Enhanced remineralisation ability and antibacterial properties of sol-gel glass ionomer cement modified by fluoride containing strontium-based bioactive glass or strontium-containing fluorapatite.

OBJECTIVES: This study aimed to compare two types of bioactive additives which were strontium-containing fluorinated bioactive glass (SrBGF) or strontium-containing fluorapatite (SrFA) added to sol-gel derived glass ionomer cement (SGIC). The objective was to develop antibacterial and mineralisation properties, using bioactive additives, to minimize the occurrence of caries lesions in caries disease. METHODS: Synthesized SrBGF and SrFA nanoparticles were added to SGIC at 1 wt% concentration to improve antibacterial properties against S. mutans, promote remineralisation, and hASCs and hDPSCs viability. Surface roughness and ion-releasing behavior were also evaluated to clarify the effect on the materials. Antibacterial activity was measured via agar disc diffusion and bacterial adhesion. Remineralisation ability was assessed by applying the material to demineralised teeth and subjecting them to a 14-day pH cycle, followed by microCT and SEM-EDS analysis. RESULTS: The addition of SrFA into SGIC significantly improved its antibacterial property. SGIC modified with either SrBGF or SrFA additives could similarly induce apatite crystal precipitation onto demineralised dentin and increase dentin density, indicating its ability to remineralise dentin. Moreover, this study also showed that SGIC modified with SrBGF or SrFA additives had promising results on the in vitro cytotoxicity of hASC and hDPSC. SIGNIFICANT: SrFA has superior antibacterial property as compared to SrBGF while demonstrating equal remineralisation ability. Furthermore, the modified SGIC showed promising results in reducing the cytotoxicity of hASCs and hDPSCs, indicating its potential for managing caries.

Zinc and Strontium-Substituted Bioactive Glass Nanoparticle/Alginate Composites Scaffold for Bone Regeneration.

The global population is growing older and entering an aging society. Aging results in severe tissue disorder and organ dysfunction. Bone-related injuries are particularly significant. The need for alternative bone replacement materials for human implants has grown over the past few decades. Alginate has the potential for use as a cell scaffold for bone tissue engineering due to its high bio-compatibility. To improve the bioactivity of alginate scaffolds, zinc- and strontium-containing sol-gel-derived bioactive glass nanoparticles (Zn-Sr-BGNPs) with sizes ranging from 100 to l40 nm were incorporated. Zn-Sr-BGNPs synthesized through the sol-gel process have a high sur-face-to-volume ratio, homogeneity, and purity, resulting in faster degradation. The therapeutic bivalent ions released from Zn-Sr-BGNPs strengthen the cell scaffold and improve the stimulation of the production and development of bone cells. Zn-Sr-BGNPs with different Zn to Si nominal ratios of 0, 1, and 1.5 were mixed with alginate in this research. The ratio of Zn in Zn-Sr-BGNPs and the ratio of Zn-Sr-BGNPs in scaffolds impact the pore size, swelling, and biological properties of synthesized composite scaffolds. The surface area and pore volume of a 1:1 1Zn-Sr-BGNP:Alg composite scaffold were 22.58 m2/g and 0.055 cm3/g, respectively. The incorporation of Zn-Sr-BGNPs improved the mechanical performance of the scaffolds up to 4.73 ± 0.48 MPa. The swelling rate decreased slightly from 2.12 (pure Alg) to 1.50 (1Zn-Sr-BGNP:Alg (1:1)). The 1Zn-Sr-BGNP:Alg (1:1) composite scaffold promoted bioactivity through apatite layer formation, increased bone cell proliferation via the dissolution products released from the scaffold, enhanced calcium deposition, and facilitated cell attachment. Thus, 1Zn-Sr-BGNP:Alg (1:1) composite scaffold is proposed as a possible artificial bone scaffold in bone tissue regeneration.

Assessment of Physical/Mechanical Performance of Dental Resin Sealants Containing Sr-Bioactive Glass Nanoparticles and Calcium Phosphate.

