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Biomaterials (v.31, #22)

Editorial board (pp. ifc).

Towards ultraporous poly(l-lactide) scaffolds from quaternary immiscible polymer blends by N. Virgilio; P. Sarazin; B.D. Favis (pp. 5719-5728).
Ultraporous poly(l-lactide) (PLLA) scaffolds were prepared by melt-processing quaternary ethylene propylene diene rubber/poly(ε-caprolactone)/polystyrene/poly(l-lactide) (EPDM/PCL/PS/PLLA) 45/45/5/5 %vol. polymer blends modified with a PS-b-PLLA diblock copolymer. The morphology consists of a PS+PLLA+copolymer sub-blend layer forming at the interface of the EPDM and PCL phases. Quiescent annealing and interfacial modification using the block copolymer are used to control the blend microstructure. The ultraporous structure is subsequently obtained by selectively extracting the EPDM, PS and PCL phases. The PLLA scaffolds modified with the PS-b-PLLA copolymer present themselves as fully interconnected porous networks with asymmetric channel walls, one side being smooth while the other is covered with an array of submicron-sized PLLA droplets. They are prepared with a high degree of control over the pore size, with averages ranging from 5μm to over 100μm and a specific surface from 9.1 to 23.1m2/g of PLLA, as annealing is carried out from 0 to 60min. The void volume reaches values as high as 95% and in all cases the shape and dimensions of the scaffolds are maintained with a high level of integrity. The proposed method represents a comprehensive approach towards the design and generation of porous PLLA scaffolds based on complex morphologies from melt-processed multiphase polymer systems.

Keywords: Polylactic acid; Porosity; Microstructure; Interface; Scaffold

Cell architecture–cell function dependencies on titanium arrays with regular geometry by Claudia Matschegewski; Susanne Staehlke; Ronny Loeffler; Regina Lange; Feng Chai; Dieter P. Kern; Ulrich Beck; Barbara J. Nebe (pp. 5729-5740).
Knowledge about biocomplexity of cell behavior in dependence on topographical characteristics is of clinical relevance for the development of implant designs in tissue engineering. The aim of this study was to find out cell architecture–cell function dependencies of human MG-63 osteoblasts on titanium (Ti) arrays with regular geometry. We compared cubic pillar structures (SU-8, dimension 3 × 3 × 5 and 5 × 5 × 5 μm) with planar samples. Electrochemical surface characterization revealed a low amount of surface energy (including polar component) for the pillar-structured surfaces, which correlated with a reduced initial cell adhesion and spreading. Confocal microscopy of cell’s actin cytoskeleton revealed no stress fiber organization instead, the actin was concentrated in a surface geometry-dependent manner as local spots around the pillar edges. This altered cell architecture resulted in an impaired MG-63 cell function – the extracellular matrix proteins collagen-I and bone sialo protein (BSP-2) were synthesized at a significantly lower level on SU-8 pillar structures; this was accompanied by reduced β3-integrin expression. To find out physicochemical factors pertaining to geometrically microstructured surfaces and their influence on adjoining biosystems is important for the development of biorelevant implant surfaces.

Keywords: Microstructure; Electrochemistry; Osteoblast; Actin; Cell adhesion; Extracellular matrix

Bradykinin forming capacity of oversulfated chondroitin sulfate contaminated heparin in vitro by Albert Adam; Nicolas Montpas; David Keire; Anik Désormeaux; Nancy J. Brown; François Marceau; Benjamin Westenberger (pp. 5741-5748).
Oversulfated chondroitin sulfate (OSCS) contaminated heparin has been associated with severe anaphylactoid reaction (AR), mainly in dialysed patients. Although attributed to bradykinin (BK) released during contact system activation by OSCS, no definitive evidence exists until now for a BK release during incubation of contaminated heparin with human plasma. In this study, we investigated the kinin forming capacity of OSCS and OSCS contaminated heparin when incubated in vitro with a pool of human plasma. At 100 μg/mL, OSCS liberates BK in a profile similar but not identical to dextran sulfate, a well known activator of the plasma contact system. The results have highlighted that the quantity of BK accumulated during contact system activation depends not only on the concentration of OSCS but also on the plasma dilution and the presence of an angiotensin converting enzyme inhibitor. We demonstrate a highly significant correlation between the concentration of OSCS present in the contaminated heparin and BK released concentration. In conclusion, for the first time, we show that OSCS contaminated heparin incubated with human plasma has the capacity to liberate BK at a concentration that could explain the role of this inflammatory peptide in the pathophysiology of AR associated with OSCS contaminated heparin.The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Keywords: Heparin; Oversulfated chondroitin sulfate; Bradykinin; Anaphylactoid reaction

