Osteoblastic Cell Line Research Articles

Zibibbo Grape Seeds’ Polyphenolic Profile: Effects on Bone Turnover and Metabolism

Background: The consumption of seeds as food has become increasingly common due to their numerous health benefits. Among these, the seeds of the Zibibbo grape from Pantelleria, a native species of southern Italy, remain largely unexplored and are usually considered waste material from viticulture. Nevertheless, Zibibbo grape seeds may offer health benefits, particularly for the elderly and people with metabolic disorders, due to their potential content of beneficial compounds such as polyphenols. Methods: The Zibibbo grape seeds extract (ZSE) was characterized using UV-visible spectrophotometry and HPLC chromatography. The antioxidant activity of ZSE was measured by different colorimetric assays and Electronic Paramagnetic Resonance (EPR). Additionally, specific in vitro tests were conducted on human osteoblast cell lines (Saos-2 and MG63) aiming to evaluate the ZSE’s effects on bone turnover and metabolism. Western blotting was used to assess the impact on specific proteins and pathways related to bone health. Results: The ZSE contained almost ~3 mg/mL of carbohydrates and phenolic compounds, including rutin (~6.4 ppm) and hesperidin (~44.6 ppm). The extracts exhibited an antioxidant activity greater than 90% across all tests performed. Moreover, the Zibibbo seed extracts exerted a significant proliferative effect on the Saos-2 cell human osteoblast-like cell line, also modulating the phosphorylation of specific kinases involved in cell health and metabolism. Conclusions: Zibibbo grape seeds are rich in phenolic compounds, especially flavonoids with strong antioxidant and free radical scavenging properties. ZSE demonstrated beneficial effects on bone metabolism and osteoblast proliferation, suggesting potential for counteracting osteodegenerative conditions like osteoporosis. If confirmed through further studies, Zibibbo grape seed phenolic compounds could serve as an adjunctive therapy for osteoporosis, helping to slow aging and bone degeneration.

Fabrication and comprehensive evaluation of Zr-based bulk metallic glass matrix composites for biomedical applications

AbstractThe aim of this study is to fabricate Zr-based bulk metallic glass matrix composites (BMG-MCs) for biomedical usage and subject them to a comprehensive and farreaching analysis with respect to their mechanical properties, biocorrosion resistance, biocompatibility, and interactions with biofilms that all may arise from their chemical compositions and unusual disordered internal structure. In this study, we fabricate Zr40Ti15Cu10Ni10Be25, Zr50Ti15Cu10Ni10Be25, and Zr40Ti15Cu10Ni5Si5Be25 alloys and confirm their glassy matrix nature through differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) analyses. The mechanical properties, assessed via nanoindentation, demonstrate the high hardness, strength, and elasticity of the produced materials. Corrosion resistance is investigated in simulated body fluid, with Zr-based BMG-MCs exhibiting superior performance compared to conventional biomedical materials, including 316L stainless steel and Ti6Al4V alloy. Biocompatibility is assessed using human fetal osteoblastic cell line hFOB 1.19, revealing low levels of cytotoxicity. The study also examines the potential for biofilm formation, a critical factor in the success of biomedical implantation, where bacterial infection is a major concern. Our findings suggest, as never reported before, that Zr-based BMG-MCs, with their unique composite glassy structure and excellent physicochemical properties, are promising candidates for various biomedical applications, potentially offering improved performance over traditional metallic biomaterials.

9263 Characterization Of A Novel Oral Small Molecule PTH1R Agonist: Proof Of Concept For An Alternative To Injectable Peptide-Based Therapy For Hypoparathyroidism