The aim of this study was to assess the chemical/mechanical properties of ion-releasing dental sealants containing strontium-bioactive glass nanoparticles (Sr-BGNPs) and monocalcium phosphate monohydrate (MCPM). Two experimental sealants, TS1 (10 wt% Sr-BGNPs and 2 wt% MCPM) and TS2 (5 wt% Sr-BGNPs and 4 wt% MCPM), were prepared. Commercial controls were ClinproXT (CP) and BeautiSealant (BT). The monomer conversion (DC) was tested using ATR−FTIR (n = 5). The biaxial flexural strength (BFS) and modulus (BFM) were determined (n = 5) following 24 h and 7 days of immersion in water. The Vickers surface microhardness (SH) after 1 day in acetic acid (conc) versus water was tested (n = 5). The bulk and surface calcium phosphate precipitation in simulated body fluid was examined under SEM-EDX. The ion release at 4 weeks was analyzed using ICP-MS (n = 5). The DC after 40 s of light exposure of TS1 (43%) and TS2 (46%) was significantly lower than that of CP (58%) and BT (61%) (p < 0.05). The average BFS of TS1 (103 MPa), TS2 (123 MPa), and BT (94 MPa) were lower than that of CP (173 MPa). The average BFM and SH of TS1 (2.2 GPa, 19 VHN) and TS2 (2.0 GPa, 16 VHN) were higher than that of CP (1.6 GPa, 11 VHN) and BT (1.3 GPa, 12 VHN). TS1 showed higher Ca, P, and Sr release than TS2. Bulk calcium phosphate precipitation was detected on TS1 and TS2 suggesting some ion exchange. In conclusion, the DC of experimental sealants was lower than that of commercial materials, but their mechanical properties were within the acceptable ranges. The released ions may support remineralizing actions.

Dose-dependent effects of neem crude extract on human dental pulp cell and murine osteoblast viability and mineralization.

Neem has been used as a medicine due to its beneficial properties such as anti-microbial effects. Neem products for oral application are on the rise. Before recommendation for therapeutic use in human, its effects on cellular activities need to be examined. Therefore, the aim of this study was to test the effects of the ethanolic neem crude extract on dental pulp cells and osteoblasts in terms of cell viability, mineralization, and gene expressions. The ethanolic neem extract derived from dry neem leaves was subjected to chemical identification using GC-MS. Human dental pulp stem cells (hDPSCs) and pre-osteoblasts (MC3T3) were treated with various concentrations of the neem crude extract. Cell viability, mineralization, and gene expressions were investigated by MTT assay, real-time PCR, and alizarin red S assay, respectively. Statistical analysis was performed by one-way ANOVA followed by Dunnett test. GC-MS detected several substance groups such as sesquiterpene. Low to moderate doses of the neem crude extract (4 - 16 µg/ml) did not affect hDPSC and MC3T3 viability, while 62.5 µg/ml of the neem extract decreased MC3T3 viability. High doses of the neem crude extract (250 - 1,000 µg/ml) significantly reduced viability of both cells. The neem crude extract at 1,000 µg/ml also decreased viability of differentiated hDPSC and MC3T3 and their mineralization. Furthermore, 4 µg/ml of neem inhibited viability of differentiated hDPSC. There is no statistical difference in gene expressions related to cell differentiation. In conclusion, the neem crude extract affected cell viability and mineralization. Cell viability altered differently depending on the doses, cell types, and cell stages. The neem crude extract did not affect cell differentiation. Screening of its effect in various aspects should be examined before the application for human use.

Dose-dependent effects of neem crude extract on human dental pulp cell and murine osteoblast viability and mineralization