The influence of poly- N-[(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide on fibrin polymerization, cross-linking and clot structure by Benjamin F.L. Lai; Yuquan Zou; Donald E. Brooks; Jayachandran N. Kizhakkedathu (pp. 5749-5758).
Poly- N-[(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide (PDMDOMA) is a neutral synthetic water-soluble polymer. In this report, we evaluated the influence of PDMDOMA on blood hemostasis by studying the fibrin polymerization process, the three-dimensional clot structure, and the mechanical properties and fibrinolysis. PDMDOMA altered the normal fibrin polymerization by changing the rate of protofibril aggregation and resulting in a 5-fold increase in the overall turbidity. Fibrin clots formed in presence of PDMDOMA exhibited thinner fibers with less branching which resulted in a more porous and heterogeneous clot structure in scanning electron micrographs. The overall strength and rigidity of the whole blood clot also decreased up to 10-fold. When a combination of plasminogen and tissue-plasminogen activators were included in clotting reactions, fibrin clots formed in the presence of PDMDOMA exhibited highly shortened clot lysis times and was supported by the enhanced clot lysis measured by thromboelastography in whole blood. Further evidence of the altered clot structure and clot cross-linking was obtained from the significant decrease ind-dimer levels measured from degraded plasma clot. Thus, PDMDOMA may play an important role as an antithrombotic agent useful in prophylactic treatments for thrombosis by modulating fibrin clot structure to enhance fibrinolysis.

Keywords: Antithrombotic materials; Blood coagulation; Fibrin polymerization; Fibrinolysis; Synthetic polymers

The role of multiple toll-like receptor signalling cascades on interactions between biomedical polymers and dendritic cells by Behnaz Shokouhi; Cevayir Coban; Vasif Hasirci; Erkin Aydin; Anandhan Dhanasingh; Nian Shi; Shohei Koyama; Shizuo Akira; Martin Zenke; Antonio S. Sechi (pp. 5759-5771).
Biomaterials are used in several health-related applications ranging from tissue regeneration to antigen-delivery systems. Yet, biomaterials often cause inflammatory reactions suggesting that they profoundly alter the homeostasis of host immune cells such as dendritic cells (DCs). Thus, there is a major need to understand how biomaterials affect the function of these cells. In this study, we have analysed the influence of chemically and physically diverse biomaterials on DCs using several murine knockouts. DCs can sense biomedical polymers through a mechanism, which involves multiple TLR/MyD88-dependent signalling pathways, in particular TLR2, TLR4 and TLR6. TLR–biomaterial interactions induce the expression of activation markers and pro-inflammatory cytokines and are sufficient to confer on DCs the ability to activate antigen-specific T cells. This happens through a direct biomaterial–DC interaction although, for degradable biomaterials, soluble polymer molecules can also alter DC function. Finally, the engagement of TLRs by biomaterials profoundly alters DC adhesive properties. Our findings could be useful for designing structure–function studies aimed at developing more bioinert materials. Moreover, they could also be exploited to generate biomaterials for studying the molecular mechanisms of TLR signalling and DC activation aiming at fine-tuning desired and pre-determined immune responses.

