In Situ Synthesis and Self-Assembly of Peptide-PEG Conjugates: A Facile Method for the Construction of Fibrous Hydrogels.

Peptide-based hydrogels have gained considerable attention as a compelling platform for various biomedical applications in recent years. Their attractiveness stems from their ability to seamlessly integrate diverse properties, such as biocompatibility, biodegradability, easily adjustable hydrophilicity/hydrophobicity, and other functionalities. However, a significant drawback is that most of the functional self-assembling peptides cannot form robust hydrogels suitable for biological applications. In this study, we present the synthesis of novel peptide-PEG conjugates and explore their comprehensive hydrogel properties. The hydrogel comprises double networks, with the first network formed through the self-assembly of peptides to create a ß-sheet secondary structure. The second network is established through covalent bond formation via N-hydroxysuccinimide chemistry between peptides and a 4-arm PEG to form a covalently linked network. Importantly, our findings reveal that this hydrogel formation method can be applied to other peptides containing lysine-rich sequences. Upon encapsulation of the hydrogel with antimicrobial peptides, the hydrogel retained high bacterial killing efficiency while showing minimum cytotoxicity toward mammalian cells. We hope that this method opens new avenues for the development of a novel class of peptide-polymer hydrogel materials with enhanced performance in biomedical contexts, particularly in reducing the potential for infection in applications of tissue regeneration and drug delivery.

Si and Ca individually and combinatorially target enhanced MC3T3-E1 subclone 4 early osteogenic marker expression.

This study tests the hypothesis that silicon and calcium ions combinatorially target gene expression during osteoblast differentiation. MC3T3-E1 subclone 4 osteoblast progenitors (transformed mouse calvarial osteoblasts) were exposed to Si(4+) (from Na(2)SiO(3)) and Ca(2+) (from CaCl(2):H(2)O) ion treatments both individually (0.4 mM each + control treatment) and combinatorially (0.4 mM Si(4+) + 0.4 mM Ca(2+) + control treatment) and compared to control treated (α-minimum essential medium, 10% fetal bovine serum, and 1% penicillin-streptomycin) cells. Cell proliferation studies showed no significant increase in cell density between treatments over 5 days of culture. Cellular differentiation studies involved addition of ascorbic acid (50 mg/L) for all treatments. Relative gene expression was determined for collagen type 1 (Col(I)α1/Col(I)α2), core-binding factor a (cbfa1/Runx2), and osteocalcin (OCN), which indicated osteoblast progenitor differentiation into a mineralizing phenotype. Increased Si(4+) or Ca(2+) ion treatments enhanced Col(I)α1, Col(I)α2, Runx2, and OCN expression, while increased Si(4+) + Ca(2+) ion treatments enhanced OCN expression. Moreover, it was found that a Si(4+)/Ca(2+) ratio of unity was optimal for maximal expression of OCN. Collagen fiber bundles were dense, elongated, and thick within extracellular matrices (ECM) exposed to Si(4+) and Si(4+) + Ca(2+) treatments, while collagen fiber bundles were sparse, short, and thin within Ca(2+) and control treated ECM. These results indicated that individual ions enhance multiple osteogenic gene expression, while combined ion treatments enhance individual gene expression. In addition, these results indicated that Si(4+) enhanced osteoblast gene expression and ECM formation at higher levels than Ca(2+). These results support the larger concept that ions (possibly released from bioactive glasses) could control bone formation by targeting osteoblast marker expression.

Silicon Oxynitrophosphide Nanoscale Coating Enhances Antioxidant Marker-Induced Angiogenesis During in vivo Cranial Bone-Defect Healing.