Abstract Neem has been used as a medicine due to its beneficial properties such as anti-microbial effects. Neem products for oral application are on the rise. Before recommendation for therapeutic use in human, its effects on cellular activities need to be examined. Therefore, the aim of this study was to test the effects of the ethanolic neem crude extract on dental pulp cells and osteoblasts in terms of cell viability, mineralization, and gene expressions. The ethanolic neem extract derived from dry neem leaves was subjected to chemical identification using GC-MS. Human dental pulp stem cells (hDPSCs) and pre-osteoblasts (MC3T3) were treated with various concentrations of the neem crude extract. Cell viability, mineralization, and gene expressions were investigated by MTT assay, real-time PCR, and alizarin red S assay, respectively. Statistical analysis was performed by one-way ANOVA followed by Dunnett test. GC-MS detected several substance groups such as sesquiterpene. Low to moderate doses of the neem crude extract (4 - 16 µg/ml) did not affect hDPSC and MC3T3 viability, while 62.5 µg/ml of the neem extract decreased MC3T3 viability. High doses of the neem crude extract (250 - 1,000 µg/ml) significantly reduced viability of both cells. The neem crude extract at 1,000 µg/ml also decreased viability of differentiated hDPSC and MC3T3 and their mineralization. Furthermore, 4 µg/ml of neem inhibited viability of differentiated hDPSC. There is no statistical difference in gene expressions related to cell differentiation. In conclusion, the neem crude extract affected cell viability and mineralization. Cell viability altered differently depending on the doses, cell types, and cell stages. The neem crude extract did not affect cell differentiation. Screening of its effect in various aspects should be examined before the application for human use.

Resumo O Neem tem sido utilizado como medicamento devido às suas propriedades benéficas, tais como os efeitos antimicrobianos. Os produtos Neem para aplicação oral estão a aumentar. Antes da recomendação para uso terapêutico no ser humano, os seus efeitos nas atividades celulares precisam ser examinados. Por conseguinte, o objectivo deste estudo era testar os efeitos do extracto bruto de neem etanólico nas células de polpa dentária e osteoblastos em termos de viabilidade celular, mineralização e expressões genéticas. O extracto de neem etanólico derivado de folhas secas de neem foi sujeito a identificação química utilizando GC-MS. As células estaminais de polpa dentária humana (hDPSCs) e os pré-osteoblastos (MC3T3) foram tratados com várias concentrações do extrato bruto de neem. A viabilidade celular, mineralização, e expressões genéticas foram investigadas pelo ensaio MTT, PCR em tempo real, e o ensaio S vermelho de alizarina, respectivamente. A análise estatística foi realizada por ANOVA unidirecional seguida pelo teste Dunnett. O GC-MS detectou vários grupos de substâncias como o esquisterpeno. Doses baixas a moderadas do extracto bruto de neem (4 - 16 µg/ml) não afetaram a viabilidade do hDPSC e MC3T3, enquanto 62,5 µg/ml do extracto de neem diminuiu a viabilidade do MC3T3. Doses elevadas do extrato bruto de neem (250 - 1.000 µg/ml) reduziram significativamente a viabilidade de ambas as células. O extrato bruto de neem a 1.000 µg/ml também diminuiu a viabilidade de hDPSC e MC3T3 diferenciados e a sua mineralização. Além disso, 4 µg/ml de neem inibiu a viabilidade do hDPSC diferenciado. Não há diferença estatística nas expressões genéticas relacionadas com a diferenciação celular. Em conclusão, o extrato bruto do neem afetou a viabilidade celular e a mineralização. A viabilidade celular alterou-se diferentemente dependendo das doses, tipos de células, e fases celulares. O extrato bruto do neem não afetou a diferenciação celular. O rastreio do seu efeito em vários aspectos deve ser examinado antes da aplicação para uso humano.

Duration and timing of bisphosphonate treatment as factors determining osteoblast mineralization.

Background: Alendronate is a drug for the treatment of excessive bone resorption. Alendronate reduces osteoblast viability and mineralization in a dose-dependent manner. Other views of alendronate could affect mineralization. Therefore, the effects of timing and duration of alendronate treatment on mineralization were investigated. Materials and methods: MC3T3 cells were treated with alendronate at different time periods ranging from 1 to 3 weeks. Mineralization was quantified using image analysis. Cell viability was measured by Thiazolyl Blue Tetrazolium Bromide assay. Results: At low concentration (1 µM), 2-3 weeks of alendronate addition decreased mineralized area and staining intensity. Longer exposure to alendronate had more effect than short exposure. At moderate to high concentrations (5-10 µM), every alendronate treatment timing inhibited mineralization. Week 3 treatment of alendronate showed less reduction of mineralization than other time treatments. Furthermore, 10 µM alendronate reduced osteoblast viability at week 1, week 1-2, and week 1-3. Conclusion: Timing and duration of alendronate addition inhibited total mineralization. Longer period of treatment reduced mineralization more than short period of treatment. In addition, reduction of total mineralization depended upon both osteoblast viability and function.