Keywords: Biomaterials; Biocompatibility; Immune response; Dendritic cells; Inflammation; TLR

The functional behavior of a macrophage/fibroblast co-culture model derived from normal and diabetic mice with a marine gelatin–oxidized alginate hydrogel by Qiong Zeng; Weiliam Chen (pp. 5772-5781).
Tissues/cells-mediated biodegradable material degradation is epitomized by the constantly changing tissues/cell–implant interface, implicating the constant adaptation of the tissues/cells. Macrophages and fibroblasts are multi-functional cells highly involved in the interactions; the two cell types modulates the behaviors of each other, but their combinatorial functional behavior in the presence of interactive bioactive wound dressings has not been adequately examined. The activity is further complicated by the implantation of biodegradable materials, such as hydrogels commonly utilized as wound dressings, in a pathological environment and this is exemplified by the macrophages with a diabetic pathology producing an alternative cytokine profile which is implicated in wound healing delay. In this study, an in situ gelable formable/conformable hydrogel formulated from modified alginate and marine gelatin was used as a model biodegradable interactive wound dressing to elucidate the combinatorial behavior of macrophages/fibroblasts derived from both normal and diabetic hosts. Cell proliferation, migration and distribution were first characterized; this was followed by simultaneous quantitative detection of 40 inflammatory cytokines and chemokines by a protein microarray. The results showed that the macrophages/fibroblasts co-culture promoted fibroblasts proliferation and migration in the presence of the hydrogel; moreover, the expressions of inflammatory cytokines and chemokines were altered when compared with the corresponding fibroblasts or macrophages monocultures. The inflammatory cytokines patterns between the normal and diabetic hosts were considerably different.

Keywords: Wound dressing; Hydrogel; Diabetes; Fibroblast; Macrophage; Co-culture

In vitro responses of human bone marrow stromal cells to a fluoridated hydroxyapatite coated biodegradable Mg–Zn alloy by Jianan Li; Yang Song; Shaoxiang Zhang; Changli Zhao; Fan Zhang; Xiaonong Zhang; Lei Cao; Qiming Fan; Tingting Tang (pp. 5782-5788).
Bone-like fluoridated hydroxyapatite (FHA) coatings were prepared on Mg-6 wt.%Zn substrates using electrochemical method. Human bone marrow stromal cells (hBMSCs) were utilized to investigate the cellular biocompatibility of Mg-6 wt.%Zn alloy after surface modification. The adhesion of hBMSCs was evaluated using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). The proliferation of the cells was also measured by carrying out the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. And the alkaline phosphatase activity (ALP) was assessed to evaluate the early stage of differentiation. Lastly, reverse transcription-polymerase chain reaction (RT-PCR) test was taken. It was found that the hBMSCs displayed better cell functions on the bioactive FHA coated alloy, compared to the bare Mg-6 wt.%Zn alloy. The in vitro results indicated that the bioactive FHA coating can improve the interfacial bioactivity of Mg-6 wt.%Zn substrate, specifically, both on biodegradation behavior control and good cellular proliferation and differentiation.

Keywords: Magnesium; Biodegradation; Fluorine–fluoride; In vitro test

Controlling dispersion of axonal regeneration using a multichannel collagen nerve conduit by Li Yao; Godard C.W. de Ruiter; Huan Wang; Andrew M. Knight; Robert J. Spinner; Michael J. Yaszemski; Anthony J. Windebank; Abhay Pandit (pp. 5789-5797).
Single channel conduits are used clinically in nerve repair as an alternative to the autologous nerve graft. Axons regenerating across single channel tubes, however, may disperse resulting in inappropriate target reinnervation. This dispersion may be limited by multichannel nerve conduits as they resemble the structure of nerve multiple basal lamina tubes. In this study, we investigated the influence of channel number on the axonal regeneration using a series of 1-, 2-, 4-, and 7-channel collagen conduits and commercial (NeuraGen ®) single channel conduits. Nerve conduits were implanted in rats with a 1 cm gap of sciatic nerve. After four months, quantitative results of regeneration were evaluated with nerve morphometry and the accuracy of regeneration was assessed using retrograde tracing: two tracers being applied simultaneously to tibial and peroneal nerves to determine the percentage of motor neurons with double projections. Recovery of function was investigated with compound muscle action potential recordings and ankle motion analysis. We showed that the fabricated 1-channel and 4-channel conduits are superior to other types of conduits in axonal regeneration. Simultaneous tracing showed a significantly lower percentage of motor neurons with double projections after 2- and 4-channel compared with 1-channel conduit repair. This study shows the potential influence of multichannel guidance on limiting dispersion without decreasing quantitative results of regeneration.