Abstract Disclosure: J. Zhang: Employee; Self; Septerna Inc.. I. Gomez: None. C. He: Employee; Self; Septerna Inc. J. Wang: Employee; Self; Septerna Inc. X. Du: Employee; Self; Septerna Inc. K. Nguyen: Employee; Self; Septerna Inc. K. Alvarez: Employee; Self; Septerna Inc. A.M. Mirza: Employee; Self; Septerna Inc.. Hypoparathyroidism (HP), a rare endocrine disorder characterized by insufficient levels of parathyroid hormone (PTH), leads to hypocalcemia and hyperphosphatemia. Most HP cases arise due to damage of the parathyroid glands during thyroid or neck surgery. HP symptoms include severe muscle cramps, tingling, burning and numbness, memory loss, and headaches, all leading to a decreased quality of life. Calcium and vitamin D supplementation do not fully ameliorate the disease and may contribute to renal disease. Chronic HP increases the risk of major complications, such as calcium depositions in the brain, eye, and kidneys. Similarly, it can lead to low bone turnover and increased bone mineral density with associated increased bone mineralization. In comparison to standard of care and traditional injectable peptide-based therapies, oral small molecules may have multiple advantages clinically. However, there are no oral small molecules currently in development. PTH functions through activation of the parathyroid hormone 1 receptor (PTH1R), a class B G protein-coupled receptor. The main target tissues for PTH1R activation include bone and kidney. In the kidney, PTH increases tubular reabsorption of calcium and promotes renal excretion of phosphate. In bone, PTH stimulates bone turnover and mobilizes calcium into the circulation to maintain serum calcium. Here we describe the characterization of SP-1462, a novel, potent, and selective oral small molecule, and show that, like the PTH peptides, it acts on PTH1R as an agonist and elicits similar downstream functions in vitro and in vivo. To assess PTH1R engagement in the major targeted organs, kidney and bone, primary human renal proximal epithelial cells (RPTEC) and a human osteoblast cell line (Saos-2) were used, respectively. RPTECs treated with PTH and SP-1462 showed significant changes in transporter genes (SLC22A2 and SLC13A1), immune related genes (CCL2 & DPP4), and matrix reorganization genes (ITGA5 and COL1A1). Saos-2 cells treated with PTH and SP-1462 both significantly affected the expression of Wnt signaling genes (DKK1 and TCF7) and the expression of coupling genes that bridge anabolic/catabolic activities (M-CSF, RANKL, HDAC4, and OPG). Importantly, oral administration of a single dose of SP-1805 in a rat surgical thyroparathyroidectomy (TPTx) model resulted in significant upregulation of serum calcium levels for a period of 24 hours in a dose dependent manner at 3, 10 and 30 mg/kg. The window of calcium upregulation is comparable to injectable PTH peptides currently in development to treat hypoparathyroidism. These data suggest that oral small molecule PTH1R agonists engage PTH pathways similar to native PTH and have the potential to replace injectable PTH peptides to treat PTH-related disorders, including hypoparathyroidism. Presentation: 6/3/2024

6821 Characterization Of A Novel Oral Small Molecule PTH1R Agonist: Proof Of Concept For An Alternative To Injectable Peptide-Based Therapy For Hypoparathyroidism

Abstract Disclosure: J. Zhang: None. I. Gomez: None. C. He: None. J. Wang: None. X. Du: None. K. Nguyen: None. K. Alvarez: None. A.M. Mirza: None. Hypoparathyroidism (HP), a rare endocrine disorder characterized by insufficient levels of parathyroid hormone (PTH), leads to hypocalcemia and hyperphosphatemia. Most HP cases arise due to damage of the parathyroid glands during thyroid or neck surgery. HP symptoms include severe muscle cramps, tingling, burning and numbness, memory loss, and headaches, all leading to a decreased quality of life. Calcium and vitamin D supplementation do not fully ameliorate the disease and may contribute to renal disease. Chronic HP increases the risk of major complications, such as calcium depositions in the brain, eye, and kidneys. Similarly, it can lead to low bone turnover and increased bone mineral density with associated increased bone mineralization. In comparison to standard of care and traditional injectable peptide-based therapies, oral small molecules may have multiple advantages clinically. However, there are no oral small molecules currently in development. PTH functions through activation of the parathyroid hormone 1 receptor (PTH1R), a class B G protein-coupled receptor. The main target tissues for PTH1R activation include bone and kidney. In the kidney, PTH increases tubular reabsorption of calcium and promotes renal excretion of phosphate. In bone, PTH stimulates bone turnover and mobilizes calcium into the circulation to maintain serum calcium. Here we describe the characterization of SP-1462, a novel, potent, and selective oral small molecule, and show that, like the PTH peptides, it acts on PTH1R as an agonist and elicits similar downstream functions in vitro and in vivo. To assess PTH1R engagement in the major targeted organs, kidney and bone, primary human renal proximal epithelial cells (RPTEC) and a human osteoblast cell line (Saos-2) were used, respectively. RPTECs treated with PTH and SP-1462 showed significant changes in transporter genes (SLC22A2 and SLC13A1), immune related genes (CCL2 & DPP4), and matrix reorganization genes (ITGA5 and COL1A1). Saos-2 cells treated with PTH and SP-1462 both significantly affected the expression of Wnt signaling genes (DKK1 and TCF7) and the expression of coupling genes that bridge anabolic/catabolic activities (M-CSF, RANKL, HDAC4, and OPG). Importantly, oral administration of a single dose of SP-1805 in a rat surgical thyroparathyroidectomy (TPTx) model resulted in significant upregulation of serum calcium levels for a period of 24 hours in a dose dependent manner at 3, 10 and 30 mg/kg. The window of calcium upregulation is comparable to injectable PTH peptides currently in development to treat hypoparathyroidism. These data suggest that oral small molecule PTH1R agonists engage PTH pathways similar to native PTH and have the potential to replace injectable PTH peptides to treat PTH-related disorders, including hypoparathyroidism. Presentation: 6/3/2024

Osteoking prevents bone loss and enhances osteoblastic bone formation by modulating the AGEs/IGF-1/β-catenin/OPG pathway in type 2 diabetic db/db mice.

