Electrospun polycaprolactone/hydroxyapatite/ZnO nanofibers as potential biomaterials for bone tissue regeneration.

Fabricating a bioartificial bone graft possessing structural, mechanical and biological properties mimicking the real bone matrix is a major challenge in bone tissue engineering. Moreover, the developed materials are prone to microbial invasion leading to biomaterial centered infections which might limit their clinical translation. In the present study, biomimetic nanofibrous scaffolds of Poly ɛ-caprolactone (PCL)/nano-hydroxyapatite (nHA) were electrospun with 1wt%, 5wt%, 10wt%, 15wt% and 30wt% of zinc oxide (ZnO) nanoparticles in order to understand the optimal concentration range of (ZnO) nanoparticles balancing both biocompatibility and osteoregeneration. The developed nanofibrous scaffolds were successfully characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDAX), contact angle, fourier transform infrared spectroscopy (FTIR), wide-angle X-Ray diffraction (WAXD), brunaueremmett Teller (BET) surface area and tensile testing. Biocompatibility of the developed scaffolds at in vitro level was evaluated by culturing MG-63 cells and investigating the impact on cell viability, proliferation, protein adsorption, alkaline phosphatase (ALP) activity and biomineralization. The PCL/nHA scaffolds exhibited a 1.2-fold increase in cell viability and proliferation, while incorporation of ZnO nanoparticles to PCL/nHA imparted antimicrobial activity to the scaffolds with a progressive increase in the antimicrobial efficacy with increasing ZnO concentration. The results of cell viability were supported by ALP activity and mineralization assay, wherein, PCL/nHA/ZnO scaffolds showed higher ALP activity and better mineralization capacity as compared to pristine PCL. Although, the PCL/nHA/ZnO scaffolds with 10, 15 and 30wt% of ZnO particles exhibited superior antimicrobial efficacy against both gram-negative (E. coli) and gram-positive (S. aureus) bacteria, a significant decrease in the cell viability and mechanical properties was observed at higher concentrations of ZnO namely 15 and 30%. Amongst the various ZnO concentrations studied optimal cell viability, antimicrobial effect and mechanical strength were observed at 10wt.% ZnO concentration. Thus, the present study revealed that the biomimetic tri-component PCL/nHA/ZnO scaffolds with ZnO concentration range of ≤ 10% could be ideal for achieving optimal biocompatibility (cell proliferation, biomineralization, and antimicrobial capacity) and mechanical stability thus making it a promising biomaterial substrate for bone tissue regeneration.

Quercetin modulates Wnt signaling components in prostate cancer cell line by inhibiting cell viability, migration, and metastases.

Epithelial-mesenchymal transition (EMT) is a plastic transition in tumor progression during which cancer cells undergo dramatic changes acquiring highly invasive properties. Transforming growth factor-ß (TGF-ß) is an inducer of EMT in epithelial cells and is obligatory for acquiring invasive phenotype in carcinoma. TGF-ß plays a vital role in metastasis and tumorigenesis in prostate cancer, and mutations in the components of Wnt signaling pathways are associated with various kinds of cancers including prostate cancer. The purpose of this study was to identify alterations in Wnt signaling pathway components involved during prostate cancer progression and to determine the effect of quercetin on TGF-ß-induced EMT in prostate cancer (PC-3) cell line. The expression of epithelial and mesenchymal markers and the components of Wnt signaling pathway were evaluated by real-time polymerase chain reaction. It was observed that quercetin prevented TGF-ß-induced expression of vimentin and N-cadherin and increased the expression of E-cadherin in PC-3 cells, thus preventing TGF-ß-induced EMT. Furthermore, the relative expression of Twist, Snail, and Slug showed that quercetin significantly decreased TGF-ß-induced expression of Twist, Snail, and Slug. In the present study, the expression of epithelial markers were found to be upregulated in naive state and downregulated in induced state whereas the mesenchymal markers were found to be downregulated in naive state and upregulated in induced state. Thus, our study concludes that quercetin may prevent prostate cancer metastasis by regulating the components of Wnt pathway.