Keywords: Collagen; Nerve regeneration; Nerve conduit; Neural tissue engineering; In vivo

Gingival mucosa regeneration in athymic mice using in vitro engineered human oral mucosa by Mahmoud Rouabhia; Patricia Allaire (pp. 5798-5804).
Our goal was to investigate in vivo tissue formation following the grafting of engineered human oral mucosa to demonstrate its usefulness in replacing mucosal defects in the oral cavity. Human gingival cells were isolated from the oral mucosa and were used in combination with a collagen scaffold to engineer oral mucosa. Structural and ultrastructural analyses revealed that the engineered mucosa had a well-organized stratified epithelium on the surface of the fibroblast-populated lamina propria tissue. Following grafting for 15 and 60 days, the engineered oral mucosa was shown to cover the recipient site with no wound contraction. The regenerated mucosa displayed an epithelium with multiple layers, including a stratum corneum where epithelial cells expressed cytokeratin Ki-67 and K-14 positive cells located in the basal and supra-basal layers. The interaction between the epithelium and the lamina propria was promoted by the formation of a basement membrane structure containing key proteins, such as laminin-5 and type IV collagen. Following the engineered mucosa grafting, the regenerated tissue was well vascularized, similar to the native mucosa. These data demonstrate the usefulness of engineered human oral mucosa as an alternative treatment for mucosal defects in the oral cavity.

Keywords: Tissue engineering; Polymer scaffold; Oral mucosa; Graft; In vivo

The nanoscale properties of bacterial inclusion bodies and their effect on mammalian cell proliferation by César Díez-Gil; Sven Krabbenborg; Elena García-Fruitós; Esther Vazquez; Escarlata Rodríguez-Carmona; Imma Ratera; Nora Ventosa; Joaquin Seras-Franzoso; Olivia Cano-Garrido; Neus Ferrer-Miralles; Antonio Villaverde; Jaume Veciana (pp. 5805-5812).
The chemical and mechanical properties of bacterial inclusion bodies, produced in different Escherichia coli genetic backgrounds, have been characterized at the nanoscale level. In regard to wild type, DnaK and ClpA strains produce inclusion bodies with distinguishable wettability, stiffness and stiffness distribution within the proteinaceous particle. Furthermore it was possible to observe how cultured mammalian cells respond differentially to inclusion body variants when used as particulate materials to engineer the nanoscale topography, proving that the actual range of referred mechanical properties is sensed and discriminated by biological systems.The data provide evidence of the mechanistic activity of the cellular quality control network and the regulation of the stereospecific packaging of partially folded protein species in bacteria. This inclusion body nanoscale profiling offers possibilities for their fine genetic tuning and the resulting macroscopic effects when applied in biological interfaces.

Keywords: Inclusion bodies; Cell growth; Nanoparticles; Stiffness; Wettability; Tissue engineering