Type 2 diabetic osteoporosis (T2DOP) is a skeletal metabolic syndrome characterized by impaired bone remodeling due to type 2 diabetes mellitus, and there are drawbacks in the present treatment. Osteoking (OK) is widely used for treating fractures and femoral head necrosis. However, OK is seldom reported in the field of T2DOP, and its role and mechanism of action need to be elucidated. Consequently, this study investigated whether OK improves bone remodeling and the mechanisms of diabetes-induced injury. We used db/db mice as a T2DOP model and stimulated MC3T3-E1 cells (osteoblast cell line) with high glucose (HG, 50 mM) and advanced glycation end products (AGEs, 100 µg/mL), respectively. The effect of OK on T2DOP was assessed using a combined 3-point mechanical bending test, hematoxylin and eosin staining, and enzyme-linked immunosorbent assay. The effect of OK on enhancing MC3T3-E1 cell differentiation and mineralization under HG and AGEs conditions was assessed by an alkaline phosphatase activity assay and alizarin red S staining. The AGEs/insulin-like growth factor-1(IGF-1)/β-catenin/osteoprotegerin (OPG) pathway-associated protein levels were assayed by western blot analysis and immunohistochemical staining. We found that OK reduced hyperglycemia, attenuated bone damage, repaired bone remodeling, increased tibial and femoral IGF-1, β-catenin, and OPG expression, and decreased receptor activator of nuclear kappa B ligand and receptor activator of nuclear kappa B expression in db/db mice. Moreover, OK promoted the differentiation and mineralization of MC3T3-E1 cells under HG and AGEs conditions, respectively, and regulated the levels of AGEs/IGF-1/β-catenin/OPG pathway-associated proteins. In conclusion, our results suggest that OK may lower blood glucose, alleviate bone damage, and attenuate T2DOP, in part through activation of the AGEs/IGF-1/β-catenin/OPG pathway.

Bone development by Hedgehog and Wnt signaling, Runx2, and Sp7.

Hedgehog and canonical Wnt signaling pathways and the transcription factors Runx2 and Sp7 are essential for osteoblast differentiation. Ihh is necessary for the commitment of perichondrial mesenchymal cells to Runx2+ osteoprogenitors and for the formation of the bone collar and primary spongiosa. Runx2 is needed for osteoblast differentiation during both endochondral and intramembranous ossification. It regulates the commitment of mesenchymal cells to osteoblast-lineage cells and their proliferation by inducing the expression of Hedgehog, Fgf, Wnt, Pthlh signaling pathway genes, and Dlx5. The Runx2-induced expression of Fgfr2 and Fgfr3 is important for the proliferation of osteoblast-lineage cells. Runx2 induces Sp7 expression and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Runx2, Sp7, and canonical Wnt signaling induce the differentiation of preosteoblasts into osteoblasts. Canonical Wnt signaling, but not Sp7, enhances the proliferation of osteoblast-lineage cells. In mature osteoblasts, Runx2 plays an important role in the expression of major bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and Bglap/Bglap2. The canonical Wnt signaling pathway is also crucial for bone formation by mature osteoblasts. Sp7 is needed for osteocytes to acquire a sufficient number of processes and a reduction in these processes results in osteocyte apoptosis and cortical porosity.

Formulation and evaluation of Phaseolus lunatus seed coat mediated silver nanoparticles mouthwash: A comprehensive study on biomedical properties and toxicological assessment