Effects of Silver Diamine Nitrate and Silver Diamine Fluoride on Dentin Remineralization and Cytotoxicity to Dental Pulp Cells: An In Vitro Study.

Silver diamine nitrate (SDN) is expected to help control caries similar to silver diamine fluoride (SDF). The aim of this study was to determine the mineral precipitation in demineralized dentin and the cytotoxicity of SDN and SDF to dental pulp cells. Demineralized dentin specimens were prepared, and SDF, SDN, or water (control) was applied. The specimens were then remineralized in simulated body fluid for 2 weeks. The mineral precipitation in the specimens was examined using FTIR-ATR, SEM-EDX, and synchrotron radiation X-ray tomographic microscopy (SRXTM). Additionally, the cytotoxicity of SDF and SDN to human dental pulp stem cells was analyzed using an MTT assay. The increase in FTIR spectra attributable to apatite formation in demineralized dentin in the SDF group was significantly higher compared to the SDN and control groups (p < 0.05). Dentinal tubule occlusion by the precipitation of silver salts was detected in both SDF and SDN groups. The mineral density as shown in SRXTM images and cytotoxicity of both SDN and SDF groups were comparable (p > 0.05). In conclusion, SDF demonstrated superior in vitro apatite formation compared to SDN. However, the degree of mineral precipitation and cytotoxic effects of both were similar.

The inhibitory effect of a novel neem paste against cariogenic bacteria.

BACKGROUND: Dental caries is a major oral health problem, which associates with cariogenic bacteria. Streptococcus mutans and Lactobacillus acidophilus are facultative anaerobic bacteria that are found in tooth decay. Accordingly, neem leaf extract was developed due to its great anti-microbial property against many bacteria. The aim of this study was to determine anti-cariogenic properties of neem leaf extract in a novel paste preparation. MATERIAL AND METHODS: The neem extract was derived from maceration of dry neem leaves in ethanol for 48 h. The ethanolic extract was subjected to chemical identification using GC-MS. Neem pastes were prepared from ethanolic extract mixed with polyethylene glycol paste with or without zinc oxide. S. mutans and L. acidophilus test were initiated at bacterial concentration of 108 CFU/ml. The antibacterial activity was then performed by disc diffusion method following by minimum bactericidal concentration (MBC) technique. RESULTS: GC-MS result displayed 35 compounds. Compounds found in the extract were n-Hexadecanoic acid (31.18%), Hentriacontane (18%), Phytol (16.79%). Disc diffusion showed that ethanolic extract and neem pastes inhibited growth of both bacteria. For MBC, neem paste with zinc oxide at concentration of neem 0.4 mg/ml was the most effective concentration on inhibiting S. mutans growth. Neem pastes and ethanolic extract at concentration of neem 6.25 mg/ml inhibited L. acidophilus growth. CONCLUSIONS: The ethanolic neem leaf extract and novel neem pastes had antimicrobial effect on both S. mutans and L. acidophilus. By this property, neem paste could be developed for the application in dental field, i.e. pulp capping. Key words:Neem, Azadirachta indica, antimicrobial, cariogenic bacteria.

Monomer Conversion, Dimensional Stability, Biaxial Flexural Strength, Ion Release, and Cytotoxicity of Resin-Modified Glass Ionomer Cements Containing Methacrylate-Functionalized Polyacids and Spherical Pre-Reacted Glass Fillers.