Physicochemical characterisation and biological evaluation of polyvinylpyrrolidone-iodine engineered polyurethane (Tecoflex(®)).

Bacterial adhesion and encrustation are the known causes for obstruction or blockage of urethral catheters and ureteral stents, which often hinders their effective use within the urinary tract. In this in vitro study, polyvinylpyrrolidone-iodine (PVP-I) complex modified polyurethane (Tecoflex(®)) systems were created by physically entrapping the modifying species during the reversible swelling of the polymer surface region. The presence of the PVP-I molecules on this surfaces were verified by ATR-FTIR, AFM and SEM-EDAX analysis, while wettability of the films was investigated by water contact angle measurements. The modified surfaces were investigated for its suitability as a urinary tract biomaterial by comparing its lubricity and ability to resist bacterial adherence and encrustation with that of base polyurethane. The PVP-I modified polyurethane showed a nanopatterned surface topography and was highly hydrophilic and more lubricious than control polyurethane. Adherence of both the gram positive Staphylococcus aureus (by 86%; **P < 0.01) and gram-negative Pseudomonas aeruginosa (by 80%; *P < 0.05) was significantly reduced on the modified surfaces. The deposition of struvite and hydroxyapatite the major components of urinary tract encrustations were significantly less on PVP-I modified polyurethane as compared to base polyurethane, especially reduction in hydroxyapatite encrustation was particularly marked. These results demonstrated that the PVP-I entrapment process can be applied on polyurethane in order to reduce/lower complications associated with bacterial adhesion and deposition of encrustation on polyurethanes.

The biocompatibility of sulfobetaine engineered polymethylmethacrylate by surface entrapment technique.

Sulfobetaine-modified polymethylmethacrylate (PMMA) systems were created by physically entrapping the zwitterionic species on the PMMA surface. The presence of the sulfobetaine molecules on these surfaces were verified by ATR-FTIR and SEM-EDAX analysis, while wettability of the films was investigated by dynamic contact angle measurements. The short-term (4 h) adhesion of two bacterial species (gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa) on these surfaces were studied. Mouse RAW 264.7 macrophage cells were used to assess the cell adhesion and inflammatory response by quantifying the expression levels of proinflammatory cytokines namely TNFalpha and IL1beta by measuring their mRNA profiles in the cells using real-time polymerase chain reaction (RT-PCR) normalized to the house keeping gene GAPDH. Whilst mouse L-929 fibroblast cells were used to assess the propensity for the materials to support fibroblast cell adhesion. A decrease in the adhesion of S. aureus by 63% and P. aeruginosa by 49% was observed on sulfobetaine modified PMMA films after 4 h. In all the cases, sulfobetaine modified PMMA films reduced cellular adhesion events (*P < 0.05) with respect to the base materials, which could be linked to the reduced protein adsorption observed on these surfaces. The cellular inflammatory response was suppressed on sulfobetaine modified substrates as expression levels of pro-inflammatory cytokines (TNFalpha and IL1beta) was found to be up regulated on bare PMMA, while it was significantly lower on sulfobetaine modified PMMA (**P < 0.001). Thus the sulfobetaine entrapment process can be applied on polymethylmethacrylate in order to achieve low biointeractions and reduced inflammatory host responses for various biomedical and biotechnological applications.

TecoflexTM functionalization by curdlan and its effect on protein adsorption and bacterial and tissue cell adhesion.

Curdlan modified polyurethane was created by physically entrapping the former on TecoflexTM surface. ATR-FT-IR, SEM-EDAX and AFM analysis revealed the formation of stable thin curdlan layer on the film. Contact-angle measurements showed that the modified film was highly hydrophilic. Confocal laser scanning microscopy showed the existence of entrapped layer of approximately 20-25 microm in depth. Surface entrapment of curdlan minimized both protein adsorption and mouse L929 fibroblast cell adhesion relative to the control. Surface induced cellular inflammatory response was determined from the expression levels of proinflammatory cytokine TNF-alpha, by measuring their mRNA profiles in the cells using real time polymerase chain reaction (RT-PCR) normalized to the housekeeping gene GAPDH. The inflammatory response was suppressed on the modified substrate as expression of TNF-alpha mRNA was found to be up regulated on TecoflexTM, while it was significantly lower on curdlan substrate. The adhesion of S. aureus decreased by 62% on curdlan modified surface. Using such simple surface entrapment process, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in the long-term as implant.