Silver nanoparticles have promising therapeutic potential in the field of dentistry, as newly emerging oral therapeutics, in the form of mouthwashes based on silver nanoparticles, demonstrate significant potential for enhancing oral management thus the present investigation aims to formulate silver nanoparticles-based mouthwash from an aqueous extract of Phaseolus lunatus seed coat (PLSC) and evaluate its biomedical properties. The green synthesized AgNPs in the mouthwash were characterized using UV–visible spectroscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and x-ray diffraction (XRD). The formulated mouthwash was assessed for its anti-microbial activity using the agar well diffusion technique and time-kill curve assay. Its anti-inflammatory and anti-oxidant activities are assessed through egg albumin assay and hydrogen peroxide assays. The Cytotoxic effect of formulated mouthwash was assessed through a brine shrimp lethality assay and MTT assay over the human osteoblast cell line (MG-63). Furthermore, the study also assessed the toxicity effect of formulated mouthwash through zebrafish embryos. The results suggest that the green synthesized AgNPs were spherical and had an average size of 42 nm and the formulated mouthwash exhibited significant anti-microbial activity, anti-inflammatory, and antioxidant properties, making them a better candidate for oral health therapeutics. The Zebrafish embryo toxicology studies of mouthwash revealed a consistent lack of abnormalities and a good viability and hatchability rate and the cytotoxic effect shows less toxicity over brine shrimp and human osteoblast cells. In conclusion, PLSC-AgNPs mouthwash is a potential oral therapeutic option with minimal toxicity for better oral management.

The impact of 45S5 bioglass vs. β-TCP nanoparticles ratio on rheological behavior of formulated printing inks and 3D printed polycaprolactone-based scaffolds final properties

Extrusion based 3-D printing has been extensively applied to create geometrically complex composite polymer-ceramic structures as bone tissue substitute. The rheological features of the formulated bioink that regulate the printability and resolution of the printed scaffolds, rely on physicochemical properties of ink components, mainly their composition and chemical structure. The aim of this study was to evaluate the effect of different content of 45S5 bioglass (BG) and β-tricalcium phosphate (β-TCP) nanoparticles on the rheological behavior of printing inks and final composite scaffolds based on polycaprolactone (PCL)/BG/β-TCP. Ceramic nano-powders were first characterized and the composite bioinks were prepared by mixing various ratios of BG and β-TCP powders into the 50% w/v of PCL solution (β-TCP/BG: 70/30, 50/50, 30/70, and 10/90 w/w %). All formulated inks showed a thixotropic behavior and viscosity significantly increased by applying higher fraction of BG. Interestingly highly loaded β-TCP ink (β-TCP/BG: 70/30) revealed a rubbery nature with high surface tension which reduced final scaffolds resolution. All printed scaffolds possessed highly porous structure with interconnected pores. However, porosity percentage and shape stability improved by increasing BG content which was accompanied with lower mechanical strength and superior biodegradation and bioactivity of scaffolds. The biological performance of the printed scaffolds was evaluated using osteoblast cell line MG-63. MTT assay and cell attachment observation by SEM confirmed that printed scaffolds are well biocompatible and properly support cell colonization and proliferation. In overall, besides more appropriate materialistic properties, scaffolds with β-TCP/BG: 30/70 composition provided the most favorable microenvironment for cell colonization and growth.

The apatite-forming ability of bioactive glasses – A comparative study in human serum and Kokubo's simulated body fluid

Currently, the apatite-forming ability evaluation of a biomaterial employs Kokubo's simulated body fluid (SBF) as a testing medium. As the conventional SBF simulates only the ionic composition and pH of human serum (HS), this study aimed to compare the apatite formation in SBF and HS on the sol-gel-derived bioglass (BG) 83S2 and commercial 45S5 as well as to verify the influence of preincubation of BG in both fluids on cytocompatibility towards human fetal osteoblast cell line hFOB 1.19. The apatite formation in HS was hindered, regardless of the type of used BG. Relatively higher apatite-forming ability was noticed for BG83S2. In direct contact test, both BGs were cytocompatible towards osteoblasts, regardless of the type of used fluid. In the case of extracts; however, only the extract obtained from BG83S2 preincubated in HS did not show the cytotoxic effect. The evaluation of apatite-forming ability in SBF appears to be a fast screening method for comparing the apatite-forming ability of various bioglasses. Nonetheless, SBF is unable to precisely imitate the morphology and crystallinity of apatite observed during the assay in HS, as well as it does not mimic the changes in profiles of osteogenic ions.

CRISPR activation of Tfeb , a master regulator of autophagy and lysosomal biogenesis, in osteoblast lineage cells increases bone mass and strength.