The aim of this study was to prepare RMGICs for pulp protection that contain polyacids functionalized with methacrylate groups (CMs) to enable light-activated polymerization without the need for toxic 2-hydroxyethyl methacrylate (HEMA) monomers. The effects of using CM liquids with 0 or 5 wt% HEMA on the physical/mechanical properties and cytotoxicity of the experimental RMGICs were assessed. Spherical pre-reacted glass fillers (SPG) were used as the powder phase. The experimental RMGICs were prepared by mixing SPG with CM liquid (0 wt% HEMA, F1) or CMH liquid (5 wt% HEMA, F2). Commercial materials (Vitrebond, VB; TheraCal LC, TC) were used for the comparisons. The degree of monomer conversion and fluoride release of both F1 and F2 were significantly lower than those of VB. F1 showed comparable biaxial flexural strength with VB but higher strength than TC. The dimensional stability (mass/volume changes) of the experimental materials was comparable with that of the commercial materials. F1 and F2 exhibited higher Sr/Ca ion release and relative cell viability than VB. The use of CMH liquid reduced the strength but enhanced the fluoride release of the experimental RMGICs. In conclusion, the experimental RMGICs showed comparable strength but lower cytotoxicity compared to the commercial RMGICs. These novel materials could be used as alternative materials for pulp protection.

Inhibitory Effects of Alendronate on Adhesion and Viability of Preosteoblast Cells on Titanium Discs.

OBJECTIVE: This study aimed to investigate the effects of alendronate (ALN; a bisphosphonate) on adhesion and viability of preosteoblasts using different cell passages on sandblasted and acid-etched (SLA) Ti surfaces. MATERIALS AND METHODS: Preosteoblast, MC3T3, cells (passage 42; P42 and passage 62; P62) were cultured with ALN (1 and 5 µM) on cell culture plate for 7 days. Cells were lifted, counted, and seeded on SLA Ti surfaces. Cells were incubated on the discs for 6 hours to examine cell adhesion by using confocal microscopy and for 24 hours to determine cell viability by using MTT assay. RESULTS: ALN interfered with cell adhesion on Ti surfaces by reducing the cell number in both cell passages. Nuclei of untreated cells showed oval shape, whereas some nuclei of ALN-treated cells demonstrated crescent and condensed appearance. ALN at 1 and 5 µM significantly decreased nuclear area and perimeter in P42, while ALN at 5 µM reduced nuclear area and perimeter in P62. After 24 hours, cells (P42) grown on Ti surfaces showed decreased cell viability when culturing with 5 µM ALN. CONCLUSION: ALN reduced cell adhesion and viability of preosteoblasts on Ti surfaces. ALN treatment seemed to exert higher inhibitory effects on nuclear shape and size as well as cell viability in lower cell passage. This led to the reduction in cell to implant surface interaction after encountering bisphosphonate treatment.

Timing of geranylgeraniol addition increases osteoblast activities under alendronate condition.

BACKGROUND: Alendronate (ALN), a nitrogen-containing bisphosphonate, is prescribed to treat bone diseases. ALN acts as an inhibitor of enzymes in the mevalonate pathway, which results in reducing osteoblast viability and mineralization. Geranylgeraniol (GGOH) is a substrate in mevalonate pathway and mediates protein prenylation in the cells. OBJECTIVE: To investigate the effects of GGOH on ALN-treated osteoblast activities in order to improve the application of GGOH. METHODS: MC3T3 cells were treated with ALN. GGOH were added at different time points. Cell activities were examined using alizarin red S, MTT assay, alkaline phosphatase (ALP) activity, and quantitative polymerase chain reaction. RESULTS: ALN decreased mineralization. In the presence of ALN, GGOH addition at the first week of culture increased mineralization compared with the addition at other time points. ALN treatment for 7 days caused a reduction in osteoblast and pre-osteoblast viability compared with untreated cells. GGOH supplement partially rescued cell viability and increased total protein in cells treated with ALN. Furthermore, GGOH significantly upregulated gene expressions of Col I, OPN, VEGF, and VEGFR2. CONCLUSION: GGOH could be best applied at the early stage of osteogenesis since GGOH helped increasing cell viability and differentiation at the first 7 day of treatment.

Assessment of bisphosphonate treated-osteoblast behaviors by conventional assays and a simple digital image analysis.