The synergistic effects of multivalent ligand display and nanotopography on osteogenic differentiation of rat bone marrow stem cells by Gagandeep Kaur; Chao Wang; Jian Sun; Qian Wang (pp. 5813-5824).
Cell–substrate interactions play a vital role in governing crucial cell functions such as adhesion, proliferation and differentiation. Surface topography and chemical properties can initiate signaling cascades modulating cell behavior. However, mimicking extracellular environment to direct cell functions through cell–surface interactions is challenging. In this report, we employed tobacco mosaic virus (TMV) as a model system to present nanotopographic features along with multivalent ligand display to study osteogenic differentiation of bone marrow stem cells (BMSCs). TMV is a rod shaped plant virus which is 300 nm in length and 18 nm in diameter. A single TMV rod comprises of 2130 identical coat proteins which assemble into the rod-like helical structure around the single strand of RNA. For the present study TMV was chemically modified with phosphate to induce calcium incorporation. Gene regulation during BMSC differentiation on TMV and TMV-phosphate (TMV-Phos) was studied over time points of 7, 14 and 21 days. We examined changes in gene expression of osteospecific genes (osteocalcin, osteopontin and runx2) which indicate that nanofeatures functionalized with phosphate groups exhibited significantly higher up regulation of osteospecific genes. Furthermore, we studied the gene regulation by coating Ti substrates with TMV and TMV-Phos. TMV-Phos coated substrates displayed higher expression of the studied genes as compared to Ti substrates. Our results imply that the differentiation capacity of BMSCs can be significantly enhanced through simple multivalent interactions with simple functional units rather than using complex biomolecules.

Keywords: Nanoparticle; Nanotopography; Bone marrow stromal cells; Mesenchymal stem cell; Virus substrate; Osteogenic differentiation

Cartilage tissue engineering using funnel-like collagen sponges prepared with embossing ice particulate templates by Hongxu Lu; Young-Gwang Ko; Naoki Kawazoe; Guoping Chen (pp. 5825-5835).
Three-dimensional porous scaffolds of collagen have been widely used for tissue engineering and regenerative medicine. In this study, we fabricated funnel-like collagen sponges with open surface pore structures by a freeze-drying method that used embossing ice particulates as a template. By controlling the size of the ice particulates and the temperature of freezing, collagen sponges with different pore structures were prepared. To investigate the effects of different pore structures on cartilage regeneration, the funnel-like collagen sponges were used to culture bovine articular chondrocytes. Scaffolds that were prepared with 400μm ice particulate templates and a freezing temperature of −3°C resulted in the best cell distribution, ECM production, and chondrogenesis. Although funnel-like collagen sponges prepared with 400μm ice particulate templates and a freezing temperature of −1°C and 720μm ice particulates and a freezing temperature of −3°C, showed even cell distribution, the cell seeding efficiencies and sGAG amount per cell were low. However, the scaffolds prepared with 400μm ice particulate templates and a freezing temperature of −5°C or −10°C showed a limited effect on the improvement of cell distribution and chondrogenesis. Control collagen sponges without ice particulates failed to support the formation of homogenous cartilage-like tissue. These results indicate that funnel-like collagen sponges were superior to control collagen sponges and that scaffolds prepared with 400μm ice particulate templates at −3°C were optimal for cartilage tissue engineering.

Keywords: Embossing ice particulate template; Funnel-like pore structure; Collagen scaffold; Porous scaffold; Cartilage tissue engineering

Intervertebral disc regeneration after implantation of a cell-free bioresorbable implant in a rabbit disc degeneration model by Michaela Endres; Alexander Abbushi; Ulrich W. Thomale; Mario Cabraja; Stefan N. Kroppenstedt; Lars Morawietz; Pablo A. Casalis; Maria L. Zenclussen; Arne-Jörn Lemke; Peter Horn; Christian Kaps; Christian Woiciechowsky (pp. 5836-5841).
Degeneration of the intervertebral disc is the most common cause of lower back pain. Interestingly, all available treatments are limited to treat the symptoms and not the underlying biologic alterations of the disc. Freeze-dried resorbable non-woven polyglycolic acid (PGA) – hyaluronan implants were used in a degenerated disc disease (DDD) model in New Zealand white rabbits. The constructs were immersed in allogenic serum and implanted into the disc defect. Animals with discectomy only served as controls. The T2-weighted/fat suppression sequence signal intensity of the operated discs as assessed by magnet resonance imaging decreased in both groups one week after the operation compared to a healthy disc. After 12 months the implanted group showed an increase of 51% in the signal intensity compared to the 1-week results whereas the signal intensity in the sham group remained on the same level from one week to 12 months. Histological and quantitative immunohistochemical examination after 12 months indicated cell migration into the defect and showed formation of disc repair tissue. In controls, repair tissue containing type II collagen was not evident. In conclusion, the implantation of polymer-based constructs after discectomy induces tissue regeneration resulting in improvement of the disc water content.

