[Study on the antimicrobial effect of new bioactive glass against common bacteria in apical periodontitis of deciduous teeth].

PURPOSE: To study the antimicrobial effect of different concentrations of new bioactive glass(BG) on common bacteria in apical periodontitis of deciduous teeth. METHODS: The diameter (mm) of the inhibitory rings formed after treatment of Enterococcus faecalis, Porphyromonas gingivalis and Clostridium nucleatum with the new bioactive glass was detected and observed by paper diffusion method, and the minimal inhibitory concentration(MIC), minimal bactericidal concentration (MBC) and minimal biofilm eradication concentration (MBEC) of E. faecalis, P. gingivalis and C. pseudomallei were determined. The mixed plaques of the three bacteria were treated with 20, 40, 60 and 80 mg/mL of the new bioactive glass for 24 h. The results were analyzed by laser confocal microscopy. The antibacterial effect of the new bioactive glass on the mixed plaque was observed by confocal laser scanning microscopy (CLSM). Statistical analysis was performed with GraphPad Prism 10.0 software. RESULTS: The new bioactive glass showed strong antibacterial potential against the common bacteria of apical periodontitis; the MBEC of the new bioactive glass on the plaque was significantly greater than MIC and MBC of Enterococcus faecalis, Porphyromonas gingivalis and Clostridium nucleatum, and as the concentration of the new bioactive glass increased, the number of dead bacteria in the mixed plaque increased, and there was significant difference from that of the blank control group (P＜0.05). CONCLUSIONS: The novel bioactive glass shows significant antibacterial efficacy against Enterococcus faecalis, Porphyromonas gingivalis and Clostridium nucleatum, which are the common bacteria in apical periodontitis of deciduous teeth.

3D Bioprinting of a Bioactive Composite Scaffold for Cell Delivery in Periodontal Tissue Regeneration.

Hydrogels have been widely applied to the fabrication of tissue engineering scaffolds via three-dimensional (3D) bioprinting because of their extracellular matrix-like properties, capacity for living cell encapsulation, and shapeable customization depending on the defect shape. However, the current hydrogel scaffolds show limited regeneration activity, especially in the application of periodontal tissue regeneration. In this study, we attempted to develop a novel multi-component hydrogel that possesses good biological activity, can wrap living cells for 3D bioprinting and can regenerate periodontal soft and hard tissue. The multi-component hydrogel consisted of gelatin methacryloyl (GelMA), sodium alginate (SA) and bioactive glass microsphere (BGM), which was first processed into hydrogel scaffolds by cell-free 3D printing to evaluate its printability and in vitro biological performances. The cell-free 3D-printed scaffolds showed uniform porous structures and good swelling capability. The BGM-loaded scaffold exhibited good biocompatibility, enhanced osteogenic differentiation, apatite formation abilities and desired mechanical strength. The composite hydrogel was further applied as a bio-ink to load with mouse bone marrow mesenchymal stem cells (mBMSCs) and growth factors (BMP2 and PDGF) for the fabrication of a scaffold for periodontal tissue regeneration. The cell wrapped in the hydrogel still maintained good cellular vitality after 3D bioprinting and showed enhanced osteogenic differentiation and soft tissue repair capabilities in BMP2- and PDGF-loaded scaffolds. It was noted that after transplantation of the cell- and growth factor-laden scaffolds in Beagle dog periodontal defects, significant regeneration of gingival tissue, periodontal ligament, and alveolar bone was detected. Importantly, a reconstructed periodontal structure was established in the treatment group eight weeks post-transplantation of the scaffolds containing the cell and growth factors. In conclusion, we developed a bioactive composite bio-ink for the fabrication of scaffolds applicable for the reconstruction and regeneration of periodontal tissue defects.

Bioactive glass in the treatment of ulcerative colitis to regulate the TLR4 / MyD88 / NF-κB pathway.