Autophagy is a recycling pathway in which damaged or dysfunctional proteins, protein aggregates, and organelles are delivered to lysosomes for degradation. Insufficiency of autophagy is thought to contribute to several age-related diseases including osteoporosis. Consistent with this, elimination of autophagy from the osteoblast lineage reduces bone formation and causes low bone mass. However, whether increasing autophagy would benefit bone health is unknown. Here, we increased expression of the endogenous Transcription Factor EB gene ( Tfeb ) in osteoblast lineage cells in vivo via CRISPR activation. Tfeb overexpression stimulated autophagy and lysosomal biogenesis in osteoblasts. Tfeb overexpressing male mice displayed a robust increase in femoral and vertebral cortical thickness at 4.5 months of age. Histomorphometric analysis revealed that the increase in femoral cortical thickness was due to increased bone formation at the periosteal surface. Tfeb overexpression also increased femoral trabecular bone volume. Consistent with these results, bone strength was increased in Tfeb overexpressing mice. Female Tfeb overexpressing mice also displayed a progressive increase in bone mass over time and at 12 months of age had high cortical thickness and trabecular bone volume. This increase in vertebral trabecular bone volume was due to elevated bone formation. Osteoblastic cultures showed that Tfeb overexpression increased proliferation and osteoblast formation. Overall, these results demonstrate that stimulation of autophagy in osteoblast lineage cells promotes bone formation and strength and may represent an effective approach to combat osteoporosis.

Regulation of Skeletal Development and Maintenance by Runx2 and Sp7.

Runx2 (runt related transcription factor 2) and Sp7 (Sp7 transcription factor 7) are crucial transcription factors for bone development. The cotranscription factor Cbfb (core binding factor beta), which enhances the DNA-binding capacity of Runx2 and stabilizes the Runx2 protein, is necessary for bone development. Runx2 is essential for chondrocyte maturation, and Sp7 is partly involved. Runx2 induces the commitment of multipotent mesenchymal cells to osteoblast lineage cells and enhances the proliferation of osteoprogenitors. Reciprocal regulation between Runx2 and the Hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathways and Dlx5 (distal-less homeobox 5) plays an important role in these processes. The induction of Fgfr2 (Fgf receptor 2) and Fgfr3 expression by Runx2 is important for the proliferation of osteoblast lineage cells. Runx2 induces Sp7 expression, and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Sp7 induces the differentiation of preosteoblasts into osteoblasts without enhancing their proliferation. In osteoblasts, Runx2 is required for bone formation by inducing the expression of major bone matrix protein genes, including Col1a1 (collagen type I alpha 1), Col1a2, Spp1 (secreted phosphoprotein 1), Ibsp (integrin binding sialoprotein), and Bglap (bone gamma carboxyglutamate protein)/Bglap2. Bglap/Bglap2 (osteocalcin) regulates the alignment of apatite crystals parallel to collagen fibrils but does not function as a hormone that regulates glucose metabolism, testosterone synthesis, and muscle mass. Sp7 is also involved in Co1a1 expression and regulates osteoblast/osteocyte process formation, which is necessary for the survival of osteocytes and the prevention of cortical porosity. SP7 mutations cause osteogenesis imperfecta in rare cases. Runx2 is an important pathogenic factor, while Runx1, Runx3, and Cbfb are protective factors in osteoarthritis development.

In Vitro Proliferation of MG-63 Cells in Additively Manufactured Ti-6Al-4V Biomimetic Lattice Structures with Varying Strut Geometry and Porosity.

Lattice structures have demonstrated the ability to provide secondary stability in orthopedic implants by promoting internal bone growth. In response to the growing prevalence of lattices in orthopedic design, we investigated the effects of porosity and unit cell geometry in additively manufactured Ti-6Al-4V biomimetic lattice structures on the osteogenesis of human MG-63 osteoblastic cell lines in vitro. We analyzed glucose consumption, alkaline phosphatase (ALP) concentration, and end-of-culture cell count as markers for osteogenic growth. Two different strut geometries were utilized (cubic and body-centered cubic), along with four different pore sizes (400, 500, 600, and 900 µm, representing 40-90% porosity in a 10 mm cube), in addition to a solid specimen. Structural characterization was performed using scanning electron microscopy. The results indicated that lattices with a 900 µm pore size exhibited the highest glucose consumption, the greatest change in ALP activity, and the highest cell count when compared to other pore sizes. Cubic 900 µm lattice structures outperformed other specimens in facilitating the maturation of viable MG-63 cells from the formation to the mineralization phase of bone remodeling, offering the most promise for osseointegration in additively manufactured titanium implants in the future. However, irrespective of a particular pore size or unit cell geometry, it was found that all the lattices were capable of promoting osteogenic growth due to surface roughness in the printed parts.

Photobiomodulation Modulates Proliferation and Gene Expression Related to Calcium Signaling in Human Osteoblast Cells.

