thus enhance osseointegration while reducing the risk of biofilm formation by the most common clinically encountered pathogens.

Chi3L1 is a therapeutic target in bone metabolism and a potential clinical marker in patients with osteoporosis

BMP2 is clinically used as an ectopic bone inducer and plays a significant role in bone development, formation, and diseases. Chitinase 3-like 1 protein (Chi3L1) is found in the skeletal system. However, Chi3L1-mediated bone metabolism and aging-related bone erosion via BMP2 signaling have not yet been demonstrated. Herein, Chi3L1 increased BMP2-induced osteoblast differentiation in mesenchymal precursor cells and human primary osteoblasts. Chi3L1KO(-/-) showed abnormal bone development, and primary osteoblasts isolated from Chi3L1KO(-/-) exhibited impaired osteoblast differentiation and maturation. Chi3L1 also potentiated BMP2 signaling and RUNX2 expression in primary osteoblasts. Chi3L1 interacted with BMPRIa, which increased the surface expression of BMPRIa and promoted BMP2 signaling to induce osteoblast differentiation. Chi3L1KO(-/-) mice showed bone formation reduced with a decrease in RUNX2 expression in calvarial defects. Chi3L1KO(-/-) mice exhibited aging-related osteoporotic bone loss with decreases in the levels of RUNX2 and OPG, while serum PYD level and osteoclast number increased. Chi3L1 increased OPG via non-canonical BMP2 signaling in osteoblasts, which suppressed osteoclastogenesis in BMMs. Furthermore, ROC analysis showed that serum Chi3L1 level clinically decreased in osteoporosis patients. Our findings demonstrate that Chi3L1 promotes bone formation, suppresses osteoclastogenesis, and prevents aging-related osteoporosis.

Moringa oleifera leaf extract induces osteogenic-like differentiation of human osteosarcoma SaOS2 cells

IntroductionMoringa oleifera is known as a ‘natural nutrition of the tropics’ because it provides vital nutritional supplements and a variety of pharmacological benefits. The focus of this study was to elucidate the dose dependent effects of Moringa oleifera leaf (MOL) extract on the growth of the human osteoblast-like osteosarcoma SaOS-2 cell line and primary osteoblast cells. MethodsTrypan blue & tetrazolium assay, intracellular ROS generation, chromatin condensation, cell cycle analysis, alkaline phosphatase (ALP), mineralization, and osteogenic gene expression were tested on both treated and untreated osteosarcoma SaOS-2 cells. ResultsAs revealed by cell viability assay, growth activity was observed at concentrations 25 and 50 μg/mL of MOL extract, whereas 100 and 200 μg/mL doses decreased the proliferation activity, resulting in ROS production and chromatin condensation. Cell cycle study revealed that MOL extract at 50 and 100 μg/mL concentrations arrested the cells in the G2/M phase. Low doses increased the ALP levels, mineralization, and expression of the bone morphogenetic protein 2 (BMP2) and runt-related transcription factor 2 (Runx2) genes in osteoblast-like SaOS-2 cells, however, high doses inhibited the proliferation properties of MOL extract. Through AutoDock Vina and iGEMDOCK 2.1, the interaction of active components of MOL, such as β-sitosterol, quercetin and kaempferol, with BMP2 and Runx2 proteins revealed a reasonable binding affinity. Moreover, these components did not show any Lipinski's rule of five violation and showed predictable pharmacokinetic properties. ConclusionThe results of the biphasic dose-response of MOL extract on the growth activity of osteoblast-like SaOS-2 cells and in silico binding interface, may provide a therapeutic and/or preventive implication in prospective drug development.

Nicotinamide Mononucleotide Alleviates Osteoblast Senescence Induction and Promotes Bone Healing in Osteoporotic Mice.