The main objective of this study was to analyze the changes in osteoblast behaviors by two different methods, a simple digital image analysis method and conventional assays. Osteoblast progenitor cells and osteoblasts were treated with alendronate (ALN; a nitrogen-containing bisphosphonate). Osteoblast behaviors such as the uptake of ALN, cell proliferation, cell differentiation, and mineralized nodule formation were examined. Quantitative assessments were conducted using a publically available ImageJ software along with conventional methods. Furthermore, ImageJ method and conventional assay for mineralized nodule formation were performed simultaneously and were compared in order to demonstrate the reliability of ImageJ analysis. Osteoblast precursors and osteoblasts responded to ALN treatments. The software could identify various colors and allowed for the quantification of staining intensity and area coverage. Both image analysis and conventional assays detected the changes in cell behaviors between treated and untreated samples. For alizarin red S assay, the staining intensity calculated by ImageJ analysis was comparable to the absorbance measured by conventional assay. These findings showed that digital image analysis along with conventional assays could be used for quantitative assessment to evaluate osteoblast alteration by drug treatment. Image analysis method is practical and might be useful for other applications in the field of biology and medical sciences. It could also be employed in a combination with the conventional assays to strengthen the data.

Geranylgeraniol reverses alendronate-induced MC3T3 cell cytotoxicity and alteration of osteoblast function via cell cytoskeletal maintenance.

BACKGROUND: Alendronate (ALN) is a bisphosphonate, which is prescribed as an anti-osteoporotic drug. ALN has been shown to increase osteoblast cell death and decrease bone mineralization. ALN inhibits a key regulatory enzyme in the mevalonate pathway, consequently reducing geranylgeranyl pyrophosphate (GGPP). Geranylgeraniol (GGOH) can be converted to GGPP. The aim of this study was to investigate the effects of exogenous GGOH on MC3T3 cell viability, cell cycle, osteoblast function, and cell cytoskeleton under ALN treatment. METHODS: MC3T3 cells and osteoblast precursors, were incubated with ALN (0-50 µmol/L) and GGOH (0-50 µmol/L). After treatment, cells were evaluated for cell viability, cell cycle, osteoblast function, and cell cytoskeleton by MTT, flow cytometry, alizarin red S assay, and fluorescent microscopy, respectively. RESULTS: ALN reduced cell viability and bone nodule formation in a dose-dependent manner. GGOH partially inhibited the negative effects of ALN on cell viability and function. ALN increased the percentages of cell apoptosis and necrosis and arrested cells in G2M phase. Co-incubation with GGOH partially reduced late cell apoptosis and rescued cell cycle arrest. Furthermore, ALN altered MC3T3 morphology and decreased cell area, actin stress fiber density as well as nuclear area. GGOH abolished the effect of ALN on cell area, actin stress fiber density, and nuclear area. CONCLUSIONS: GGOH partially inhibited negative effects of ALN on cell viability, cell cycle, function, and cell cytoskeleton. It might be an additional option for increasing osteoblast function and reducing apoptosis of osteoblasts in the condition treated with low bisphosphonate concentration.

Inhibition of macrophage viability by bound and free bisphosphonates.

INTRODUCTION: Long-term administration of bisphosphonates (BPs) may cause osteonecrosis of the jaw (BRONJ). After administration, 50% of BPs in the circulation rapidly binds to calcium phosphate of bone. Two forms, bound and free BPs, may affect cells residing in bone including macrophages. Therefore, the aim of this study was to examine the effects of bound and free BPs on macrophage viability. MATERIALS AND METHODS: Biomaterials coated with BPs were used as a model to investigate the effect of bound BPs. For free BPs, RAW cells were plated on uncoated materials and BPs were added into the media. Cell viability and number were investigated by MTT assay and nuclei staining, respectively. Furthermore, coating and washing media were collected and were used to examine cell viability. RESULTS: RAW cells grew on biomaterials for 7 days. At 3 days, free and calcium-bound BPs significantly decreased cell viability and cell number compared to control. Coating media collected from pre-incubation with BP-coated composite materials reduced macrophage cell viability. CONCLUSION: This study showed that macrophages were directly affected by bound and free BPs. The presence of macrophages is mandatory for bone healing, thus the inhibition of cell viability might serve as an etiology of BRONJ.

