3D-printed gelatin/sodium alginate/58S bioactive glass scaffolds promote osteogenesis in vitro and in vivo.

Three-dimensional (3D)-printed scaffolds are a new strategy to fabricate biomaterials for treating bone defects. Here, using a 3D-printing technique, we fabricated scaffolds consisting of gelatin (Gel), sodium alginate (SA), and 58S bioactive glass (58S BG). To evaluate mechanical properties and biocompatibility of Gel/SA/58S BG scaffolds, the degradation test, compressive strength test, and cytotoxicity test were performed. The effect of the scaffolds on cell proliferation in vitro was determined by 4',6-diamidino-2-phenylindole (DAPI) staining. To evaluate osteoinductive properties, rBMSCs were cultured on the scaffolds for 7, 14, and 21 days and the expression of osteogenesis-related genes was analyzed using qRT-PCR. To examine the bone healing properties of Gel/SA/58S BG scaffolds in vivo, we used a rat mandibular critical-size defect bone model. The scaffolds were implanted into the defect area of rat mandible and bone regeneration and new tissue formation were assessed using microcomputed tomography (microCT) and hematoxylin and eosin (H&E) staining. The results showed that Gel/SA/58S BG scaffolds had appropriate mechanical strength as a filling material for bone defects. Furthermore, the scaffolds could be compressed within certain limits and then could recover their shape. The extract of the Gel/SA/58S BG scaffold showed no cytotoxicity. In vitro, the expression levels of Bmp2, Runx2, and OCN were increased in rBMSCs cultured on the scaffolds. In vivo, microCT and H&E staining demonstrated that scaffolds induced the formation of new bone at the mandibular defect area. These results indicated that Gel/SA/58S BG scaffolds have excellent mechanical characteristics, biocompatibility, and osteoinductive properties, suggesting that it could be a promising biomaterial for the repair of bone defects.

Research on the osteogenesis and biosafety of ECM-Loaded 3D-Printed Gel/SA/58sBG scaffolds.

Employing scaffolds containing cell-derived extracellular matrix (ECM) as an alternative strategy for the regeneration of bone defects has shown prominent advantages. Here, gelatin (Gel), sodium alginate (SA) and 58s bioactive glass (58sBG) were incorporated into deionized water to form ink, which was further fabricated into composite scaffolds by the 3D printing technique. Then, rat aortic endothelial cells (RAOECs) or rat bone mesenchymal stem cells (RBMSCs) were seeded on the scaffolds. After decellularization, two kinds of ECM-loaded scaffolds (RAOECs-ECM scaffold and RBMSCs-ECM scaffold) were obtained. The morphological characteristics of the scaffolds were assessed meticulously by scanning electron microscopy (SEM). In addition, the effects of scaffolds on the proliferation, adhesion, and osteogenic and angiogenic differentiation of RBMSCs were evaluated by Calcein AM staining and reverse transcription polymerase chain reaction (RT-PCR). In vivo, full-thickness bone defects with a diameter of 5 mm were made in the mandibles of Sprague-Dawley (SD) rats to assess the bone regeneration ability and biosafety of the scaffolds at 4, 8 and 16 weeks. The osteogenic and angiogenic potential of the scaffolds were investigated by microcomputed tomography (Micro-CT) and histological analysis. The biosafety of the scaffolds was evaluated by blood biochemical indices and histological staining of the liver, kidney and cerebrum. The results showed that the ECM-loaded scaffolds were successfully prepared, exhibiting interconnected pores and a gel-like ECM distributed on their surfaces. Consistently, in vitro experiments demonstrated that the scaffolds displayed favourable cytocompatibility. In vitro osteogenic differentiation studies showed that scaffolds coated with ECM could significantly increase the expression of osteogenic and angiogenic genes. In addition, the results from mandibular defect repair in vivo revealed that the ECM-loaded scaffolds effectively promoted the healing of bone defects when compared to the pure scaffold. Overall, these findings demonstrate that both RAOECs-ECM scaffold and RBMSCs-ECM scaffold can greatly enhance bone formation with good biocompatibility and thus have potential for clinical application in bone regeneration.

MicroRNA-155 expression is associated with pulpitis progression by targeting SHIP1.