Ulcerative colitis (UC) is a chronic and recurrent intestinal disease of unknown aetiology, and the few treatments approved for UC have serious side effects. In this study, a new type of uniformly monodispersed calcium-enhanced radial mesoporous micro-nano bioactive glass (HCa-MBG) was prepared for UC treatment. We established cellular and rat UC models to explore the effects and mechanism of HCa-MBG and traditional BGs (45S5, 58S) on UC. The results showed that BGs significantly reduced the cellular expression of several inflammatory factors, such as IL-1ß, IL-6, TNF-α and NO. In the animal experiments, BGs were shown to repair the DSS-damaged colonic mucosa. Moreover, BGs downregulated the mRNA levels of the inflammatory factors IL-1ß, IL-6, TNF-α and iNOS, which were stimulated by DSS. BGs were also found to manage the expression of key proteins in NF-kB signal pathway. However, HCa-MBG was more effective than traditional BGs in terms of improving UC clinical manifestations and reducing the expression of inflammatory factors in rats. This study confirmed for the first time that BGs can be used as an adjuvant drug in UC treatment, thereby preventing UC progression.

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.

Corrigendum: Micro-Nano Bioactive Glass Particles Incorporated Porous Scaffold for Promoting Osteogenesis and Angiogenesis in vitro.

[This corrects the article DOI: 10.3389/fchem.2019.00186.].

Drug-loading three-dimensional scaffolds based on hydroxyapatite-sodium alginate for bone regeneration.

Bone tissue engineering is a promising approach for tackling clinical challenges. Osteoprogenitor cells, osteogenic factors, and osteoinductive/osteoconductive scaffolds are employed in bone tissue engineering. However, scaffold materials remain limited due to their source, low biocompatibility, and so on. In this study, a composite hydrogel scaffold composed of hydroxyapatite (HA) and sodium alginate (SA) was manufactured using three-dimensional printing. Naringin (NG) and calcitonin-gene-related peptide (CGRP) were used as osteogenic factors in the fabrication of drug-loaded scaffolds. Investigation using animal experiments, as well as scanning electron microscopy, cell counting kit-8 testing, alkaline phosphatase staining, and alizarin red-D staining of bone marrow mesenchymal stem cell culture showed that the three scaffolds displayed similar physicochemical properties and that the HA/SA/NG and HA/SA/CGRP scaffolds displayed better osteogenesis than that of the HA/SA scaffold. Thus, the HA/SA scaffold could be a biocompatible material with potential applications in bone regeneration. Meanwhile, NG and CGRP doping could result in better and more positive proliferation and differentiation.

Local delivery of FTY720 in mesoporous bioactive glass improves bone regeneration by synergistically immunomodulating osteogenesis and osteoclastogenesis.

The addition of osteoimmunology drugs to bone repair materials is beneficial to bone regeneration by regulating the local immune microenvironment. Fingolimod (FTY720) has been reported to be an osteoimmunology drug that promotes osteogenesis. However, there is no ideal biomaterial for the sustained release of FTY720 in the bone defect areas. In the present work, FTY720 loaded mesoporous bioactive glass (FTY720@MBGs) was successfully prepared based on the mesoporous properties of MBGs and electrostatic attraction. FTY720 achieved a sustained release for 7 days. The in vitro study found that FTY720@MBGs could synergistically promote osteogenesis and inhibit osteoclastogenesis due to their ability to promote macrophages toward the M2 phenotype. The in vivo study confirmed that FTY720@MBGs could significantly improve bone regeneration. This study provides new strategies for designing smart cell-instructive biomaterials that can play a role in all bone healing processes from early inflammation to bone reconstruction.

Fabrication and characterization of strontium-hydroxyapatite/silk fibroin biocomposite nanospheres for bone-tissue engineering applications.

Osteoinductive bone filling biomaterials are in high demand for effective bone defect reconstruction. In this study, we aimed to design both organic and inorganic substances containing strontium-doped hydroxyapatite/silk fibroin (SrHA/SF) biocomposite nanospheres as an osteoinductive bone defect-filling biomaterial. SrHA/SF nanospheres were prepared with different concentration of Sr using ultrasonic coprecipitation method. The nanospheres were characterized using XRD, FTIR, SEM, TEM, ICP-AES and TGA. Solid and dense SrHA/SF nanospheres with 500-700 nm size and rough surfaces were synthesized successfully. Higher crystallinity and HA/SF phase were observed with the increase in Sr-concentration. The doping of different concentration of Sr did not affect the size and surface characteristics of the nanospheres. ICP-AES data showed that Sr/Ca ratio in SrHA/SF is very close to the nominal value. Nanospheres with higher concentration of Sr did not negatively affect the biocompatibility, but enhanced viability of mesenchymal stem cells (MSCs). Moreover, SrHA/SF nanospheres showed higher osteogenic differentiation potential compared to HA/SF nanospheres as indicated by the results from ALP staining, ALP activity, and Runx2, Alp, Col-1 and Opn gene expression assay in MSCs culture. Our findings suggest this novel design of biocompatible and osteoinductive SrHA/SF biocomposite nanospheres as a potential bone defect-filling biomaterial for bone regenerative applications.