Critical-sized bone defects are challenging to heal because of the sudden and large volume of lost bone. Fixative plates are often used to stabilize defects, yet oxidative stress and delayed angiogenesis are contributing factors to poor biocompatibility and delayed bone healing. This study tests the angiogenic and antioxidant properties of amorphous silicon oxynitrophosphide (SiONPx) nanoscale-coating material on endothelial cells to regenerate vascular tissue in vitro and in bone defects. in vitro studies evaluate the effect of silicon oxynitride (SiONx) and two different SiONPx compositions on human endothelial cells exposed to ROS (eg, hydrogen peroxide) that simulates oxidative stress conditions. in vivo studies using adult male Sprague Dawley rats (approximately 450 g) were performed to compare a bare plate, a SiONPx-coated implant plate, and a sham control group using a rat standard-sized calvarial defect. Results from this study showed that plates coated with SiONPx significantly reduced cell death, and enhanced vascular tubule formation and matrix deposition by upregulating angiogenic and antioxidant expression (eg, vascular endothelial growth factor A, angiopoetin-1, superoxide dismutase 1, nuclear factor erythroid 2-related factor 2, and catalase 1). Moreover, endothelial cell markers (CD31) showed a significant tubular structure in the SiONPx coating group compared with an empty and uncoated plate group. This reveals that atomic doping of phosphate into the nanoscale coating of SiONx produced markedly elevated levels of antioxidant and angiogenic markers that enhance vascular tissue regeneration. This study found that SiONPx or SiONx nanoscale-coated materials enhance antioxidant expression, angiogenic marker expression, and reduce ROS levels needed for accelerating vascular tissue regeneration. These results further suggest that SiONPx nanoscale coating could be a promising candidate for titanium plate for rapid and enhanced cranial bone-defect healing. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Amorphous Silicon Oxynitrophosphide-Coated Implants Boost Angiogenic Activity of Endothelial Cells.

Lack of osteointegration is a major cause of aseptic loosening and failure of implants used in bone replacement. Implants coated with angiogenic biomaterials can improve osteointegration and potentially reduce these complications. Silicon- and phosphorus-based materials have been shown to upregulate expression of angiogenic factors and improve endothelial cell functions. In the present study, we hypothesize that implants coated with amorphous silica-based coatings in the form of silicon oxynitrophosphide (SiONP) by using plasma-enhanced chemical vapor deposition (PECVD) technique could enhance human umbilical vein endothelial cell angiogenic properties in vitro. The tested groups were: glass coverslip (GCS), tissue culture plate, SiON, SiONP1 (O: 7.3 at %), and SiONP2 (O: 14.2 at %) implants. The SiONP2 composition demonstrated 3.5-fold more fibronectin deposition than the GCS (p < 0.001). The SiONP2 group also presented a significant improvement in the capillary tubule length and thickness compared with the other groups (p < 0.01). At 24 h, we observed at least a twofold upregulation of vascular endothelial growth factor A, hypoxia-inducible factor-1α, angiopoietin-1, and nesprin-2, more evident in the SiONP1 and SiONP2 groups. In conclusion, the studied amorphous silica-coated implants, especially the SiONP2 composition, could enhance the endothelial cell angiogenic properties in vitro and may induce faster osteointegration and healing. Impact Statement In this study, we report for the first time the significant enhancement of human umbilical vein endothelial cell angiogenic properties (in vitro) by the amorphous silica-based coatings in the form of silicon oxynitrophosphide (SiONP). The SiONP2 demonstrated 3.5-fold more fibronectin deposition than the glass coverslip and presented a significant improvement in the capillary tubule length and thickness. At 24 h, SiONP reported twofold upregulation of vascular endothelial growth factor A, hypoxia-inducible factor-1α, angiopoietin-1, and nesprin-2. The studied amorphous silica-coated implants enhance the endothelial cell angiogenic properties in vitro and may induce faster osteointegration and healing.

Rapid Regeneration of Vascularized Bone by Nanofabricated Amorphous Silicon Oxynitrophosphide (SiONP) Overlays.

Fracture healing is a complex biological process. Severe bone loss and ischemia from traumatic fractures lead to inflammation and accumulation of damaging reactive oxygen species (ROS). Fixative devices that not only provide mechanical support but also stimulate antioxidants such as superoxide dismutase (SOD1) and influence signaling pathways for extracellular matrix (ECM) mineralization, are critical for normal healing of such fractures. In this study, we report a novel biomaterial, silicon oxynitrophosphide (SiONP) that provides sustained release of ionic silicon (Si+4) and phosphorous (P) over few weeks under physiological conditions. Anti-oxidant role of Si+4 and augmented ECM mineralization by P ions lead to enhanced osteogenesis coupled with quick revascularization for rapid bone regeneration. Plasma enhanced chemical vapor deposition (PECVD) provided a conformal, well adherent and highly reproducible surface chemistry overlaid onto nanofabricated bioinspired surfaces. The Nitrogen to P and O content ratio was observed to change the dissolution rate and the release kinetics of the overlaid film. The SiONP films with optimal release kinetics promoted anti-oxidant expression via enhanced SOD1, which downstream upregulated other osteogenic markers with MC3T3-E1 cells. These surfaces also promoted angiogenesis evident by formation of thicker tubules by Human umbilical vein endothelial cells (HUVEC). In-vivo evaluation using a rat critical-sized calvarial defect model showed rapid bone-regeneration for these nanofabricated biomaterials as compared to control groups, and opens new horizon for future clinical trials of new antioxidant materials on biomedical devices that can reduce healing time, lower medical care cost, and increase the quality of newly formed bone in critical size defects.