Introduction: Photobiomodulation with low-level laser treatment can enhance bone formation by stimulating the cell division of osteoblasts and increasing the amount of protein deposition, thus encouraging the formation of new bone. The aim of this study was to evaluate the effects of photobiomodulation with a low-level laser on proliferation and gene expression related to calcium signaling in human osteoblasts. Methods: Osteoblastic cell lines of the hFOB1.19 lineage, human osteoblasts, were grown and assigned into two groups, control (C; n=78 cultured wells) and photobiomodulation (L; n=78 cultured wells) with n=6 per day of the experimental period. Cells were cultured (immature at 34 ºC), and after maturation at 37 ºC, group L cells were exposed to laser irradiation with a low-level laser device (gallium and aluminum arsenide), at a wavelength of 808 nm, a power output of 200 mW, and a power density of 200 mW/cm2. The energy delivered to the cells was 37 J/cm2, with a beam area of 0.02 mm2 and an exposure time of 5 seconds. This treatment was applied daily for a period of 13 days. Following this, the number of cells was counted, and RNA was isolated, measured, and then converted into cDNA for further quantification using a comparative Ct method with real-time polymerase chain reaction. The results were then subjected to statistical analysis through a Mann-Whitney test, with a significance level of P<0.05. Results: The cell count in the L group (37.25x10±4±22.02) was statistically higher compared to the control group (22.75x10±4±7.660) with a P value of 0.0259. The values of 2-ΔΔCt for S100A6, plasma membrane calcium ATPase (PMCA), and calmodulin genes indicated hyper-expression on the thirteenth day, while the osteocalcin gene showed hypo-expression. Conclusion: The study suggests that the photobiomodulation mechanism with a low-level laser may regulate gene expression in human osteoblasts in a dose-dependent and cumulative manner.

Effects of Three Retrograde Filling Materials on Production of Inflammatory Cytokines and Resorbing Mediators

: Root-end filling materials are typically used following endodontic surgeries, and due to their different chemical compositions, they have varying effects on the survival and differentiation of tissues around the root apex. The purpose of this in vitro study was to evaluate the effects of three retrograde filling materials—ProRoot MTA, Super ethoxy benzoic acid (EBA), and Geristore—on the production of pro-inflammatory and resorptive cytokines (RANK, RANK-L, OPG, IL-1β, and TNF-α). After mixing and setting, the experimental materials were exposed to UV rays and then applied to the prepared cells. This study used osteoblast (MG-63) and THP-1 monocyte cell lines, as well as peripheral blood monocytes (PBM). The evaluation times for RANK-L and OPG were 24 and 48 hours, while IL-1β and tumor necrosis factor (TNF-α) were evaluated at 24 hours. The RANK levels were measured using flow cytometry, and monocyte survival was assessed at different times using the MTT assay. The results were analyzed using Minitab statistical software, which indicated that the type of material significantly affected cytokine production. Exposure of monocytes to EBA resulted in a decrease in TNF-α and IL-1β secretion compared to the other groups. However, in the THP-1 cell line, there was no significant difference in the release of pro-inflammatory cytokines between the groups. Conversely, Geristore induced the highest level of RANK-L and the lowest level of OPG in cultures with the MG-63 cell line. According to the MTT assay, the viability order of the materials from early to late was Geristore &lt; EBA &lt; MTA.

Metformin enhances osteogenic differentiation of BMSC by modulating macrophage M2 polarization

Bone marrow-derived mesenchymal stem cells (BMSCs) are capable of developing into osteoblastic cell lines in vitro and regenerating bone tissue in vivo, and they are considered to be a reliable source for bone regenerative medicine. In recent years, studies have shown that the immune microenvironment is important for osteogenesis, in which macrophages are an important component of innate immunity and coordinate with stem cells. Metformin (Met), a hypoglycemic drug that exerts a powerful effect on metabolic signaling, has been shown to modulate inflammatory responses and osteogenic activity. However, whether metformin modulates macrophage polarization and subsequently affects osteogenesis remains to be elucidated. In the present study, we investigated the potential immunomodulatory capacity of metformin on macrophage inflammatory responses and phenotypic switching, and the subsequent effects on osteogenic differentiation of BMSCs. Flow cytometry and qPCR were used to study the effects of metformin on macrophage phenotypic regulation. qPCR, ALP, ARS and calcium content measurement and ALP activity assay were used to determine the effects of macrophage-secreted activators on the osteogenic differentiation of BMSCs. Our study demonstrates that metformin can improve the immune microenvironment by modulating macrophage polarization towards an anti-inflammatory phenotype, promoting an increase in a range of anti-inflammatory factors and inhibiting pro-inflammatory factors. This was characterized by increased expression of IL-10 and CD206, Arg-1 and decreased expression of IL-1β, TNF-α, IL-6 and iNOS. In addition, metformin-modulated macrophage-conditioned medium promoted osteogenic differentiation of BMSCs, increased the expression levels of genes (ALP, Runx-2, OCN, and Col-1), enhanced ALP activity, and significantly formed mineralized nodules. In conclusion, our new study elucidates that metformin can promote osteogenic differentiation of BMSCs by modulating macrophage phenotype and thereby.

