Modulation of Macrophage Polarization by Phospholipids on the Surface of Titanium.

Macrophage polarization has become increasingly important for the improvement of the biocompatibility of biomaterials. In this study, we coated Ti discs with phospholipids (phosphatidylserine/phosphatidylcholine [4:1 mole/mole]) by evaporating the solvent under vacuum, and observed the polarization of RAW 264.7 cells cultured on the discs. The coated discs were hydrated before cell culture was added. The shape of the hydrated phospholipids varied with the concentration of loaded phospholipids: a perforated layer (0.1 mM), tubules and spheres (1 mM), and spheres (10 mM). RAW 264.7 cells exhibited different morphologies, depending on the concentration of phospholipids. On the coated discs, the gene expression and protein release of TGF-ß1, VEGF, Arg-1, and TNF-α were downregulated, especially with 10 mM phospholipids. The stimulation of mRNA expression and the protein release of those genes by IL-4 and LPS were also disturbed on the phospholipid-coated discs. In conclusion, the polarization of RAW 264.7 cells was prevented by hydrated phospholipids on Ti discs.

Immunomodulation of Biomaterials by Controlling Macrophage Polarization.

Macrophages are key players in innate immune responses to foreign substances. They participate in the phagocytosis of biomaterial-derived particles, angiogenesis, recruitment of fibroblasts, and formation of granulation tissues. Most macrophage functions are achieved through the release of various cytokines and chemokines; the release profile of cytokines is dependent on the phenotype of macrophages, namely proinflammatory M1 or antiinflammatory M2. M1 and M2 macrophages coexist during an inflammatory phase, and the M1/M2 ratio is considered to be an important factor for wound-healing or tissue regeneration. This ratio depends on the chemical and physical properties of biomaterials. To obtain a favorable foreign body reaction to biomaterials, the phenotypes of the macrophages can be modulated by cytokines, antibodies, small chemicals, and microRNAs. Geometrical surface fabrication of biomaterials can also be used for modulating the phenotype of macrophages.

Effects of phosphatidylserine-containing supported lipid bilayers on the polarization of macrophages.

Placement of dental implants initiates inflammatory foreign body response, in which macrophages play a central role and affect the subsequent tissue healing process such as bone formation. The purpose of this study was to fabricate phosphatidylserine (PS)-containing supported lipid bilayers (SLBs) on a titanium surface to regulate the polarization of macrophages, a critical factor that affects following tissue healing and regeneration. The fluorescent recovery after photobleaching images showed that the percentage of PS had a critical influence on the fluidity, and 20% PS had the highest fluidity. Furthermore, more expanded and elongated cells were observed in the SLB-coated groups. transforming growth factor-ß1 and vascular endothelial growth factor, the key cytokine markers of M2 macrophage polarization, were increased in the SLB-coated groups, especially in the 20% PS group. Consistently, cells cultured on the SLB-coated titanium exhibited the distribution of CD206+ , which is a M2 macrophage specific maker. The results of this study demonstrated M2 polarization of macrophages on PS-SLB-coated titanium discs, which suggests the application of PS-SLB as an immune-regulating coating material to improve tissue reactions to dental implants. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2625-2633, 2018.

The Effects of M1 and M2 Macrophages on Odontogenic Differentiation of Human Dental Pulp Cells.

