Electrospun Tamarindus indica-loaded antimicrobial PMMA/cellulose acetate/PEO nanofibrous scaffolds for accelerated wound healing: In-vitro and in-vivo assessments.

In this work, Tamarindus indica (T. indica)-loaded crosslinked poly(methyl methacrylate) (PMMA)/cellulose acetate (CA)/poly(ethylene oxide) (PEO) electrospun nanofibers were designed and fabricated for wound healing applications. T. indica is a plant extract that possesses antidiabetic, antimicrobial, antioxidant, antimalarial and wound healing properties. T. indica leaves extract of different concentrations were blended with a tuned composition of a matrix comprised of PMMA (10 %), CA (2 %) and PEO (1.5 %), and were electrospun to form smooth, dense and continuous nanofibers as illustrated by SEM investigation. In vitro evaluation of T. indica-loaded nanofibers on normal human skin fibroblasts (HBF4) revealed a high compatibility and low cytotoxicity. T. indica-loaded nanofibers significantly increased the healing activity of scratched HBF4 cells, as compared to the free plant extract, and the healing activity was significantly enhanced upon increasing the plant extract concentration. Moreover, T. indica-loaded nanofibers demonstrated significant antimicrobial activity in vitro against the tested microbes. In vivo, nanofibers resulted in a superior wound healing efficiency compared to the control untreated animals. Hence, engineered nanofibers loaded with potent phytochemicals could be exploited as an effective biocompatible and eco-friendly antimicrobial biomaterials and wound healing composites.

A PMMA bone cement with improved antibacterial function and flexural strength.

A novel non-leaching antibacterial bone cement has been developed and evaluated. An antibacterial furanone derivative was synthesized and covalently coated onto the surface of alumina filler particles, followed by mixing into a conventional poly(methyl methacrylate) bone cement. Flexural strength and bacterial viability were used to evaluate the modified cements. Effects of coated antibacterial moiety content, coated alumina filler particle size and loading were investigated. Results showed that almost all the modified cements showed higher flexural strength (up to 10%), flexural modulus (up to 18%), and antibacterial activity (up to 67% to S. aureus and up to 84% to E. coli), as compared to original poly(methyl methacrylate) cement. Increasing antibacterial moiety and filler loading significantly enhanced antibacterial activity. On the other hand, increasing coated filler particle size decreased antibacterial activity. Increasing antibacterial moiety content and particle size did not significantly affect flexural strength and modulus. Increasing filler loading did not significantly affect flexural modulus but reduced flexural strength. Antibacterial agent leaching tests showed that it seems no leachable antibacterial component from the modified experimental cement to the surrounding environment. Within the limitations of this study, the modified poly(methyl methacrylate) bone cement may potentially be developed into a clinically useful bone cement for reducing in-surgical and post-surgical infection.

Multifunctional bone cement for synergistic magnetic hyperthermia ablation and chemotherapy of osteosarcoma.

Myelosuppression, gastrointestinal toxicity and hypersensitivities always accompany chemotherapy of osteosarcoma (OS). In addition, the intricate karyotype of OS, the lack of targeted antitumor drugs and the bone microenvironment that provides a protective alcove for tumor cells reduce the therapeutic efficacy of chemotherapy. Here, we developed a multifunctional bone cement loaded with Fe3O4 nanoparticles and the antitumor drug doxorubicin (DOX/Fe3O4@PMMA) for synergistic MH ablation and chemotherapy of OS. The localized intratumorally administered DOX/Fe3O4@PMMA can change from liquid into solid at the tumor site via a polyreaction. The designed multifunctional bone cement was constructed with Fe3O4 nanoparticles, PMMA, and an antitumor drug approved by the U.S. Food and Drug administration (FDA). The injectability, magnetic hyperthermia (MH) performance, controlled drug release profile, and synergistic therapeutic effect of DOX/Fe3O4@PMMA in vitro were investigated in detail. Furthermore, the designed DOX/Fe3O4@PMMA controlled the release of DOX, enhanced the apoptosis of OS tissue, and inhibited the proliferation of tumor cells, demonstrating synergistic MH ablation and chemotherapy of OS in vivo. The biosafety of DOX/Fe3O4@PMMA was also evaluated in detail. This strategy significantly reduced surgical time, avoided operative wounds and prevented patient pain, showing a great clinical translational potential for OS treatment.

Square prism micropillars on poly(methyl methacrylate) surfaces modulate the morphology and differentiation of human dental pulp mesenchymal stem cells.