Keywords: Degenerative disc disease; Polyglycolic acid; Discectomy; Cell-free implant; Disc regeneration

The magnetophoretic mobility and superparamagnetism of core-shell iron oxide nanoparticles with dual targeting and imaging functionality by Faquan Yu; Lei Zhang; Yongzhuo Huang; Kai Sun; Allan E. David; Victor C. Yang (pp. 5842-5848).
With the goal to achieve highly efficacious MRI-monitored magnetic targeting, a novel drug carrier with dual nature of superior magnetophoretic mobility and superparamagnetism was synthesized. This carrier was specially designed in a core-shell structure. The core was achieved by utilizing a strategy of self-assembly of oppositely charged ultrafine superparamagnetic iron oxide nanoparticles previously prepared. The final particles were formed by coating such cores with carboxymethyldextran (CMD) polymer. By exclusion of non-magnetic materials from the interior part of the particles, this structure maximized the amount of magnetic material and thus yielded a superior magnetophoretic mobility. Such a strategy avoids the challenge of superparamagnetism loss, which would be caused by cores exceeding a critical domain size. Coating the self-assembled core enables the magnetic carrier to be stable upon usage and storage and to be readily linked with drug molecules for therapeutic applications. In vitro characterization showed that these nanoparticles displayed a 3- to 4-fold enhancement in magnetophoretic mobility, and a markedly improved stability when stored in 50% serum as a comparison of conventional iron oxide-based magnetic nanoparticles. Preliminary in vivo studies revealed that the nanoparticles also function well as a contrast enhancer for MR imaging of brain glioma. This technology could lead to the development of a new paradigm of magnetic carriers that meet with the needs of various clinical applications.

Keywords: Magnetophoresis; Superparamagnetism; Core-shell structure; MRI; Iron oxide

The effect of TAT conjugated platinum nanoparticles on lifespan in a nematode Caenorhabditis elegans model by Juewon Kim; Takuji Shirasawa; Yusei Miyamoto (pp. 5849-5854).
We have shown that platinum nanoparticle species (nano-Pt) is a superoxide dismutase/catalase mimetic that scavenges superoxide and hydrogen peroxide. In Caenorhabditis elegans, nano-Pt functions as an effective antioxidant that induces an extension in lifespan and strong resistance against excessive oxidative stress. Our study with C. elegans was the first trial to use nano-Pt as a bio-active substance. However, a high concentration of nano-Pt was required for these survival effects, probably due to limited membrane permeability. Here, we show that the conjugation of nano-Pt with an HIV-1 TAT fusion protein C-terminally linked to a peptide with high affinity for platinum improves internalization, eliciting a similar level of antioxidant effects at one hundredth the concentration of unconjugated nano-Pt. This approach is a potential method to facilitate translocation of bio-active nanoparticles into living organisms and could be a model assay for estimate the effects of antioxidant in living organism.

Keywords: Nano particle; Platinum; Biomimetic material; Metal surface treatment; Surface modification; Drug delivery