Clopidogrel eluting electrospun polyurethane/polyethylene glycol thromboresistant, hemocompatible nanofibrous scaffolds.

Biomaterials used as blood-contacting material must be hemocompatible and exhibit lower thrombotic potential while maintaining hemostasis and angiogenesis. With the aim of developing thromboresistant, hemocompatible nanofibrous scaffolds, polyurethane/polyethylene glycol scaffolds incorporated with 1, 5, and 10 wt% Clopidogrel were fabricated and evaluated for their physiochemical properties, biocompatibility, hemocompatibility, and antithrombotic potential. The results of physicochemical characterization revealed the fabrication of nanometer-sized scaffolds with smooth surfaces. The incorporation of both polyethylene glycol and Clopidogrel to polyurethane enhanced the hydrophilicity and water uptake potential of polyurethane/polyethylene glycol/Clopidogrel scaffolds. The dynamic mechanical analysis revealed the enhancement in mechanical strength of the polyurethane/polyethylene glycol scaffolds on incorporation of Clopidogrel. The polyurethane/polyethylene glycol/Clopidogrel scaffolds showed a tri-phasic drug release pattern. The results of hemocompatibility assessment demonstrated the excellent blood compatibility of the polyurethane/polyethylene glycol/Clopidogrel scaffolds, with the developed scaffolds exhibiting lower hemolysis, increased albumin and plasma protein adsorption while reduction in fibrinogen adsorption. Further, the platelet adhesion was highly suppressed and significant increase in coagulation period was observed for Clopidogrel incorporated scaffolds. The results of cell adhesion and cell viability substantiate the biocompatibility of the developed nanofibrous scaffolds with the HUVEC cell viability on polyurethane/polyethylene glycol, polyurethane/polyethylene glycol/Clopidogrel-1, 5, and 10% at day 7 found to be 12.35, 13.36, 14.85, and 4.18% higher as compared to polyurethane scaffolds, and the NIH/3T3 cell viability found to be 35.27, 70.82, 36.60, and 7.95% higher as compared to polyurethane scaffolds, respectively. Altogether the results of the study advocate the incorporation of Clopidogrel to the polyurethane/polyethylene glycol blend in order to fabricate scaffolds with appropriate antithrombotic property, hemocompatibility, and cell proliferation capacity and thus, might be successfully used as antithrombotic material for biomedical application.

Surface engineering of polycaprolactone by biomacromolecules and their blood compatibility.

Improving blood compatibility of biodegradable polymers is an area of intensive research in blood contacting devices. In this study, curdlan sulphate and heparin-modified poly (caprolactone) (PCL) hybrids were developed by physically entrapping these molecules on the PCL surface. This modification technique was performed by reversible gelation of the PCL surface region following exposure to a solvent and nonsolvent mixture. The presence of these biomacromolecules on the PCL surface was verified by atomic force microscopy (AFM) and scanning electron microscopy and energy dispersive X-ray analysis (SEM-EDAX) analysis, while wettability of the films was investigated by dynamic contact angle measurements. The blood compatibilities of the surface-modified films were examined using in vitro platelet and leukocyte adhesion and thrombus formation. Mouse RAW 264.7 macrophage cells were used to assess the cell adhesion and inflammatory response to the modified surface by quantifying mRNA expression levels of proinflammatory cytokines namely TNF-α and IL-1ß using real-time polymerase chain reaction (RT-PCR). A lower platelet and leukocyte adhesion and activation was observed on the modified films incubated with whole human blood for 2 h. The thrombus formation on the PCL was significantly decreased upon immobilization of both curdlan sulphate (39%, *p<0.05) and heparin (28%, *p<0.01) when compared to bare PCL (80%). All of these results revealed that improved blood compatibility was obtained by surface entrapment of both curdlan sulphate (CURS) and heparin (HEP) onto PCL films. Both PCL-CURS and PCL-HEP films reduced RAW 264.7 macrophage cell adhesion (*p<0.05) with respect to the base unmodified PCL. The cellular inflammatory response was suppressed on the modified substrates. The mRNA expression levels of proinflammmatory cytokines (TNF-α and IL-1ß) were upregulated on bare PCL, while it was significantly lower on PCL-CURS and PCL-HEP substrates (**p<0.001). Thus, this biomacromolecule entrapment process can be applied on PCL in order to achieve improved blood compatibility and reduced inflammatory host response for its future blood contacting applications.