Use of soluble factors is the most common strategy to induce osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro, but it may raise potential side effects in vivo. The topographies of the substrate surfaces affect cell behavior, and this could be a promising approach to guide stem cell differentiation. Micropillars have been reported to modulate cellular and subcellular shape, and it is particularly interesting to investigate whether these changes in cell morphology can modulate gene expression and lineage commitment without chemical induction. In this study, poly(methyl methacrylate) (PMMA) films were decorated with square prism micropillars with different lateral dimensions (4, 8 and 16 µm), and the surface wettability of the substrates was altered by oxygen plasma treatment. Both, pattern dimensions and hydrophilicity, were found to affect the attachment, proliferation, and most importantly, gene expression of human dental pulp mesenchymal stem cells (DPSCs). Decreasing the pillar width and interpillar spacing of the square prism pillars enhanced cell attachment, cell elongation, and deformation of nuclei, but reduced early proliferation rate. Surfaces with 4 or 8 µm wide pillars/gaps upregulated the expression of early bone-marker genes and mineralization over 28 days of culture. Exposure to oxygen plasma increased wettability and promoted cell attachment and proliferation but delayed osteogenesis. Our findings showed that surface topography and chemistry are very useful tools in controlling cell behavior on substrates and they can also help create better implants. The most important finding is that hydrophobic micropillars on polymeric substrate surfaces can be exploited in inducing osteogenic differentiation of MSCs without any differentiation supplements.

Precision-porous templated scaffolds of varying pore size drive dendritic cell activation.

Scaffold based systems have shown significant potential in modulating immune responses in vivo. While there has been much attention on macrophage interactions with tissue engineered scaffolds for tissue regeneration, fewer studies have looked at the effects of scaffold design on the response of immune cells-that is, dendritic cells (DCs). Here, we present the effects of varying pore size of poly (2-hydroxyethyl methacrylate) (pHEMA) and poly(dimethylsiloxane) (PDMS, silicone) scaffolds on the maturation and in vivo enrichment of DCs. We employ a precision templating method to make 3-D porous polymer scaffolds with uniformly defined and adjustable architecture. Hydrophilic pHEMA and hydrophobic PDMS scaffolds were fabricated in three pore sizes (20, 40, 90 µm) to quantify scaffold pore size effects on DCs activation/maturation in vitro and in vivo. In vitro results showed that both pHEMA and PDMS scaffolds could promote maturation in the DC cell line, JAWSII, that resembled lipopolysaccharide (LPS)-activated/matured DCs (mDCs). Scaffolds with smaller pore sizes correlate with higher DC maturation, regardless of the polymer used. In vivo, when implanted subcutaneously in C57BL/6J mice, scaffolds with smaller pore sizes also demonstrated more DCs recruitment and more sustained activation. Without the use of DC chemo-attractants or chemical adjuvants, our results suggested that DC maturation and scaffold infiltration profile can be modulated by simply altering the pore size of the scaffolds.

Elution of High Dose Amphotericin B Deoxycholate From Polymethylmethacrylate.

Fungal periprosthetic joint infections are rare, devastating complications of arthroplasty. There is conflicting evidence as to the efficacy of amphotericin B elution from cement spacers. The purpose of this study was to determine whether concentrations of amphotericin B released from bone cement over time would be efficacious in treating a periprosthetic infection. A continuous flow chamber was used to evaluate the in vitro release of amphotericin from cement beads containing 7.5% amphotericin. Following polymerization, 3.3% of the initially loaded amphotericin B was detected. The peak mean concentration eluted from the bone cement was 0.33 µg/mL at 8 hours. The AUC0-24 was 2.79 µg/mL/h; 0.20% of the amphotericin B was released. In conclusion, amphotericin B is released from bone cement at a clinically useful concentration.

Resolution, sensitivity, and in vivo application of high-resolution computed tomography for titanium-coated polymethyl methacrylate (PMMA) dental implants.