Combating the accumulated senescent cells and the healing of osteoporotic bone fractures in the older remains a significant challenge. Nicotinamide mononucleotide (NMN), a precursor of NAD+, is an excellent candidate for mitigating aging-related disorders. However, it is unknown if NMN can alleviate senescent cell induction and enhance osteoporotic bone fracture healing. Here we show that NMN treatment partially reverses the effects of tumor necrosis factor-alpha (TNF-α) on human primary osteoblasts (HOBs): senescent cell induction, diminished osteogenic differentiation ability, and intracellular NAD+ and NADH levels. Mechanistically, NMN restores the mitochondrial dysfunction in HOBs induced by TNF-α evidenced by increased mitochondrial membrane potential and reduced reactive oxidative species and mitochondrial mass. NMN also increases mitophagy activity by down-regulating P62 expression and up-regulating light chain 3B-II protein expression. In addition, the cell senescence protective effects of NMN on HOBs are mitigated by a mitophagy inhibitor (Bafilomycin A1). In vivo, NMN supplementation attenuates senescent cell induction in growth plates, partially prevents osteoporosis in an ovariectomized mouse model, and accelerates bone healing in osteoporotic mice. We conclude that NMN can be a novel and promising therapeutic candidate to enhance bone fracture healing capacity in the older.

E-Cigarette Vapour Condensate Reduces Viability and Impairs Function of Human Osteoblasts, in Part, via a Nicotine Dependent Mechanism.

Cigarette consumption negatively impacts bone quality and is a risk-factor for the development of multiple bone associated disorders, due to the highly vascularised structure of bone being exposed to systemic factors. However, the impact on bone to electronic cigarette (e-cigarette) use, which contains high doses of nicotine and other compounds including flavouring chemicals, metal particulates and carbonyls, is poorly understood. Here, we present the first evidence demonstrating the impact of e-cigarette vapour condensate (replicating changes in e-cigarette liquid chemical structure that occur upon device usage), on human primary osteoblast viability and function. 24 h exposure of osteoblasts to e-cigarette vapour condensate, generated from either second or third generation devices, significantly reduced osteoblast viability in a dose dependent manner, with condensate generated from the more powerful third generation device having greater toxicity. This effect was mediated in-part by nicotine, since exposure to nicotine-free condensate of an equal concentration had a less toxic effect. The detrimental effect of e-cigarette vapour condensate on osteoblast viability was rescued by co-treatment with the antioxidant N-Acetyl-L-cysteine (NAC), indicating toxicity may also be driven by reactive species generated upon device usage. Finally, non-toxic doses of either second or third generation condensate significantly blunted osteoblast osteoprotegerin secretion after 24 h, which was sustained for up to 7 days. In summary we demonstrate that e-cigarette vapour condensate, generated from commonly used second and third generation devices, can significantly reduce osteoblast viability and impair osteoblast function, at physiologically relevant doses. These data highlight the need for further investigation to inform users of the potential risks of e-cigarette use on bone health, including, accelerating bone associated disease progression, impacting skeletal development in younger users and to advise patients following orthopaedic surgery, dental surgery, or injury to maximise bone healing.

390 Assessing Biocompatibility &amp; Mechanical Testing of 3D-Printed PEEK Versus Milled PEEK in Maxillofacial Surgical Applications