White spot lesions: Does etching really matter?

AIM: The clinical significance of acid etching prior to orthodontic bonding is controversial. In the present study, we evaluated the effect of 15 seconds of acid etching on enamel demineralization. METHODS: Twenty-seven human molars were sectioned and assigned to two groups. Under standardized conditions, the enamel surfaces were imaged using FluoreCam to obtain baseline data. Group 1 was etched using 37% phosphoric acid for 15 seconds, rinsed with water, and then imaged again; group 2 was only rinsed with water. Water rinse was collected for calcium chemical analysis using inductively-coupled plasma auger electron spectrometry. Both groups were subjected to 9 days of pH cycling, after which final FluoreCam images were obtained. RESULTS: Group 1 showed a significant increase in lesion area (P=.012), decrease in light intensity (P=.009), and decrease in impact (P=.007) after acid etching. The amount of calcium that leached out over the 15 seconds was 14 ppm ±2.4 (0.35 mmol/L±0.06). Following pH cycling, there was no statistically-significant between-group difference in overall enamel demineralization. CONCLUSION: Initial demineralization caused by 15 seconds of acid etching does not increase enamel susceptibility to further demineralization. This suggests that acid etching does not increase the risk of developing white spot lesions during orthodontics.

Ionic silicon improves endothelial cells' survival under toxic oxidative stress by overexpressing angiogenic markers and antioxidant enzymes.

Oxidative stress, induced by harmful levels of reactive oxygen species, is a common occurrence that impairs proper bone defect vascular healing through the impairment of endothelial cell function. Ionic silicon released from silica-based biomaterials, can upregulate hypoxia-inducible factor-1α (HIF-1α). Yet it is unclear whether ionic Si can restore endothelial cell function under oxidative stress conditions. Therefore, we hypothesized that ionic silicon can help improve human umbilical vein endothelial cells' (HUVECs') survival under toxic oxidative stress. In this study, we evaluated the ionic jsilicon effect on HUVECs viability, proliferation, migration, gene expression, and capillary tube formation under normal conditions and under harmful hydrogen peroxide levels. We demonstrated that 0.5-mM Si4+ significantly enhanced angiogenesis in HUVECs under normal condition (p < 0.05). HUVECs exposed to 0.5-mM Si4+ presented a morphological change, even without the bed of Matrigel, and formed significantly more tube-like structures than the control (p < 0.001). In addition, 0.5-mM Si4+ enhanced cell viability in HUVECs under harmful H2 O2 levels. HIF-1α, vascular endothelial growth factor-A, and vascular endothelial growth factor receptor-2 were overexpressed more than twofold in silicon-treated HUVECs, under normal and toxic H2 O2 conditions. Moreover, the HUVECs were treated with 0.5-mM Si4+ overexpressed superoxide dismutase-1 (SOD-1), catalase-1 (Cat-1), and nitric oxide synthase-3 (NOS3) under normal and oxidative stress environment (p < 0.01). A computational model was used for explaining the antioxidant effect of Si4+ in endothelial cells and human periosteum cells by SOD-1 enhancement. In conclusion, we demonstrated that 0.5-mM Si4+ can recover the HUVECs' viability under oxidative stress conditions by reducing cell death and upregulating expression of angiogenic and antioxidant factors.

Role of Hydrogen and Nitrogen on the Surface Chemical Structure of Bioactive Amorphous Silicon Oxynitride Films.