OBJECTIVES: The aims of this study were (i) to determine the spatial resolution and sensitivity of micro- versus nano-computed tomography (CT) techniques and (ii) to validate micro- versus nano-CT in a dog dental implant model, comparative to histological analysis. MATERIAL AND METHODS: To determine spatial resolution and sensitivity, standardized reference samples containing standardized nano- and microspheres were prepared in polymer and ceramic matrices. Thereafter, 10 titanium-coated polymer dental implants (3.2 mm in Ø by 4 mm in length) were placed in the mandible of Beagle dogs. Both micro- and nano-CT, as well as histological analyses, were performed. RESULTS: The reference samples confirmed the high resolution of the nano-CT system, which was capable of revealing sub-micron structures embedded in radiodense matrices. The dog implantation study and subsequent statistical analysis showed equal values for bone area and bone-implant contact measurements between micro-CT and histology. However, because of the limited sample size and field of view, nano-CT was not rendering reliable data representative of the entire bone-implant specimen. CONCLUSIONS: Micro-CT analysis is an efficient tool to quantitate bone healing parameters at the bone-implant interface, especially when using titanium-coated PMMA implants. Nano-CT is not suitable for such quantification, but reveals complementary morphological information rivaling histology, yet with the advantage of a 3D visualization.

Effects of Ti, PMMA, UHMWPE, and Co-Cr wear particles on differentiation and functions of bone marrow stromal cells.

This study investigates the roles of orthopedic biomaterial particles [Ti-alloy, poly(methyl methacrylate) (PMMA), ultrahigh-molecular-weight polyethylene (UHMWPE), Co-Cr alloy] on the differentiation and functions of bone marrow stromal cells (BMSCs). Cells were isolated from femurs of BALB/c mice and cultured in complete osteoblast-induction medium in presence of micron-sized biomaterial particles at various doses. 3-(4,5)-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase assay were performed for cell proliferation and cytotoxicity. Differentiation and function of osteoblasts were evaluated by alkaline phosphatase (ALP), osteocalcin, RANKL, OSX, and Runx2 expressions. Murine interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α in culture media were determined by enzyme-linked immunosorbent assay. Challenge with low doses of Ti, UHMWPE, or Co-Cr particles markedly promoted the bone marrow cell proliferation while high dose of Co-Cr significantly inhibited cell growth (p < 0.05). Cells challenged with low dose of PMMA or UHMWPE particles (0.63 mg/mL) exhibited strong ALP activity, whereas Ti and Co-Cr groups showed minimal effects (p < 0.05). UHMWPE and Ti particles also promoted higher expression of proinflammatory cytokines. Real-time polymerase chain reaction data suggested that cells treated with low dose (0.5 mg/mL) particles resulted in distinctly diminished RANKL expression compared to those exposed to high concentrated (3 mg/mL) particles. In conclusion, various types of wear debris particles behaved differently in the differentiation, maturation, and functions of osteogenic cells; and the particulate debris-interacted BMSCs may play an important role in the pathogenesis and process of the debris-associated aseptic prosthetic loosening.

Calcite as a bone substitute. Comparison with hydroxyapatite and tricalcium phosphate with regard to the osteoblastic activity.

Close to the bone mineral phase, the calcic bioceramics, such as hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP), are commonly used as substitutes or filling materials in bone surgery. Besides, calcium carbonate (CaCO3) is also used for their excellent biocompatibility and bioactivity. However, the problem with the animal-origin aragonite demands the new technique to synthesize pure calcite capable of forming 3D bone implant. This study aims to manufacture and evaluate a highly-pure synthetic crystalline calcite with good cytocompatibility regarding to the osteoblasts, comparing to that of HA and ß-TCP. After the manufacture of macroporous bioceramic scaffolds with the identical internal architecture, their cytocompatibility is studied through MC3T3-E1 osteoblasts with the tests of cell viability, proliferation, vitality, etc. The results confirmed that the studied process is able to form a macroporous material with a controlled internal architecture, and this synthesized calcite is non-cytotoxic and facilitate the cell proliferation. Indeed requiring further improvement, the studied calcite is definitely an interesting alternative not only to coralline aragonite but also to calcium phosphate ceramics, particularly in bone sites with the large bone remodelling.

In vitro assessment of poly(methylmethacrylate)-based bone cement containing magnetite nanoparticles for hyperthermia treatment of bone tumor.

Poly(methylmethacrylate) (PMMA)-based cements containing magnetite (C-PMMA/Fe(3)O(4)) is useful in hyperthermia treatment for bone tumor. We have prepared C-PMMA/Fe(3)O(4) by incorporating Fe(3) O(4) powders of different diameters (means of 300, 35, and 11 nm) into the polymerization reaction of methyl methacrylate monomer to develop a new bone cement with high heating efficiencies in alternating current (AC) magnetic fields. Further, we have investigated the in vitro heating capability of the cements in different AC magnetic fields. The mechanical strength and biocompatibility of the resultant cements were also assessed. Their heat generation strongly depends on the magnetite nanoparticle sizes and applied magnetic fields. The cement containing Fe(3)O(4) with mean diameter around 35 nm exhibited the highest heating capability in AC magnetic fields of 120 and 300 Oe at 100 kHz while that with mean diameter around 11 nm exhibited optimum heating capability in AC magnetic fields of 40 Oe at 600 kHz. The incorporation of Fe(3)O(4) into cement-30 wt % of the total amount of cement-did not significantly change the compressive strength of cement, and the proliferation of rat fibroblast Rat-1 cells on cement discs was not inhibited. Our investigations are useful for designing new PMMA/Fe(3)O(4) bone cement with high heating efficiencies and biocompatibilities for bone tumor treatments.