The biocompatibility of sulfobetaine engineered poly (ethylene terephthalate) by surface entrapment technique.

Sulfobetaine-modified poly(ethylene terephthalate) (PET) systems were created by physically entrapping the zwitterionic species on the PET surface. The presence of the sulfobetiane molecules on these surfaces were verified by ATR-FTIR and SEM-EDAX analysis, while wettability of the films was investigated by water contact angle measurements. The blood compatibility of the modified films was evaluated by platelet adhesion in human platelet-rich plasma (PRP). The adhesion and inflammatory response of Mouse RAW 264.7 macrophage cells were studied. The surface induced cellular inflammatory response was determined by quantifying the expression levels of proinflammatory cytokines namely TNF-alpha and IL-1beta by measuring their mRNA profiles in the cells using real time polymerase chain reaction normalized to the housekeeping gene GAPDH. L-929 fibroblast cells were used to assess the propensity of the materials to support the fibroblast cell adhesion. A lower platelet adhesion and activation were observed on the sulfobetaine-modified PET film incubated in PRP after 2h when compared to control. The modified film reduced cellular adhesion events ( p<0.05) with respect to the base material, which could be linked to the reduced protein adsorption observed on this surface. The cellular inflammatory response was suppressed on sulfobetaine-modified substrate. Expression levels of pro-inflammmatory cytokines (TNF-alpha and IL-1beta) was found to be upregulated on bare PET, while it was significantly lower on modified PET ( p<0.001). Thus the sulfobetaine entrapment process can be applied on PET in order to achieve low biointeractions and reduced inflammatory host response for various biomedical and biotechnological applications.

In vivo modulation of foreign body response on polyurethane by surface entrapment technique.

Implanted polymeric materials, such as medical devices, provoke the body to initiate an inflammatory reaction, known as the foreign body response (FBR), which causes several complications. In this study, polyurethane (Tecoflex®, PU) surface modified with the nonionic surfactant Tween80® (PU/T80) and the cell adhesive PLL-RGD peptide (PU/PLL-RGD) by a previously described entrapment technique were implanted in the peritoneal cavity of Wistar rats for 30 days. Implants were retrieved and examined for tissue reactivity and cellular adherence by various microscopic and analytical techniques. Surface-induced inflammatory response was assessed by real-time PCR based quantification of proinflammatory cytokine transcripts, namely, TNF-α and IL-1ß, normalized to housekeeping gene GAPDH. Cellular adherence and their distribution profile were assessed by microscopic examination of H&E stained implant sections. It was observed that PU/PLL-RGD followed by the bare PU surface exhibited severe inflammatory and fibrotic response with an average mean thickness of 19 and 12 µm, respectively, in 30 days. In contrast, PU/T80 surface showed only a cellular monolayer of 2-3 µm in thickness, with a mild inflammatory response and no fibrotic encapsulation. The PU/PLL-RGD peptide-modified substrate promoted an enhanced rate of macrophage cell fusion to form foreign body giant cell (FBGCs), whereas FBGCs were rarely observed on Tween80®-modified substrate. The expression levels of proinflammatory cytokines (TNF-α and IL-1ß) were upregulated on PU/PLL-RGD surface followed by bare PU, whereas the cytokine expressions were significantly suppressed on PU/T80 surface. Thus, our study highlights modulation of foreign body response on polyurethane surfaces through surface entrapment technique in the form of differential responses observed on PLL-RGD and Tween80® modified surfaces with the former effective in triggering tissue cell adhesion thereby fibrous encapsulation, while the later being mostly resistant to this phenomenon.