Silicon oxynitride (Si-O-N) is a new biomaterial in which its O/N ratio is tunable for variable Si release and its subsequent endocytotic incorporation into native hydroxyapatite for enhanced bone healing. However, the effect of nitrogen and hydrogen bonding on the formation and structure of hydroxyapatite is unclear. This study aims to uncover the roles of H and N in tuning Si-O-N surface bioactivity for hydroxyapatite formation. Conformal Si-O-N films were fabricated by plasma-enhanced chemical vapor deposition (PECVD) onto Ti/Si substrates. Fourier transform infrared spectroscopy (FTIR) and Rutherford backscattering spectrometry (RBS) analysis indicated increased Si-H and N-H bonding with increased N content. Surface energy decreased with increased N content. X-ray absorbance near edge structure (XANES) analysis showed tetrahedral coordination in O-rich films and trigonal coordination in N-rich films. O-rich films exhibited a 1:1 ratio of 2p3/2 to 2p1/2 electron absorbance, while this ratio was 1.73:1 for N-rich films. Both Si and N had a reduced partial charge for both O- and N-rich films, whereas O maintained its partial charge for either film. O-rich films were found to exhibit random bonding SizOxNy, while N-rich films exhibited random mixing: [Si-Si]-[Si-O]-[Si-N]. Thus, hydrogen bonding limits random nitrogen bonding in Si-O-N films via surface Si-H and N-H bonding. Moreover, increased nitrogen content reduces the partial charge of constituent elements and changes the bonding structure from random bonding to random mixing.

Enhanced osteoprogenitor elongated collagen fiber matrix formation by bioactive glass ionic silicon dependent on Sp7 (osterix) transcription.

Bioactive glasses release ions, those enhance osteoblast collagen matrix synthesis and osteogenic marker expression during bone healing. Collagen matrix density and osteogenic marker expression depend on osteogenic transcription factors, (e.g., Osterix (OSX)). We hypothesize that enhanced expression and formation of collagen by Si(4+) depends on enhanced expression of OSX transcription. Experimental bioactive glass (6P53-b) and commercial Bioglass(TM) (45S5) were dissolved in basal medium to make glass conditioned medium (GCM). ICP-MS analysis was used to measure bioactive glass ion release rates. MC3T3-E1 cells were cultured for 20 days, and gene expression and extracellular matrix collagen formation was analyzed. In a separate study, siRNA was used to determine the effect of OSX knockdown on impacting the effect of Si(4+) on osteogenic markers and matrix collagen formation. Each bioactive glass exhibited similar ion release rates for all ions, except Mg(2+) released by 6P53-b. Gene expression results showed that GCM markedly enhanced many osteogenic markers, and 45S5 GCM showed higher levels of expression and collagen matrix fiber bundle density than 6P53-b GCM. Upon knockdown of OSX transcription, collagen type 5, alkaline phosphatase, and matrix density were not enhanced as compared to wild type cells. This study illustrates that the enhancement of elongated collagen fiber matrix formation by Si(±) depends on OSX transcription. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2604-2615, 2016.

Amorphous Silica: A New Antioxidant Role for Rapid Critical-Sized Bone Defect Healing.

Traumatic fractures cause structurally unstable sites due to severe bone loss. Such fractures generate a high yield of reactive oxygen species (ROS) that can lead to oxidative stress. Excessive and prolonged ROS activity impedes osteoblast differentiation and instigates long healing times. Stimulation of antioxidants such as superoxide dismutase (SOD1), are crucial to reduce ROS, stimulate osteogenesis, and strengthen collagen and mineral formation. Yet, no current fixative devices have shown an ability to enhance collagen matrix formation through antioxidant expression. This study reports plasma-enhanced chemical vapor deposition based amorphous silicon oxynitride (Si(ON)x) as a potential new fracture healing biomaterial that adheres well to the implant surface, releases Si(+4) to enhance osteogenesis, and forms a surface hydroxyapatite for collagen mineral attachment. These materials provide a sustained release of Si(+4) in physiological environment for extended times. The dissolution rate partially depends on the film chemistry and can be controlled by varying O/N ratio. The presence of Si(+4) enhances SOD1, which stimulates other osteogenic markers downstream and leads to rapid mineral formation. In vivo testing using a rat critical-sized calvarial defect model shows a more rapid bone-regeneration for these biomaterials as compared to control groups, that implies the clinical significance of the presented biomaterial.

Enhanced interfacial adhesion and osteogenesis for rapid "bone-like" biomineralization by PECVD-based silicon oxynitride overlays.