The therapeutic efficacy of conjugated linoleic acid – Paclitaxel on glioma in the rat by Xi-Yu Ke; Bo-Jun Zhao; Xin Zhao; Ying Wang; Yue Huang; Xiao-Mei Chen; Bing-Xiang Zhao; Shan-Shan Zhao; Xuan Zhang; Qiang Zhang (pp. 5855-5864).
Considering the effects of conjugated linoleic acid (CLA) on anti-tumor and anti-angiogenic in brain tumor, synergistic anti-tumor activity with taxane as well as potential activity for transporting chemotherapeutic agents across the blood–brain barrier (BBB), the purpose of this study was to synthesize CLA–paclitaxel (CLA–PTX) conjugate which could reach to the brain tissue and target brain tumor. The CLA was covalently linked to PTX. The conjugate was stable in PBS and rat plasma in vitro and had no microtubule assembly activity in solution and slight effect of arresting cell cycle progression at the G2-M phase. The in vitro cytotoxicity of conjugate was lower than that of PTX ( p < 0.05). The conjugate showed higher cellular uptake efficiency on C6 glioma cells. The entire pharmacokinetic index revealed the significant enhancement of the conjugate pharmacokinetics compared with that in PTX ( p < 0.01). The conjugate, unlike PTX, could distribute in brain tissue and retained higher concentrations throughout 360 h. The anti-tumor efficacy in brain tumor-bearing rats after administering conjugate was significantly higher than that after giving Taxol ( p < 0.01). In conclusion, this CLA–PTX conjugate showed great potential to become a new prodrug of PTX and the methodology can be applied to other anticancer drugs.

Keywords: Conjugated linoleic acid (CLA); Paclitaxel (PTX); Conjugate; Blood–brain barrier; Brain glioma-bearing rats; Anti-tumor efficacy

Teicoplanin-loaded borate bioactive glass implants for treating chronic bone infection in a rabbit tibia osteomyelitis model by Xin Zhang; WeiTao Jia; YiFei Gu; Wei Xiao; Xin Liu; DePing Wang; ChangQing Zhang; WenHai Huang; Mohamed N. Rahaman; Delbert E. Day; Nai Zhou (pp. 5865-5874).
The treatment of chronic osteomyelitis (bone infection) remains a clinical challenge. In this work, pellets composed of a chitosan-bonded mixture of borate bioactive glass particles (<50μm) and teicoplanin powder (antibiotic), were evaluated in vitro and in vivo for treating chronic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA) in a rabbit model. When immersed in phosphate-buffered saline, the pellets showed sustained release of teicoplanin over 20–30 days, while the bioactive glass converted to hydroxyapatite (HA) within 7 days, eventually forming a porous HA structure. Implantation of the teicoplanin-loaded pellets in a rabbit tibia osteomyelitis model resulted in the detection of teicoplanin in the blood for about 9 days. The implants converted to a bone-like HA graft, and supported the ingrowth of new bone into the tibia defects within 12 weeks of implantation. Microbiological, histological and scanning electron microscopy techniques showed that the implants provided a cure for the bone infection. The results indicate that the teicoplanin-loaded borate bioactive glass implant, combining sustained drug release with the ability to support new bone ingrowth, could provide a method for treating chronic osteomyelitis.

Keywords: Osteomyelitis; Bone infection; Bone regeneration; Borate bioactive glass; Drug release

Osteogenic induction of hBMSCs by electrospun scaffolds with dexamethasone release functionality by Albino Martins; Ana Rita C. Duarte; Susana Faria; Alexandra P. Marques; Rui L. Reis; Nuno M. Neves (pp. 5875-5885).
Electrospun structures were proposed as scaffolds owing to their morphological and structural similarities with the extracellular matrix found in many native tissues. These fibrous structures were also proposed as drug release systems by exploiting the direct dependence of the release rate of a drug on the surface area. An osteogenic differentiation factor, dexamethasone (DEX), was incorporated into electrospun polycaprolactone (PCL) nanofibers at different concentrations (5, 10, 15 and 20 wt.% polymer), in a single-step process. The DEX incorporated into the polymeric carrier is in amorphous state, as determined by DSC, and does not influence the typical nanofibers morphology. In vitro drug release studies demonstrated that the dexamethasone release was sustained over a period of 15 days. The bioactivity of the released dexamethasone was assessed by cultivating human bone marrow mesenchymal stem cells (hBMSCs) on 15 wt.% DEX-loaded PCL NFMs, under dexamethasone-absent osteogenic differentiation medium formulation. An increased concentration of alkaline phosphatase and deposition of a mineralized matrix was observed. Phenotypic and genotypic expression of osteoblastic-specific markers corroborates the osteogenic activity of the loaded growth/differentiation factor. Overall data suggests that the electrospun biodegradable nanofibers can be used as carriers for the sustained release of growth/differentiation factors relevant for bone tissue engineering strategies.