Abstract Aim PEEK (poly-ether-ether-ketone) is an innovative thermoplastic polymeric biomaterial used in Maxillofacial Surgery. As opposed to metallic implants, PEEK implants can be 3D-printed specifically for patients and do not suffer stress shielding effects- a major cause of implant failure. This investigation aimed to compare mechanical integrity and biocompatibility of modern 3D-printed PEEK implants against traditionally produced Milled-PEEK implants and metallic biomaterials e.g., Titanium. Method Fused-deposition-modelling and non-CNC Milling were used to produce ASTM/ISO-designed PEEK samples. Samples were designed and produced using the Apium P155 FDM-3D-printer, Fusion 360 and Simplify3D software. Fracture Toughness KIc, Tensile Strength, Vickers micro-hardness and 3-point flexural strength tests were performed. Cell adhesion on sandblasted 3D-printed PEEK was evaluated by culturing primary human endothelial cells and osteoblasts (HUVEC, HOBS) and evaluating cell growth. Results Milled PEEK had higher tensile strength (p=0.022), higher flexural strength (p&amp;lt;0.0001) and higher fracture toughness (p=0.003) than FDM-printed PEEK. However, FDM-printed PEEK demonstrated higher microhardness than Milled PEEK (p=0.014). Both FDM and Milled PEEK showed mechanical strength values in normal ranges of cortical bone, and close to ranges of alternative bone-replacement biomaterials. Both HOBS and HUVEC demonstrated strong adhesion to PEEK with &amp;gt;95% viability. Conclusions 3D-printing of PEEK is an emerging technology with advantages over milling and metallic biomaterials in implant production. Nonetheless, this manufacturing modality may produce 3D-printed PEEK devices with lower mechanical resistance parameters compared to milled PEEK, but with values compatible with natural bone. Sandblasting is a modality to create uniform micro-rough surfaces potentially enhancing osseointegration of PEEK implants with host tissue.

The Effects of 5% 5-Aminolevulinic Acid Gel and Red Light (ALAD-PDT) on Human Fibroblasts and Osteoblasts.

This study aimed to evaluate the effects of a new photodynamic protocol (ALAD-PDT), consisting of 5% 5-aminolevulinic acid-gel and 630 nm-LED, already used for antibacterial effects in the treatment of periodontitis, on human gingival fibroblasts (HGF) and primary human osteoblasts (HOB). HGF and HOB were incubated with different ALAD concentrations for 45 min, and subsequently irradiated with 630 nm-LED for 7 min. Firstly, the cytotoxicity at 24 h and proliferation at 48 and 72 h were assessed. Then the intracellular content of the protoporphyrin IX (PpIX) of the ROS and the superoxide dismutase (SOD) activity were investigated at different times. Each result was compared with untreated and unirradiated cells as the control. Viable and metabolic active cells were revealed at any concentrations of ALAD-PDT, but only 100-ALAD-PDT significantly enhanced the proliferation rate. The PpIX fluorescence significantly increased after the addition of 100-ALAD, and decreased after the irradiation. Higher ROS generation was detected at 10 min in HGF, and at 30 min in HOB. The activity of the SOD enzyme augmented at 30 min in both cell types. In conclusion, ALAD-PDT not only showed no cytotoxic effects, but had pro-proliferative effects on HGF and HOB, probably via ROS generation.

Mast cell chymase has a negative impact on human osteoblasts

Mast cells have been linked to osteoporosis and bone fractures, and in a previous study we found that mice lacking a major mast cell protease, chymase, develop increased diaphyseal bone mass. These findings introduce the possibility that mast cell chymase can regulate bone formation, but the underlying mechanism(s) has not previously been investigated. Here we hypothesized that chymase might exert such effects through a direct negative impact on osteoblasts, i.e., the main bone-building cells. Indeed, we show that chymase has a distinct impact on human primary osteoblasts. Firstly, chymase was shown to have pronounced effects on the morphological features of osteoblasts, including extensive cell contraction and actin reorganization. Chymase also caused a profound reduction in the output of collagen from the osteoblasts, and was shown to degrade osteoblast-secreted fibronectin and to activate pro-matrix metallopeptidase-2 released by the osteoblasts. Further, chymase was shown to have a preferential impact on the gene expression, protein output and phosphorylation status of TGFβ-associated signaling molecules. A transcriptomic analysis was conducted and revealed a significant effect of chymase on several genes of importance for bone metabolism, including a reduction in the expression of osteoprotegerin, which was confirmed at the protein level. Finally, we show that chymase interacts with human osteoblasts and is taken up by the cells. Altogether, the present findings provide a functional link between mast cell chymase and osteoblast function, and can form the basis for a further evaluation of chymase as a potential target for intervention in metabolic bone diseases.

Promotion of angiogenesis in vitro by Astragalus polysaccharide via activation of TLR4 signaling pathway.