Structurally unstable fracture sites require metal fixative devices, which have long healing times due to their lack of osteoinductivity. Bioactive glass coatings lack in interfacial bonding, delaminate, and have reduced bioactivity due to the high temperatures used for their fabrication. Here, we test the hypothesis that low-temperature PECVD amorphous silica can enhance adhesion to the underlying metal surface and that N incorporation enhances osteogenesis and rapid biomineralization. A model Ti/TiO2-SiOx interface was formed by first depositing Ti onto Si wafers, followed by surface patterning, thermal annealing to form TiO2, and depositing SiOx/Si(ON)x overlays. TEM micrographs showed conformal SiOx layers on Ti/TiO2 overlays while XPS data revealed the formation of an elemental Ti-O-Si interface. Nanoscratch testing verified strong SiOx bonding with the underlying TiO2 layers. In vitro studies showed that the surface properties changed significantly to reveal the formation of hydroxycarbonate apatite within 6 h, and Si(ON)x surface chemistry induced osteogenic gene expression of human periosteal cells and led to a rapid "bone-like" biomineral formation within 4 weeks. XANES data revealed that the incorporation of N increased the surface HA bioactivity by increasing the carbonate to phosphate ratio. In conclusion, silicon oxynitride overlays on bone-implant systems enhance osteogenesis and biomineralization via surface nitrogen incorporation.

Decreasing NF-κB expression enhances odontoblastic differentiation and collagen expression in dental pulp stem cells exposed to inflammatory cytokines.

Inflammatory response in the dental pulp can alter the collagen matrix formation by dental pulp stem cells and lead to a delay or poor healing of the pulp. This inflammatory response is mediated by cytokines, including interleukin-1ß and tumor necrosis factor-α. In this study, it is hypothesized that suppressing the actions of these inflammatory cytokines by knocking down the activity of transcription factor Nuclear Factor-κB will lead to dental pulp stem cell differentiation into odontoblasts and the production of collagen. Here, the role of Nuclear Factor-κB signaling and its reduction was examined during odontogenic behavior in the presence of these cytokines. The results showed a significant increase in Nuclear Factor-κB gene expression and p65 protein expression by interleukin-1ß and tumor necrosis factor-α. Nuclear Factor-κB activation in the presence of these cytokines decreased significantly in a dose-dependent manner by a Nuclear Factor-κB inhibitor (MG132) and p65 siRNA. Down-regulation of Nuclear Factor-κB activity also enhanced the gene expression of the odontoblastic markers (dentin sialophosphoprotein, Nestin, and alkaline phosphatase) and displayed an odontoblastic cell morphology indicating the promotion of odontogenic differentiation of dental pulp stem cells. Finally, dental pulp stem cells exposed to reduced Nuclear Factor-κB activity resulted in a significant increase in collagen (I)-α1 expression in the presence of these cytokines. In conclusion, a decrease in Nuclear Factor-κB in dental pulp stem cells in the presence of inflammatory cytokines enhanced odontoblastic differentiation and collagen matrix formation.

Human periosteum cell osteogenic differentiation enhanced by ionic silicon release from porous amorphous silica fibrous scaffolds.

Current synthetic grafts for bone defect filling in the sinus can support new bone formation but lack the ability to stimulate or enhance osteogenic healing. To promote such healing, osteoblast progenitors such as human periosteum cells must undergo osteogenic differentiation. In this study, we tested the hypothesis that degradation of porous amorphous silica fibrous (PASF) scaffolds can enhance human periosteum cell osteogenic differentiation. Two types of PASF were prepared and evaluated according to their densities (PASF99, PASF98) with 99 and 98% porosity, respectively. Silicon (Si) ions were observed to rapidly release from both scaffolds within 24 h in vitro. PASF99 Si ion release rate was estimated to be nearly double that of PASF98 scaffolds. Mechanical tests revealed a lower compressive strength in PASF99 as compared with PASF98. Osteogenic expression analysis showed that PASF99 scaffolds enhanced the expression of activating transcription factor 4, alkaline phosphatase, and collagen (Col(I)α1, Col(I)α2). Scanning electron microscopy showed cellular and extracellular matrix (ECM) ingress into both scaffolds within 16 days and the formation of Ca-P precipitates within 85 days. In conclusion, this study demonstrated that PASF scaffolds enhance human periosteum cell osteogenic differentiation by releasing ionic Si, and structurally supporting cellular and ECM ingress.

Osteoconductive bio-based meshes based on poly(hydroxybutyrate-co-hydroxyvalerate) and poly(butylene adipate-co-terephthalate) blends.