An Exploration of the Role of Osteoclast Lineage Cells in Bone Tissue Engineering.

Bone defects because of age, trauma, and surgery, which are exacerbated by medication side effects and common diseases such as osteoporosis, diabetes, and rheumatoid arthritis, are a problem of epidemic scale. The present clinical standard for treating these defects includes autografts and allografts. Although both treatments can promote robust regenerative outcomes, they fail to strike a desirable balance of availability, side effect profile, consistent regenerative efficacy, and affordability. This difficulty has contributed to the rise of bone tissue engineering (BTE) as a potential avenue through which enhanced bone regeneration could be delivered. BTE is founded upon a paradigm of using biomaterials, bioactive factors, osteoblast lineage cells (ObLCs), and vascularization to cue deficient bone tissue into a state of regeneration. Despite promising preclinical results, BTE has had modest success in being translated into the clinical setting. One barrier has been the simplicity of its paradigm relative to the complexity of biological bone. Therefore, this paradigm must be critically examined and expanded to better account for this complexity. One potential avenue for this is a more detailed consideration of osteoclast lineage cells (OcLCs). Although these cells ostensibly oppose ObLCs and bone regeneration through their resorptive functions, a myriad of investigations have shed light on their potential to influence bone equilibrium in more complex ways through their interactions with both ObLCs and bone matrix. Most BTE research has not systematically evaluated their influence. Yet contrary to expectations associated with the paradigm, a selection of BTE investigations has demonstrated that this influence can enhance bone regeneration in certain contexts. In addition, much work has elucidated the role of many controllable scaffold parameters in both inhibiting and stimulating the activity of OcLCs in parallel to bone regeneration. Therefore, this review aims to detail and explore the implications of OcLCs in BTE and how they can be leveraged to improve upon the existing BTE paradigm.

Zinc-doped phosphate coatings for enhanced corrosion resistance, antibacterial properties, and biocompatibility of AZ91D Mg alloy

Magnesium and its alloys have been foreseen as promising bone-implant owing to good biocompatibility, high strength, mechanical properties, and biodegradability to replace the conventional bio-metals (Titanium, stainless-steel, Co-Cr alloys) in orthopedic applications. However, high corrosion rate and high degradation rates adversely effects like abrupt evolution of H2 gas and localized alkalization in a physiological environment limit its medical applications. To overcome these issues, phosphate-based surface coating is developed on Mg-alloy to resist the high biodegradation and corrosion rate utilizing magnesium substrate as source of magnesium ion through a single-step hydrothermal process. Controlling the fast corrosion rate and localized alkalization would reduce the antibacterial character of magnesium-based implants therefore, the simple phosphate-based coatings has been induced by doping it with zinc ions due to its physiological tolerance and excellent antibacterial efficacy. The doping of zinc ions may provide a sustained drug-free antibacterial characteristic for potential application in orthopedics. The synthesized phosphate-based coating was characterized using advanced techniques like Scanning Electron Microscopy, X-ray Diffraction, and wettability test, followed by comprehensive electrochemical testing to assess its corrosion behavior. The in vitro immersion test in simulated body fluid (SBF) indicates that deposition of phosphate and zinc doped phosphate coatings reduces the degradation rate consequently minimized the fast dissolution of Mg2+ ions and OH- in physiological environment. Additionally, cytotoxicity and biocompatibility were evaluated using Alamar Blue and live/dead assays on the MC3T3-E1 mouse osteoblast cell line. These assays demonstrated good cell viability, indicating the coatings possess favorable cytocompatibility and support cellular metabolic activity.

Zinc phosphate glass microspheres promoted mineralization and expression of BMP2 in MC3T3-E1 cells.