During the implantation of functional tissue-engineered constructs for treating bone defects, a functional vascular network is critical for the survival of the construct. One strategy to achieve rapid angiogenesis for this application is the co-culture of outgrowth endothelial cells (OECs) and primary human osteoblasts (POBs) within a scaffold prior to implantation. In the present study, we aim to investigate whether Astragalus polysaccharide (APS) promotes angiogenesis or vascularization via the TLR4 signaling pathway in a co-culture of OECs and POBs. The co-cultures were treated with various concentrations of APS for 24 h and, subsequently, another 7 days, followed by CD31 staining and analysis of micro-vessel-formation areas using software. Additionally, APS (0.4mg/ml for 24 h) was added to monocultures of OECs or POBs for evaluating proliferation, apoptosis, angiogenesis, osteogenesis, TLR4 signaling pathway, and inflammatory cytokine release. We found that APS promoted angiogenesis in the co-culture at the optimal concentration of 0.4mg/ml. TLR4 activation by APS up-regulated the expression level of TLR4/MyD88 and enhanced angiogenesis and osteogenesis in monocultures of OECs and POBs. The levels of E-selectin adhesion molecules, three cytokines (IL-6, TNF-α, and IFN-γ), and VEGF and PDGF-BB, which can induce angiogenesis, increased significantly (p < .05) following APS treatment. Therefore, APS appears to promote angiogenesis and ossification in the co-culture system via the TLR4 signaling pathway. PRACTICAL APPLICATIONS: This study demonstrates that APS may promote angiogenesis and osteocyte proliferation in OEC and POB co-culture systems through the MyD88-dependent TLR4 signaling pathway. APS might represent a potential therapeutic strategy in tissue-engineered bone implantation for the treatment of large bone defects; additionally, it has the advantage of safety, as it exhibits low or no side effects. In the future, it is expected to be used in vitro for the construction of tissue-engineered bone and in vivo after implantation in patients with bone defects for promoting rapid vascularization and ossification of tissue-engineered bone and early fusion with the recipient's bone. In addition, as a food additive, Astragalus membranaceus can be used as a tonic material in patients recovering from a fracture for promoting blood-vessel formation at the fracture site and fracture recovery. Combining traditional Chinese medicine with tissue engineering can provide further strategies for promoting the development of regenerative medicine.

Bone Sialoprotein Immobilized in Collagen Type I Enhances Bone Regeneration In vitro and In vivo.

The use of bioactive molecules is a promising approach to enhance the bone healing properties of biomaterials. The aim of this study was to define the role of bone sialoprotein (BSP) immobilized in collagen type I in various settings. In vitro studies with human primary osteoblasts in mono- or in co-culture with endothelial cells demonstrated a slightly increased gene expression of osteogenic markers as well as an increased proliferation rate in osteoblasts after application of BSP immobilized in collagen type I. Two critical size bone defect models were used to analyze bone regeneration. BSP incorporated in collagen type I increased bone regeneration only marginally at one concentration in a calvarial defect model. To induce the mechanical stability, three-dimensional printing was used to produce a stable porous cylinder of polylactide. The cylinder was filled with collagen type I and immobilized BSP and implanted into a femoral defect of critical size in rats. This hybrid material was able to significantly induce bone regeneration. Our study clearly shows the osteogenic effect of BSP when combined with collagen type I as carrier and thereby offers various approaches and options for its use as bioactive molecule in bone substitute materials.