Micro-Nano Bioactive Glass Particles Incorporated Porous Scaffold for Promoting Osteogenesis and Angiogenesis in vitro.

Constructing the interconnected porous biomaterials scaffolds with osteogenesis and angiogenesis capacity is extremely important for efficient bone tissue engineering. Herein, we fabricated a bioactive micro-nano composite scaffolds with excellent in vitro osteogenesis and angiogenesis capacity, based on poly (lactic-co-glycolic acid) (PLGA) incorporated with micro-nano bioactive glass (MNBG). The results showed that the addition of MNBG enlarged the pore size, increased the compressive modulus (4 times improvement), enhanced the physiological stability and apatite-forming ability of porous PLGA scaffolds. The in vitro studies indicated that the PLGA-MNBG porous scaffold could enhance the mouse bone mesenchymal stem cells (mBMSCs) attachment, proliferation, and promote the expression of osteogenesis marker (ALP). Additionally, PLGA-MNBG could also support the attachment and proliferation of human umbilical vein endothelial cells (HUVECs), and significantly enhanced the expression of angiogenesis marker (CD31) of HUVECs. The as-prepared bioactive PLGA-MNBG nanocomposites scaffolds with good osteogenesis and angiogenesis probably have a promising application for bone tissue regeneration.

3D printing mesoporous bioactive glass/sodium alginate/gelatin sustained release scaffolds for bone repair.

Drug delivery and release are a major challenge fabricating bone tissue engineering. In this study, we fabricated new sustained release hydrogel scaffolds composited of mesoporous bioactive glass, sodium alginate and gelatin by a three-dimensional printing technique. Naringin and calcitonin gene-related peptide were used as drugs to prepare drug-loaded scaffolds by direct printing or surface absorption. The physicochemical properties of the scaffolds and the drug release profiles of the two drug-loading models were investigated. We also examined the biocompatibility of the scaffolds, as well as the effect of the released medium on the proliferation and osteogenic differentiation of human osteoblast-like MG-63 cell. The results showed that the scaffolds had a high porosity (approximately 80%) with an interconnected cubic pore structure, rough surface morphology, bioactivity and strong biocompatibility. Furthermore, the naringin or calcitonin gene-related peptide co-printed into the scaffold displayed a steady sustained release behaviour for up to 21 days without an initial burst release, while both naringin and calcitonin gene-related peptide absorbed onto the surface of the scaffold were completely released within two days. MG-63 cells cultured with the extraction containing released drugs displayed promoted cell proliferation and the expression of osteogenesis-related genes more effectively compared with the drug-free extractions. Therefore, these results demonstrate that the developed mesoporous bioactive glass/sodium alginate/gelatin sustained release scaffolds provide a potential application for bone tissue engineering.

Size-Dependent Mechanism of Intracellular Localization and Cytotoxicity of Mono-Disperse Spherical Mesoporous Nano- and Micron-Bioactive Glass Particles.

Mono-disperse spherical mesoporous nano- and micro- bioactive glass particles (NMBGs) can find potential use in bone tissue engineering. However, their size-dependent interaction with osteoblasts has never been studied. Herein, the proliferation, morphology, cytoskeleton organization and apoptosis of MC3T3-E1 osteoblasts are studied in response to the NMBGs with varying sizes (from 61 to 1085 nm) at different concentrations. Generally, smaller NMBGs at a lower dose show weaker cytotoxicity compared to the larger particles and higher doses, arising from a novel size-dependent mechanisrm of intracellular localization of NMBGs observed by electron and confocal microscopy. Specifically, NMBGs pass through perinuclear membrane of the cells to initiate endocytosis. Once internalized, the sizes of NMBGs are found to play a significant role in determining their intracellular localization. When the NMBGs are smaller than 174 nm, they are transported via the lysosomal pathway and phagocytized in lysosomes, resulting in little cytotoxicity at later time points. On the contrary, larger NMBGs (over 174 nm) escape from the lysosomes after endocytosis, and are localized inside the intra-cytoplasmic vacuoles or randomly in the cytoplasm of cells. Their lysosomal escape may damage the lysosomes, inducing cell apoptosis and thus the greater cytotoxicity.