Alteration of macrophage viability, differentiation, and function by bisphosphonates.

BACKGROUND: A serious adverse effect of long-term bisphosphonate (BP) administration is bisphosphonate-related osteonecrosis of the jaw (BRONJ). Among different proposed pathogenesis, suppression of immune cells is gaining interest. Because monocytes/macrophages could get access to BP since residing in the blood and bone microenvironment, the aim of this study was to analyze the behaviors of macrophages after BP treatments in vitro. METHODS: THP-1 cell, an established human monocytic cell model, was used in this study. The effects of BPs, alendronate (ALN) and zoledronic acid (ZA), on macrophage viability, differentiation, and function were investigated. MTT, morphological analysis, flow cytometry, quantitative PCR, and gelatin zymography assay were performed. RESULTS: BPs impaired macrophage viability at almost all concentration tested (1-100 µM). Cell morphology was altered in the presence of 100 µM BPs. Furthermore, differentiating macrophage viability was also affected by both ALN and ZA at 100 and 10-100 µM, respectively. At high concentration (100 µM), ZA caused a reduction in cell differentiation. On the contrary, ALN and ZA increased matrix metalloproteinase mRNA expressions and activities at low doses (1-10 µM). CONCLUSION: BPs directly acted on macrophage by reducing macrophage survival, inducing morphological alterations, impairing differentiation from monocytes to macrophages, and affecting macrophage function at both mRNA and activity levels.

In-vitro responses of T lymphocytes to poly(butylene succinate) based biomaterials.

BACKGROUND: Polybutylene succinate (PBSu) and PBSu/ß-tricalcium phosphate (TCP) composites are biocompatible and good candidates as bone graft materials. However, little is known about the responses of T lymphocytes to these biomaterials, which play an important role in the success of bone grafting. METHODS: Activated T lymphocytes were cultured onto 32 mm diameter films (PBSu/TCP films), that had previously been placed in 6-well culture plates, for 8, 24 and 72 hours. A plastic-well culture plate was used as a control surface. The effects of PBSu-based biomaterials on T lymphocytes were examined by the using flow cytometry and reverse-transcription polymerase chain reaction. RESULTS: These biomaterials were non-toxic to T lymphocytes, allowing their normal DNA synthesis and activation. All materials induced only transient activation of T lymphocytes, which existed no longer than 72 hours. Proportions of four main CD4/CD8 T lymphocyte subpopulations were not affected by these biomaterials. Moreover, PBSu and PBSu/TCP significantly suppressed the expression of IL-1ß and IL-6 genes by 15-35% and 21-26%, respectively. In contrast, a PBSu/TCP composite (at PBSu:TCP=60:40) significantly stimulated the expression of IL-10 and IL-13 genes by 17% and 19%, respectively. CONCLUSIONS: PBSu and PBSu/TCP composites were non-toxic to T lymphocytes and did not induce unfavorable responses of T lymphocytes. The tested biomaterials down-regulated key proinflammatory cytokine genes and up-regulated anti-inflammatory cytokine genes in T lymphocytes. These suggest that the biomaterials studied are good candidates as bone graft materials.

Osteoinduction of stem cells by collagen peptide-immobilized hydrolyzed poly(butylene succinate)/ß-tricalcium phosphate scaffold for bone tissue engineering.