[Effects of rat serum containing Chinese herbal medicine Sangen Decoction on osteoclastogenesis and bone resorption of osteoclasts induced by polymethylmethacrylate particles].

OBJECTIVE: To investigate the effects of Sangen Decoction, a compound Chinese herbal medicine, on osteoclastogenesis and bone resorption function of osteoclasts induced by polymethylmethacrylate particles in vitro. METHODS: Macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) were used to induce differentiation of bone marrow-derived macrophages (BMMs) towards osteoclasts. BMMs and polymethylmethacrylate particles with ratio of 1:3 were added to the 24-well plate and 96-well plate with bone slices respectively. A total of 50 male SD rats were divided into 5 groups randomly with each group containing 10 rats. After being treated with different drugs, serum samples of rats in each group were extracted, i.e., the blank serum, Western medicine (ibandronate) serum and high-, medium-, and low-dose Sangen Decoction serum and were added to the medium respectively. The tartrate-resistant acid phosphatase (TRAP) staining was used to identify the differentiation of BMMs and for counting of osteoclasts. Area of lacuna induced by osteoclast bone resorption on the bone slices was measured by computer image processing. RESULTS: Numbers of osteoclasts of treatment groups were less than that of blank group by TRAP staining (P<0.05); numbers of osteoclasts of positive control group and high-dose Sangen Decoction group were much lower than those of medium- and low-dose Sangen Decoction groups (P<0.05), and no difference was found between Western medicine group and high-dose Sangen Decoction group (P>0.05). In bone resorption assay, area of lacuna of blank group was larger than those of treatment groups (P<0.05); areas of lacuna of Western medicine group and high-dose Sangen Decoction group were much smaller than those of medium- and low-dose Sangen Decoction groups (P<0.05), and no difference was found between Western medicine group and high-dose Sangen Decoction group (P>0.05). CONCLUSION: Sangen Decoction can inhibit osteoclastogenesis induced by polymethylmethacrylate particles as well as bone resorption function of osteoclasts.

Low-modulus PMMA bone cement modified with castor oil.

Some of the current clinical and biomechanical data suggest that vertebroplasty causes the development of adjacent vertebral fractures shortly after augmentation. These findings have been attributed to high injection volumes as well as high Young's moduli of PMMA bone cements compared to that of the osteoporotic cancellous bone. The aim of this study was to evaluate the use of castor oil as a plasticizer for PMMA bone cements. The Young's modulus, yield strength, maximum polymerization temperature, doughing time, setting time and the complex viscosity curves during curing, were determined. The cytotoxicity of the materials extracts was assessed on cells of an osteoblast-like cell line. The addition of up to 12 wt% castor oil decreased yield strength from 88 to 15 MPa, Young's modulus from 1500 to 446 MPa and maximum polymerization temperature from 41.3 to 25.6°C, without affecting the setting time. However, castor oil seemed to interfere with the polymerization reaction, giving a negative effect on cell viability in a worst-case scenario.

Ceramic and PMMA particles differentially affect osteoblast phenotype.