Regulation of cellular behaviors of fibroblasts related to wound healing by sol-gel derived bioactive glass particles.

Sol-gel derived bioactive glass (BG) holds great potential in the application of skin repair. However, the specific regulation of BG on skin cells is still unclear and demands more investigation. Herein, we synthesized sol-gel derived BGs with different compositions (60S, 70S, 80S, and 90S) and found 90S BGs (90 mol % SiO2 , 6 mol % CaO, 4 mol % P2 O5 ) exhibited the best supportiveness for the proliferation of normal human foreskin fibroblasts. Thus, 90S BG particles were used as a model to systematically study the wound healing related cellular response of fibroblasts to BGs. Time-lapse imaging revealed a promoted fibroblast motility stimulated by 90S BG particles. Results on the expression of extracellular matrix (ECM) related genes illustrated that 90S BG particles modulated the synthesis capacity for critical ECM molecules including type I collagen, type III collagen, fibronectin, and tenascin-C. Moreover, the myofibroblastic differentiation of fibroblasts was greatly inhibited by 90S BG particles. Further analysis on the intracellular signaling pathways demonstrated that 90S BG particles down-regulated the collagen synthesis and fibroblast-to-myofibroblast differentiation via TGF-ß1-Smad2 signaling, evidenced by the decreased expression levels of TGF-ß receptor I and its downstream effector Smad2. Our study provided a further understanding of the specific regulation of 90S BG particles on fibroblasts, which may guide the future design of BG based wound dressing and benefit the clinical application of BG particles in skin repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2420-2429, 2016.

Acute toxicity and in vivo biodistribution of monodispersed mesoporous bioactive glass spheres in intravenously exposed mice.

The use of biomaterials from laboratories to clinics requires exhaustive and elaborate studies involving the biodistribution, clearance, and biocompatibility of biomaterials for in vivo biomedical applications. This study aimed to evaluate the acute toxicity and biodistribution of intravenously administrated sub-micrometer mesoporous bioactive glass spheres (SMBGs) in mice. The lethal dose 50 (LD50) of SMBGs was higher than 250 mg/kg. The acute toxicity was evaluated at 14 days after intravenous injection of SMBGs at 20, 100 and 180 mg/kg in ICR mice. The mortality, coefficients of major organs, hematology data and blood biochemical indexes revealed the low in vivo toxicity of SMBGs at all doses. However, the histological examination showed lymphocytic infiltration and granuloma formation in hepatocyte and megakaryocyte hyperplasia in the spleen at high dose. The silicon content analysis using ICP-OES and TEM results indicated that SMBGs mainly distributed in the resident macrophages of the liver and spleen, and could be cleared from the body more than 2 weeks. These findings can be important for the toxicity assessment of sub-micrometer particles and the development of bioactive glass based drug delivery system for biomedical applications.

Angiogenesis stimulated by novel nanoscale bioactive glasses.

The ability of biomaterials to induce rapid vascular formation is critical in tissue regeneration. Combining recombinant angiogenic growth factors with bioengineered constructs have proven to be difficult due to several issues, including the instability of recombinant proteins, the need for sustained delivery and the dosage of factors. New formulations of bioactive glass, 58S nanosized bioactive glass (58S-NBG), have been reported to enhance wound healing in animal models better than the first generation of 45S5 Bioglass. Therefore, we investigated the effects of extracts of 58S-NBG and 80S-NBG on cultures of human umbilical vein endothelial cells (HUVECs). Cell viability was assessed by MTS assay. In vitro angiogenesis was measured using an ECM gel tube formation assay, and levels of mRNAs for five angiogenic related genes were measured by qRT-PCR. Extracts of 58S-NBG and 80S-NBG stimulated the proliferation of HUVECs, accelerated cell migration, up-regulated expression of the vascular endothelial growth factor, basic fibroblast growth factor, their receptors, and endothelial nitric oxide synthase, resulting in enhanced tube formation in vitro. The enhanced angiogenic response correlated with increased levels of Ca and Si in the extracts of 58S-NBG and 80S-NBG. The ability of 58S-NBG and 80S-NBG to stimulate angiogenesis in vitro provides alternative approaches for stimulating neovascularization of tissue-engineered constructs.