INTRODUCTION: M1 (classically activated) and M2 (alternatively activated) macrophages are known to play primary roles in inflammation and tissue regeneration. To investigate the role of macrophages in dentin regeneration, this study examined the effects of M1 and M2 macrophages on the odontogenic/osteogenic differentiation of human dental pulp cells (HDPCs) using the conditioned media (CM) of the activated human monocyte cell line THP-1. METHODS: M1 and M2 macrophages were induced by lipopolysaccharide/interferon-γ and interleukin-4, respectively, and the phenotypes were confirmed by flow cytometry. Macrophage CM was prepared at 2-day intervals for a period of 6 days, which included the first 2 days of activation. The CM obtained on days 4 (M1CM-4 day and M2CM-4 day) and 6 (M1CM-6 day and M2CM-6 day) were tested for their ability to promote the alkaline phosphatase (ALP) activity of HDPCs. M2CM-4 day was also examined for its effects on the messenger RNA expression of dentin sialophosphoprotein and osteocalcin genes and the matrix mineralization of HDPCs. Tumor necrosis factor alpha and transforming growth factor beta 1 (TGF-ß1) in M1CM and M2CM, respectively, were quantified by an enzyme-linked immunosorbent assay. To verify the role of TGF-ß1, M2CM-4 day was pretreated by a TGF-ß blocking antibody and was examined for its effect on the ALP activity of HDPCs. RESULTS: M2CM-4 day and M2CM-6 day enhanced the ALP activity of HDPCs (P < .05). Furthermore, M2CM-4 day promoted the messenger RNA expression of the dentin sialophosphoprotein gene and matrix mineralization (P < .05), whereas M1CM did not affect ALP activity. The enzyme-linked immunosorbent assay detected large amounts of TGF-ß1 in M2CM-4 day and M2CM-6 day. The TGF-ß blocking antibody suppressed the ALP-enhancing activity of M2CM-4 day (P < .05). Furthermore, the same amount of TGF-ß1 as in M2CM-4 day increased ALP activity to a similar level as M2CM-4day-treated HDPCs. CONCLUSIONS: The CM of M2 macrophages enhanced the odontogenic/osteogenic differentiation of HDPCs. M1CM did not affect the ALP activity of HDPCs at least in the absence of M1-type inducers. The effects of M2CM on HDPCs were likely caused by TGF-ß1. Therefore, M2 macrophages are expected to support dentin regeneration in dental pulps.

Modulation of the anti-inflammatory effects of phosphatidylserine-containing liposomes by PEGylation.

Inhibiting liposome uptake by macrophages using polyethylene glycol (PEG) surface modifications is a widely used approach for extending the half-life of liposomes circulating in the blood. However, the biological effects of PEGylated liposomes on macrophages have not yet been thoroughly investigated. The purpose of this study was to examine the effects of PEGylated phosphatidylserine-containing liposomes (PEG-PSLs) on the expression of two inflammation-associated cytokines, tumor necrosis factor-α (TNF-α) and transforming growth factor-ß (TGF-ß), in the murine macrophage-like cell line RAW 264.7. Previous studies have demonstrated that PSLs inhibit TNF-α secretion and enhance TGF-ß synthesis in macrophages by mimicking apoptotic cells. We found that PEGylation differentially affected the TNF-α and TGF-ß levels. The PSL-mediated inhibitory effect on TNF-α secretion was enhanced by PEGylation, and PEG-PSLs decreased TGF-ß levels compared with non-PEGylated PSLs. Fluorescence-activated cell sorting analysis demonstrated that 1% PEGylation disturbed the incorporation of PSLs into macrophages. The interference of uptake is thought to extend the binding interaction between PS to PS receptors for PSL-mediated inhibition of TNF-α expression. Together, these findings indicate that PEG-PSLs can prevent TNF-α secretion without increasing TGF-ß levels in macrophages, and they support the potential clinical use of PEG-PSLs as anti-inflammatory agents with a relatively low potential to induce tissue fibrosis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1479-1486, 2017.

Effects of phosphatidylserine-containing liposomes on odontogenic differentiation of human dental pulp cells.

Phosphatidylserine (PS) is known to enhance biomineralization due to the ability to accumulate calcium ions. In this study, the effects of PS on odontogenic differentiation and mineralization of human dental pulp cells (HDPCs) were investigated using phosphatidylserine-containing liposomes (PSLs). PSL was slightly cytotoxic at 125 µM in growth medium, and ALP activity was up-regulated in the PSL-treated HDPCs at non-cytotoxic concentrations. Mineralization was also enhanced by PSL, while mRNA expressions of DSPP and OCN genes were slightly attenuated. The mRNA expression of Runx2 was not altered by PSL. It is thus likely that PSL selectively affected odontogenic differentiation processes of HDPC. Finally, the interaction between PSL and HDPC was investigated by staining with annexin V-FITC in PSL-treated HDPC. It was found that PS was gradually incorporated into HDPC cytoplasm for several days. The results of this study suggest that PSL is able to stimulate dentin formation in dental pulps.