Effect of Enterococcus faecalis OG1RF on human calvarial osteoblast apoptosis

BackgroundEnterococcus faecalis is a dominant pathogen in the root canals of teeth with persistent apical periodontitis (PAP), and osteoblast apoptosis contributes to imbalanced bone remodelling in PAP. Here, we investigated the effect of E. faecalis OG1RF on apoptosis in primary human calvarial osteoblasts. Specifically, the expression of apoptosis-related genes and the role of anti-apoptotic and pro-apoptotic members of the BCL-2 family were examined.MethodsPrimary human calvarial osteoblasts were incubated with E. faecalis OG1RF at multiplicities of infection corresponding to infection time points. Flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, caspase-3/-8/-9 activity assay, polymerase chain reaction (PCR) array, and quantitative real-time PCR were used to assess osteoblast apoptosis.ResultsE. faecalis infection increased the number of early- and late-phase apoptotic cells and TUNEL-positive cells, decreased the mitochondrial membrane potential (ΔΨm), and activated the caspase-3/-8/-9 pathway. Moreover, of all 84 apoptosis-related genes in the PCR array, the expression of 16 genes was upregulated and that of four genes was downregulated in the infected osteoblasts. Notably, the mRNA expression of anti-apoptotic BCL2 was downregulated, whereas that of the pro-apoptotic BCL2L11, HRK, BIK, BMF, NOXA, and BECN1 and anti-apoptotic BCL2A1 was upregulated.ConclusionsE. faecalis OG1RF infection triggered apoptosis in human calvarial osteoblasts, and BCL-2 family members acted as regulators of osteoblast apoptosis. Therefore, BCL-2 family members may act as potential therapeutic targets for persistent apical periodontitis.

Effects of the Interleukin-6 Receptor Blocker Sarilumab on Metabolic Activity and Differentiation Capacity of Primary Human Osteoblasts.

Interleukin (IL-) 6 is a key factor in the inflammatory processes of rheumatoid arthritis. Several biologic agents target the IL-6 signaling pathway, including sarilumab, a monoclonal antibody that blocks the IL-6 receptor and inhibits IL-6-mediated cis- and trans-signaling. A careful analysis of the IL-6 signaling blockade should consider not only inflammatory processes but also the regenerative functions of IL-6. The purpose of this study was to investigate whether inhibition of the IL-6 receptors affects differentiation of human primary osteoblasts (hOB). The effects of sarilumab on viability and the differentiation capacity in unstimulated osteoblasts as well as after stimulation with various IL-6 and sIL6-R concentrations were determined. Sarilumab treatment alone did not affect the differentiation or induction of inflammatory processes in hOB. However, the significant induction of alkaline phosphatase activity which was observed after exogenous IL-6/sIL-6R costimulation at the highest concentrations was reduced back to baseline levels by the addition of sarilumab. The IL-6 receptor blockade also decreased gene expression of mediators required for osteogenesis and bone matrix maintenance. Our results demonstrate that concomitant administration of the IL-6 receptor blocker sarilumab can inhibit IL-6/sIL-6R-induced osteogenic differentiation.

Osteoblasts in a Perfusion Flow Bioreactor-Tissue Engineered Constructs of TiO2 Scaffolds and Cells for Improved Clinical Performance.

Combining biomaterial scaffolds with cells serves as a promising strategy for engineering critical size defects; however, homogenous cellular growth within large scaffolds is challenging. Mechanical stimuli can enhance bone regeneration by modulating cellular growth and differentiation. Here, we compare dynamic seeding in a perfusion flow bioreactor with static seeding for a synthetic bone scaffold for up to 21 days using the cell line MC3T3-E1 and primary human osteoblast, confocal laser scanning microscopy, and real-time reverse transcriptase-polymerase chain reaction. The secretion of bone-related proteins was quantified using multiplex immunoassays. Dynamic culture improved cellular distribution through the TiO2 scaffold and induced a five-fold increase in cell number after 21 days. The relative mRNA expression of osteopontin of MC3T3-E1 was 40-fold enhanced after 7 and 21 days at a flow rate of 0.08 mL/min, and that of collagen type I alpha I expression was 18-fold after 21 days. A flow rate of 0.16 mL/min was 10-fold less effective. Dynamic culture increased the levels of dickkopf-related protein 1 (60-fold), osteoprotegrin (29-fold), interleukin-6 (23-fold), interleukin-8 (36-fold), monocyte chemoattractant protein 1 (28-fold) and vascular endothelial growth factor (6-fold) in the medium of primary human osteoblasts after 21 days compared to static seeding. The proposed method may have clinical potential for bone tissue engineering.