Synthesis of radial mesoporous bioactive glass particles to deliver osteoactivin gene.

Mesoporous bioactive glasses (MBGs) can be used as carriers for biomolecule delivery with improved functions. Although there are a great number of studies on drug delivery by MBGs, until now little work has been done to investigate the DNA gene transfection effect of MBGs. In this study, radial mesoporous bioactive glasses (rMBGs) were prepared by sol-gel process combined with a micro-emulsion method. The surface was further modified by amino groups in order to improve its affinity for DNA. Our study showed that rMBGs have good apatite-forming ability and cellular biocompatibility. In addition, rMBGs can enter cells in a time- and dose-dependent manner, and mainly localize in the cytoplasm. Agarose gel electrophoresis demonstrated that pOA-EGFP (containing the osteoactivin and the green fluorescent protein fusion gene) can be completely absorbed and protected from DNase I degradation by the aminated rMBGs. Additionally, the plasmid can be successfully expressed in cells transfected by rMBGs.

Investigation of emulsified, acid and acid-alkali catalyzed mesoporous bioactive glass microspheres for bone regeneration and drug delivery.

Acid-catalyzed mesoporous bioactive glass microspheres (MBGMs-A) and acid-alkali co-catalyzed mesoporous bioactive glass microspheres (MBGMs-B) were successfully synthesized via combination of sol-gel and water-in-oil (W/O) micro-emulsion methods. The structural, morphological and textural properties of mesoporous bioactive glass microspheres (MBGMs) were characterized by various techniques. Results show that both MBGMs-A and MBGMs-B exhibit regularly spherical shape but with different internal porous structures, i.e., a dense microstructure for MBGMs-A and internally porous structure for MBGMs-B. (29)Si NMR data reveal that MGBMs have low polymerization degree of silica network. The in vitro bioactivity tests indicate that the apatite formation rate of MBGMs-B was faster than that of MBGMs-A after soaking in simulated body fluid (SBF) solution. Furthermore, the two kinds of MBGMs have similar storage capacity of alendronate (AL), and the release behaviors of AL could be controlled due to their unique porous structure. In conclusion, the microspheres are shown to be promising candidates as bone-related drug carriers and filling materials of composite scaffold for bone repair.

[Growth ability of human dental pulp cells on three bioactive scaffolds].

OBJECTIVE: To investigate the proliferation and differentiation of the human dental pulp cells (hDPCs) on the bioactive scaffolds. METHODS: Primary HDPCs were harvested from impacted third molars of healthy adult individuals (18-25 years of age) by enzyme digestion, expanded and cultured. The cells used for this investigation were the 4 th passage. Immunohistochemical methods were used to verify that the cells were dental pulp cells. The expression of stromal precursor antigen-1 (STRO-1) was determined by flow cytometry. Three different types of scaffolds were used: collagen (COL), collagen / bioglass (COL-BG) and collagen / bioglass / polycaprolactone (COL-BG-PCL). Cell proliferation on the scaffolds was determined using a MTT assay at hour 6, on days 1, 3, 5, 7, 14 and 21. On day 14, the scaffolds were stained with the alkaline phosphatase (ALP) staining kit. RESULTS: The tested cells had STRO-1 positive cells. The proliferation of HDPCs was significantly higher on the COL-BG scaffold and COL-BG-PCL scaffold as compared with COL scaffold (P<0.05). Especially on days 14 and 21, the optical density value of bioglass composite scaffold were 5 times that of the COL scaffold. The ALP positive staining area was observed more extensively on the COL-BG scaffold and COL-BG-PCL scaffold than on the COL scaffold. CONCLUSION: As comparison with the COL scaffold, HDPCs' proliferation and differentiation present more activity on the COL-BG and COL-BG-PCL scaffolds.