Poly(butylene adipate-co-terephthalate) (PBAT) and Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) are biopolymers that have the potential to be used in applications of bone healing. In this study, it is hypothesized that the polymer blend has the combined strength and osteoconductivity to support osteoblast collagen formation. PBAT (PBAT 100), and a blend with 20% PHBV (PBAT 80) were extruded in the form of fibers and then knitted in the form of mesh. These were tested in the warp as well as weft direction for the tensile properties; these showed that the weft direction had higher performance than the warp. The individual fibers were kept in phosphate buffered saline (PBS) over the period of 8 weeks and were tested for the storage and loss modulus using a dynamic mechanical analyser (DMA). The results indicated that mechanical relaxation strength showed a decrease and then an increase. In vitro osteoconductivity studies were done by using differentiating osteoblasts (MC3T3-E1 subclone 4 cells). Environmental Scanning Electron Microscopy (ESEM) showed that pre-soaking the samples in α-MEM for two weeks resulted in cell attachment and growth. X-ray diffraction (XRD) was used to determine the change in structure of polymers due to in vitro degradation for two weeks. Raman spectroscopy showed that all scaffolds supported the formation of a collagenous network over the scaffold surfaces. For a combination of knittable manufacturing, mechanical performance and osteoconductivity, blends offer an effective route.

Combinatorial effect of Si4+, Ca2+, and Mg2+ released from bioactive glasses on osteoblast osteocalcin expression and biomineralization.

Osteocalcin (OCN) expression is an essential osteogenic marker of successful bone regeneration therapies. This study hypothesizes that Si(4+) and Ca(2+) combinatorial released by bioactive glass enhance osteoblast biomineralization through up-regulation of OCN expression; and Mg(2+) release delays such enhancement. Osteoblasts (MC3T3-E1) were treated with ionic products of bioactive glass dissolution (6P53-b experimental bioactive glass and 45S5 commercial Bioglass™). Results showed that gene expressions, including OCN and its up-stream transcription factors (Runx2, ATF4, MSX1, SP7/OSX), growth factors and signaling proteins (BMP2, BMP6, SMAD3), were enhanced in both 45S5 and 6P53-b glass conditioned mediums (GCMs). This up-regulation led to enhanced mineral formation by 45S5 glass conditioned mediums ([GCM], Si(4+)+Ca(2+)) after 20 days, and by 45S5 GCM and 6P53-b GCM (Si(4+)+Ca(2+)+Mg(2+)) after 30 days. In examining the extracellular matrix generated by cells when exposed to each GCM, it was found that 45S5 GCM had slightly elevated levels of mineral content within ECM as compared to 6P53-b GCM after 30 days while control treatments exhibited no mineral content. The formation of well-defined mineralized nodules (distinct PO4(3-) [960 cm(-1)] and CO3(2-) [1072 cm(-1)] peaks from Raman Spectra) was observed for each GCM as the soluble glass content increased. In examining the individual and combined ion effects between Si(4+), Ca(2+), and Mg(2+), it was found Mg(2+) down-regulates OCN expression. Thus, ions released from both 45S5 and 6P53-b bioactive glasses up-regulate OCN expression and biomineralization while 6P53-b GCM Mg(2+) release down-regulated OCN expression and delayed osteoblast biomineralization. These results indicate that Si(4+), Ca(2+), and Mg(2+) combinatorially regulate osteoblast OCN expression and biomineralization.

The ionic products of bioactive glass particle dissolution enhance periodontal ligament fibroblast osteocalcin expression and enhance early mineralized tissue development.

This study resulted in enhanced collagen type 1 and osteocalcin expression in human periodontal ligament fibroblasts (hPDLF) when exposed to bioactive glass conditioned media that subsequently may promote early mineralized tissue development. Commercial Bioglass™ (45S5) and experimental bioactive coating glass (6P53-b), were used to make a glass conditioned media (GCM) for comparison to control medium. ICP-MS analysis showed increased concentrations of Ca(2+), PO(4) (3-), Si(4+), and Na(+), for 45S5 GCM and Mg(2+), K(+), Ca(2+), PO(4)(3-), Si(4+), and Na(+) for 6P53-b GCM (relative to control medium). Differentiating hPDLF cultures exposed to 45S5 and 6P53-b GCM showed enhanced expression of collagen type 1 (Col1α1, Col1α2), osteocalcin, and alkaline phosphatase gene expression. These GCM also enhanced osteocalcin protein expression. After 16 d of culture, 45S5 and 6P53-b GCM treated cells showed regions of deep red Alizarin staining, indicating increased Ca within their respective extracellular matrices (ECM), while control-treated cells did not exhibit these features. SEM analysis showed more developed ECM in GCM treated cultures, indicated by multiple tissue layering and abundant collagen fiber bundle formation, while control treated cells did not exhibit these features. SEM analysis showed polygonal structures suggestive of CaP in 45S5 GCM treated cultures. These results indicate the osteogenic potential of bioactive coating glass in periodontal bone defect filling applications.