Surface Characterization and Human Stem Cell Behaviors of Zirconia Implant Disks Biomimetic-Treated in Simulated Body Fluid.

PURPOSE: This study investigated the effects of biomimetic deposition on a zirconia surface in simulated body fluid (SBF) and assessed the proliferation and differentiation of human bone marrow mesenchymal stem cells on the SBF-treated zirconia disks. MATERIALS AND METHODS: Corrected SBF was prepared according to Kokubo's recipe. Eighty yttrium oxide-stabilized tetragonal zirconia polycrystalline disks were prepared and divided into two groups: (1) the test group with SBF-treated disks and (2) the control group with nontreated disks. Zirconia disks were soaked in SBF for 1, 4, 7, and 14 days at 36.5°C, and the hydroxyapatite (HA) precipitation was verified by analyzing the surface morphology. For more in-depth validation of HA formation, the surface roughness, composition, and crystallization of the 7-day treated disks were analyzed. Human bone marrow mesenchymal stem cells were used to further evaluate cell proliferation, alkaline phosphatase activity, and osteoblast gene expression on the 7-day treated zirconia disks. RESULTS: Disks showed different surface morphologies after soaking for different time periods. As the SBF soaking time increased, the amount of HA coverage increased gradually, uniformly covering the disks by day 7. There was no difference in surface roughness between the two groups (P > .05). Cell proliferation was higher on the SBF-treated disks (P < .05). At 9 days, alkaline phosphatase activity was higher on the SBF-treated disks (P < .05). Alkaline phosphatase staining was significant on the SBF-treated disks (P < .05). A gene study revealed that alkaline phosphatase and osteocalcin showed higher expression in SBF-treated disks (P < .05); however, collagen type I and runt-related transcription factor 2 did not show significant differences between the two groups (P > .05). CONCLUSION: This study demonstrated that biomimetic deposition has an effect on the formation of HA on zirconia disks. The cell attachment, proliferation, and differentiation of SBF-treated zirconia disks was superior to that of nontreated disks, which indicates that SBF-treated zirconia implants have long-term clinical value.

Optimization of growth inducing factors for colony forming and attachment of bone marrow-derived mesenchymal stem cells regarding bioengineering application.

PURPOSE: These days, mesenchymal stem cells (MSCs) have received worldwide attention because of their potentiality in tissue engineering for implant dentistry. The purpose of this study was to evaluate various growth inducing factors in media for improvement of acquisition of bone marrow mesenchymal stem cells (BMMSCs) and colony forming unit-fibroblast (CFU-F). MATERIALS AND METHODS: The mouse BMMSCs were freshly obtained from female C3H mouse femur and tibia. The cells seeded at the density of 10(6)/dish in media supplemented with different density of fetal bovine serum (FBS), 1α, 25-dihydroxyvitamin (VD3) and recombinant human epidermal growth factor (rhEGF). After 14 days, CFU-F assay was conducted to analyze the cell attachment and proliferation, and moreover for VD3, the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was additionally conducted. RESULTS: The cell proliferation was increased with the increase of FBS concentration (P<.05). The cell proliferation was highest at the density of 20 ng/mL rhEGF compared with 0 ng/mL and 200 ng/mL rhEGF (P<.05). For VD3, although the colony number was increased with the increase of its concentration, the difference was not statistically significant (P>.05). CONCLUSION: FBS played the main role in cell attachment and growth, and the growth factor like rhEGF played the additional effect. However, VD3 did not have much efficacy compare with the other two factors. Improvement of the conditions could be adopted to acquire more functional MSCs to apply into bony defect around implants easily.