Degradable phosphate glasses have shown favorable properties for tissue engineering. By changing the composition of the glasses, the degradation rate, and ion release are controllable. Zinc oxide can function as a glass network modifier and has been shown to play a positive role in bone formation. Also, phosphate glasses can easily be processed into microspheres, which can be used as microcarriers. This study aims to develop zinc phosphate glasses microspheres and explore the optimized size and composition for applications in bone tissue engineering. Zinc-titanium-calcium-sodium phosphate glasses with 0, 1, 3, 5, or 10 mol % zinc oxide were prepared and processed into microspheres. The smaller microspheres ranged in size from 50 to 106 μm, while the larger ones ranged from 106 to 150 μm. The characteristics of glasses were examined. The osteoblastic cell line MC3T3-E1 was cultured on the surface of microspheres and the cell viability was examined. To evaluate osteogenic differentiation, Alizarin Red S staining, quantitative reverse transcription polymerase chain reaction, and western blot analysis were performed after 14 days. Different sizes of zinc phosphate glass microspheres were successfully made. The glass microspheres with <10 mol % zinc oxide were able to support the adhesion and proliferation of MC3T3-E1 cell lines. The relative gene expression of BMP2 was significantly upregulated in the smaller glass microspheres containing 3 mol % zinc oxide (26-fold, p < .001) and both sizes of microspheres containing 5 mol % zinc oxide (smaller: 27-fold, p < .001; larger: 35-fold, p < .001). Additionally, cluster formation was observed in glass microspheres after 14 days, and the mineralization of MC3T3-E1 cell lines was promoted. Based on these findings, the glass microspheres containing 3-5 mol % of zinc oxide can promote osteogenic differentiation for MC3T3-E1 cells.

Biological Effects of Double-Layered Hydroxyapatite and Zirconium Oxide Depositions on Titanium Surfaces.

This study aimed to confirm the synergy effect of these two materials by evaluating osteoblast and antibacterial activity by applying a double-layered hydroxyapatite(HA) zirconium oxide(ZrO2) coating to titanium. The specimens used in this study were divided into four groups: a control group (polished titanium; group T) and three experimental groups: Group TH (RF magnetron sputtered HA deposited titanium), Group Z (ZrO2 ALD deposited titanium), and Group ZH (RF magnetron sputtered HA and ZrO2 ALD deposited titanium). The adhesion of Streptococcus mutans (S.mutans) to the surface was assessed using a crystal violet assay. The adhesion, proliferation, and differentiation of MC3T3-E1 cells, a mouse osteoblastic cell line, were assessed through a WST-8 assay and ALP assay. Group Z showed a decrease in the adhesion of S. mutans (p < 0.05) and an improvement in osteoblastic viability (p < 0.0083). Group TH and ZH showed a decrease in adhesion of S. mutans (p < 0.05) and an increase in osteoblastic cell proliferation and cell differentiation (p < 0.0083). Group ZH exhibited the highest antibacterial and osteoblastic differentiation. In conclusion double-layered HA and ZrO2 deposited on titanium were shown to be more effective in inhibiting the adhesion of S. mutans, which induced biofilm formation, and increasing osteoblastic differentiation involved in osseointegration by the synergistic effect of the two materials.

Strategies for the Codelivery of Osteoclasts and Mesenchymal Stem Cells in 3D-Printable Osteochondral Scaffolds.

Osteochondral defects, characterized by structural compromises to articular cartilage and subchondral bone, can cause pain and lead to progressive cartilage damage and eventual osteoarthritis. Unfortunately, repairing these defects remains difficult because of the poor regenerative properties of cartilage and complex mechanical demands of the joint. As such, the field of tissue engineering aims to develop multiphasic implants that replace pathological cartilage and bone tissue and restore mechanical functionality to the joint. Recent bone physiology investigations have demonstrated that osteoclast (OC) lineage cells are inextricably involved in osteoblastic bone formation through an extensive network of anabolic signaling pathways, and so the codelivery OC and osteoblast (OB) lineage cells within scaffolds is being actively explored for bone tissue engineering purposes. However, it remains unclear how these cells can be incorporated into the design of multiphasic osteochondral scaffolds to potentially enhance subchondral bone formation and subsequent implant osseointegration. To explore this question, we examined direct surface seeding and hydrogel encapsulation as potential scaffold cellularization strategies. First, we examined how OC precursor cells and peripheral blood monocytes (PBMCs) influence early-stage bone matrix development and osteogenesis in 2D coculture. Then, we evaluated the osteogenic potential of mesenchymal stem cells (MSCs) and PBMCs cocultures encapsulated within a gelatin methacrylate (GelMA) hydrogel system. Our findings demonstrate that coculturing PBMCs with MSCs in 2D cultures significantly enhanced cell proliferation, early bone matrix deposition, and the formation of cell clusters by Day 28. However, we observed no significant difference in type I collagen deposition between GelMA hydrogel scaffolds cultured in basal and OC conditions during the same period. In addition, we found that the GelMA hydrogel system with MSC/PBMC cocultures in OC conditions exhibited decreased osteogenic activity by Day 28. Collectively, our findings support the osteogenic potential of OC-lineage cells in 2D culture conditions, and the potential benefits of surface-seeding for the codelivery of OC-lineage cells and MSCs in osteo-scaffolds for enhanced osteochondral regeneration and broader bone tissue engineering purposes.