There is increasing evidence that wear debris particles present in periprosthetic tissues have direct effects on osteoblasts. The nature of the cell response varies with the chemistry of the particle and the number of particles. Most studies have used Ti, Ti-6Al-4V, and ultrahigh molecular weight polyethylene (UHMWPE) particles since these materials are most frequently used in implants and as a result, these particles predominate in peri-prosthetic tissues. Ceramics have also been used successfully as load-bearing surfaces in implants for years, although it is unknown how wear debris from these surfaces may contribute to aseptic bone loss. Further, particles resulting from polymethylmethacrylate (PMMA) cements used for fixation may also be involved in aseptic loosening of implants, but how these particles may affect bone formation is unknown. In the present study, we examined whether aluminum oxide (Al2O3), zirconium oxide (ZrO2), and PMMA particles exert effects on osteoblast proliferation, phenotypic expression, and local factor production, and if so, whether the effects were specific to the particle type. ZrO2 particles were produced in a custom-made axial mixer in which ZrO2 containers were filled with ZrO2 bars and 95% ethanol and then rotated continuously at room temperature. PMMA particles were prepared in a ZrO2 roller mill. Al2O3 was produced and provided by Aesculap AG. Particles were endotoxin-free with equivalent circle diameters <3 microm; Al2O3 particles were significantly smaller than ZrO2 or PMMA particles. Particle suspensions were added to confluent cultures of MG63 osteoblast-like cells after diluting them 1:100, 1:10, and 1:1 with culture medium. Cells were incubated with the particles for 24 h. Transmission electron microscopy showed that MG63 cells phagocytosed Al2O3 particles and exhibited ultrastructural changes consistent with cytotoxicity. This was supported by biochemical changes as well. Proliferation, alkaline phosphatase activity, and TGF-beta1 levels were decreased. ZrO2 and PMMA particles increased proliferation and alkaline phosphatase specific activity. The effect of ZrO2 on alkaline phosphatase was targeted to matrix vesicles, the effect of PMMA was greater on the cells. All particles increased prostaglandin E2 production. These results show that Al2O3, ZrO2, and PMMA particles elicit direct effects on osteoblasts and that cell response depends on the particle type. None of the particles tested had the same effect as noted previously for UHMWPE: increased proliferation and decreased alkaline phosphatase. These results may indicate that the response of peri-prosthetic tissues to wear particles may be modulated by the relative contributions of the various particle types present.

No effect of methacrylate-based bone cement CMW 1 on the plasmatic phase of coagulation, red blood cells and endothelial cells in vitro.

The compatibility of a methacrylate-based bone cement (CMW 1, DePuy International Ltd, England) used for the fixation of joint prostheses was evaluated on plasma, an erythrocyte suspension and cultured human endothelial cells. The extract of the cement was tested, following 1 hour and 7 days of curing. After the contact in vitro of the extract with plasma, activated partial thromboplastin time, antithrombin III, thrombin-antithrombin complexes and fibrin degradation products were assayed. Hemolytic activity was tested by adding the cement extracts to a suspension of erythrocytes. After 4 hours of incubation at 37 degrees C, the hemoglobin concentration was determined on the supernatants by the colorimetric method. The effect of the cement on tissue factor and thrombomodulin production was evaluated on human umbilical vein endothelial cell cultures. Tissue factor was determined in cell lysates by enzyme immunoassay, following 4 hours' incubation of cultures with the cement extract. Thrombomodulin was assayed in cell lysates by enzyme immuno assay, after 24 hours' incubation with the cement extract. The response to all trans-retinoic acid (ATRA) was tested. The cement caused no significant modifications of the coagulation tests, had no hemolytic activity, did not determine tissue factor production and did not modify thrombomodulin, compared to the negative control. The response to stimulation with ATRA was similar to that of the negative control. We conclude that the cement extract does not affect the plasmatic phase of coagulation, has no effect on erythrocytes, does not induce the expression of procoagulant activity by endothelial cells and does not impair their antithrombotic property, within the limits of the tests performed.

The effects of Pain-Free Desensitizer on dentine permeability and tubule occlusion over time, in vitro.

The purpose of this in vitro study was to evaluate the efficacy of a new resin emulsion (Pain-Free Desensitizer) treatment for dentine hypersensitivity, for its ability to decrease dentine permeability. Crown segments were prepared from extracted, unerupted human 3rd molars by horizontal sectioning to remove occlusal enamel and the roots. The specimens were allocated in one of two groups: In group 1, the dentine surface was acid-etched to simulate the patent tubules of hypersensitive dentine. In group 2, the mineralized dentine surface was polished free of smear layer using a hydroxyapatite paste and ultrasonication. The hydraulic conductance of each specimen was then measured to obtain a pretreatment of control value. After a single treatment with resin desensitizer, the permeability was remeasured at 5 min, 1 day, 1 week and 1 month. Between measurements, the specimens were stored in buffer solution to simulate the solubilizing effects of saliva. Parallel specimens were followed by SEM examination. The results showed that a single treatment with resin desensitizer produced large, immediate, reductions in dentine permeability in both acid-etched and mineralized surfaces. In the acid-etched (group 1) specimens, the permeability returned to control values within 7 days, while the permeability of the group 2 specimens remained low even after 30 days of soaking. This simple treatment for occluding dentinal tubules may provide sufficient temporary reduction in dentine permeability to permit the development of natural desensitization.