BACKGROUND: Pulpitis is a commonly seen oral inflammation condition in clinical practice, it can cause much pain for the patient and may induce infections in other systems. Much is still unknown for the pathogenic mechanism of pulpitis. In this work, we discovered that the expression of miR-155 was associated with dental pulpal inflammation both in vivo and in vitro. METHODS AND RESULTS: Our experiments of LPS stimulated odontoblast cell line MDPC-23 showed miR-155 could act as a positive regulator by increasing the production of pro-inflammatory cytokines IL-1ß and IL-6 during inflammatory responses, whereas knockdown of miR-155 can reverse the effects. Bioinformatics analysis demonstrated that SHIP1 is a direct target of miR-155 in odontoblasts, this result was further verified at both mRNA and protein level. Inhibition of miR-155 resulted in the downregulation of inflammation factors, while co-transfection of si-SHIP1 and miR-155 inhibitor promoted the inflammatory responses. Treatment with miR-155 mimic or si-SHIP1 up-regulated the protein level of p-PI3K and p-AKT. By contrast, miR-155 inhibitor exerted the opposite effects. miR-155 mimics could upregulate the gene expression of IL-1ß and IL-6. Co-transfection of LY294002 and miR-155 mimic attenuated the inflammatory responses. Consistent with in vitro results, miR-155-/- mice could alleviate inflammatory response, as well as decrease the activation of p-PI3K and p-AKT, whereas increase the activation of SHIP1. CONCLUSIONS: Our data revealed a novel role for miR-155 in regulation of dental pulpal inflammatory response by targeting SHIP1 through PI3K/AKT signaling pathway.

LncRNA H19 promotes odontoblastic differentiation of human dental pulp stem cells by regulating miR-140-5p and BMP-2/FGF9.

BACKGROUND: Increasing evidence has revealed that long non-coding RNAs (lncRNAs) exert critical roles in biological mineralization. As a critical process for dentin formation, odontoblastic differentiation is regulated by complex signaling networks. The present study aimed to investigate the biological role and regulatory mechanisms of lncRNA-H19 (H19) in regulating the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). METHODS: We performed lncRNA microarray assay to reveal the expression patterns of lncRNAs involved in odontoblastic differentiation. H19 was identified and verified as a critical factor by qRT-PCR. The gain- and loss-of-function studies were performed to investigate the biological role of H19 in regulating odontoblastic differentiation of hDPSCs in vitro and in vivo. Odontoblastic differentiation was evaluated through qRT-PCR, Western blot, and Alizarin Red S staining. Bioinformatics analysis identified that H19 could directly interact with miR-140-5p, which was further verified by luciferase reporter assay. After overexpression of miR-140-5p in hDPSCs, odontoblastic differentiation was determined. Moreover, the potential target genes of miR-140-5p were investigated and the biological functions of BMP-2 and FGF9 in hDPSCs were verified. Co-transfection experiments were conducted to validate miR-140-5p was involved in H19-mediated odontoblastic differentiation in hDPSCs. RESULTS: The expression of H19 was significantly upregulated in hDPSCs undergoing odontoblastic differentiation. Overexpression of H19 stimulated odontoblastic differentiation in vitro and in vivo, whereas downregulation of H19 revealed the opposite effect. H19 binds directly to miR-140-5p and overexpression of miR-140-5p inhibited odontoblastic differentiation of hDPSCs. H19 acted as a miR-140-5p sponge, resulting in regulated the expression of BMP-2 and FGF9. Overexpression of H19 abrogated the inhibitory effect of miR-140-5p on odontoblastic differentiation. CONCLUSION: Our data revealed that H19 plays a positive regulatory role in odontoblastic differentiation of hDPSCs through miR-140-5p/BMP-2/FGF9 axis, suggesting that H19 may be a stimulatory regulator of odontogenesis.

Biomechanical Properties of First Maxillary Molars with Different Endodontic Cavities: A Finite Element Analysis.

INTRODUCTION: The aim of this study was to compare the biomechanical properties of first maxillary molars with different endodontic cavities using the finite element method. METHODS: Three finite element analysis models of a maxillary first molar were designed and constructed with 3 different types of endodontic cavities: a traditional endodontic cavity, a conservative endodontic cavity, and an extended endodontic cavity. An intact tooth model was used for comparison. Each model was subjected to 3 different force loads directed at the occlusal surface. The stress distribution patterns and the maximum von Mises (VM) stresses were calculated and compared. RESULTS: The peak VM stress on all models was at the site of the force load. The occlusal stresses were spread in an approximate actinomorphic pattern from the force loading point, and the stress was much higher when the force load was close to the access cavity margin. The peak root VM stresses on the root-filled teeth occurred at the apex and were significantly higher than that on the intact tooth, which appeared on the pericervical dentin. The area of pericervical dentin experiencing high VM stress increased as the cavities extended and the stress became concentrated in the area between the filling materials and the dentin. CONCLUSIONS: The stress distribution on the occlusal surface were similar between the conservative endodontic cavity, the traditional endodontic cavity, and the extended endodontic cavity. With enlargement of the access cavity, the stress on the pericervical dentin increases dramatically.