Bone substitute is a therapeutic approach to treat bone abnormalities. A scaffold serves mainly as osteoconductive elements. To facilitate a better biological performance, short collagen peptide was immobilized onto hydrolyzed poly(butylene succinate)/ß-tricalcium phosphate (HPBSu/TCP) scaffolds. PBSu/TCP (80:20) scaffolds were fabricated by a supercritical CO2 technique, hydrolyzed with 0.6 M NaOH and conjugated with short collagen peptide tagged with or without red fluorescence. The surface morphology and porous structure of scaffolds were characterized by scanning electron microscopy and micro-computed tomography. Human mesenchymal stem cells were cultured onto the scaffolds and examined for osteogenic differentiation and biomineralization in vitro by means of alkaline phosphatase activity, alizarin red staining, and reverse transcription-polymerase chain reaction. The PBSu/TCP and HPBSu/TCP scaffolds were successfully prepared. Scanning electron microscopy and micro-computed tomography results showed that the porosity was distributed throughout the scaffolds with the pore sizes in the range of 250-900 µm. Fluorescence microscopy demonstrated retention of tagged short collagen peptide on the scaffold. Mesenchymal stem cells adhered and grew well on the material. Under osteogenic induction, cells cultured on the short collagen peptide -immobilized scaffold significantly produced a greater amount of alkaline phosphatase activity and positive mineralization than those cultured on the control scaffold. The present results have shown that the short collagen peptide-immobilized HPBSu/TCP scaffold enhanced osteoinduction and biomineralization of stem cell-derived osteoblasts, possibly via stimulation of alkaline phosphatase activity. This suggests the potential use of osteogenic peptide-immobilized material in bone tissue engineering for correcting bone defects.

Stem cell adhesion and proliferation on hydrolyzed poly(butylene succinate)/ß-tricalcium phosphate composites.

Although poly(butylene succinate)/ß-tricalcium phosphate (PBSu/TCP) composites are biocompatible and allow the growth and osteogenic differentiation of stem cells, cell attachment and adhesion to the PBSu-based substrates is often limited. To enhance cell adhesion and proliferation, we used a sodium hydroxide (NaOH) hydrolysis technique to generate a different degree of roughness on PBSu/TCP substrates with different PBSu:TCP ratios. The results showed that NaOH hydrolysis increased surface roughness of PBSu/TCP substrates in a concentration-dependent manner. Substrates with higher ratios of TCP:PBSu provided more porous topography after NaOH hydrolysis, with a substrate containing 40 wt % TCP (PBSu/TCP-6040) hydrolyzed with 1.5M NaOH (HPBSu/TCP-6040-1.5) showing the highest degree of roughness. As with the roughness, PBSu/TCP surface hydrophilicity was positively affected by the increasing NaOH concentration and TCP incorporation. Stem cells adhered best on HPBSu/TCP-6040-1.5 with three-dimensionally elongated cell extensions. Moreover, the HPBSu/TCP-6040-1.5 substrate most significantly facilitated stem cell actin cytoskeleton reorganization and vinculin-positive focal adhesion formation when compared with the other substrates tested. HPBSu/TCP-6040-1.5 also demonstrated the greatest increase in cell proliferation when compared with the other substrates studied. In conclusion, the results have shown that among various substrates tested, HPBSu/TCP-6040-1.5 provided the best support for stem cell adhesion and proliferation, suggesting its potential use in bone engineering.

Enhanced osteogenic activity of a poly(butylene succinate)/calcium phosphate composite by simple alkaline hydrolysis.

Bone engineering offers the prospect of alternative therapies for clinically relevant skeletal defects. Poly(butylene succinate) (PBSu) is a biodegradable and biocompatible polyester which may possess some limitations in clinical use due to its hydrophobicity. In order to overcome these limitations and increase the bioactivity, a simple and convenient surface hydrolysis of PBSu, PBSu/hydroxyapatite and PBSu/ß-tricalcium phosphate (TCP) films was performed. The resulting surfaces (i.e., HPBSu, HPBSu/HA and HPBSu/TCP) were tested for their physicochemical property, biocompatibility and osteogenic potency. The results showed that surface hydrolysis significantly increased surface roughness and hydrophilicity of the composites, with the HPBSu/TCP possessing the most pronounced results. All the materials appeared to be biocompatible and supported in vitro growth and osteoblast differentiation of hMSCs, and the alkaline hydrolysis significantly enhanced the hMSC cell proliferation and the osteogenic potency of PBSu/TCP compared with the non-hydrolyzed sample. In conclusion, the HPBSu/TCP possessed better hydrophilicity, biocompatibility and osteogenic potency in vitro, suggesting that this simple and convenient alkaline hydrolysis could be used to augment the biological property of PBSu-based composites for bone engineering in vivo.