Keywords: Drug release; Polycaprolactone; Mesenchymal stem cells; Bone tissue engineering; Molecular biology

Virus-mimetic polymeric micelles for targeted siRNA delivery by Xiao-Bing Xiong; Hasan Uludağ; Afsaneh Lavasanifar (pp. 5886-5893).
In this study, an engineered non-viral polymer based delivery systems with structural features mimicking that of viral vectors was developed and the potential of this carrier for siRNA delivery was assessed. The developed siRNA carrier was based on poly(ethylene oxide)- block-poly(ɛ-caprolactone) (PEO- b-PCL) micelles decorated with integrin αvβ3 targeting peptide (RGD4C) and/or cell penetrating peptide (TAT) on the PEO shell, and modified with a polycation (spermine) in the PCL core for siRNA binding and protection. We observed increased cellular uptake and effective endosomal escape of siRNA delivered with the peptide-functionalized micelles especially those with dual functionality (RGD/TAT-micelles) compared to unmodified micelles (NON-micelles) in MDA435/LCC6 resistant cells. Transfection of mdr1 siRNA formulated in peptide-modified micelles led to P-gp down regulation both at the mRNA and protein level. Subsequent to P-gp down regulation, increased cellular accumulation of P-gp substrate, doxorubicin (DOX), in the cytoplasm and nucleus of resistant MDA435/LCC6 cells after treatment with peptide decorated polymeric micelle/mdr1 siRNA complexes was observed. As a result, resistance to DOX was successfully reversed. Interestingly, RGD/TAT-micellar siRNA complexes produced improved cellular uptake, P-gp silencing, DOX cellular accumulation, DOX nuclear localization and DOX induced cytotoxicity in MDA435/LCC6 cells when compared to micelles decorated with individual peptides. Results of this study indicated a potential for RGD/TAT-functionalized virus-like micelles as promising carriers for efficient delivery of mdr1 siRNA to MDA435/LCC6 resistant cells as means to reverse the P-gp mediated multidrug resistance to DOX.

Keywords: Polymeric micelles; siRNA delivery; Drug targeting; Multidrug resistance; P-glycoprotein

The influence of mediators of intracellular trafficking on transgene expression efficacy of polymer–plasmid DNA complexes by Sutapa Barua; Kaushal Rege (pp. 5894-5902).
Polymer-mediated gene delivery is an attractive alternative to viral vectors, but is limited by low efficacies of transgene expression. We report that polymers possess differential efficacies for transfecting closely related human prostate cancer cells, which correlates with dramatically different intracellular fate of nanoscale cargo in these cells. Sequestration of nanoscale cargo (27 nm quantum dots and 150–250 nm polyplexes) at a single location near the microtubule organizing compartment (MTOC) in PC3–PSMA human prostate cancer cells correlated with lower polymer-mediated transgene expression compared to PC3 cells, which showed distributed localization throughout the cytoplasm. We show, for the first time, that treatment with the histone deacetylase 6 (HDAC6) inhibitor tubacin, which acetylates tubulin of microtubules in the cytoplasm, abolished quantum dot and polyplex sequestration at the perinuclear recycling compartment/microtubule organizing center (PNRC/MTOC) and increased polymer-mediated transgene expression by up to forty-fold compared to cells not treated with the HDAC6 inhibitor drug. Treatment with the class I and II HDAC inhibitor trichostatin A (TSA) demonstrated similar levels of transgene expression enhancement. These results indicate that mediators of intracellular trafficking can be employed to modulate nanoparticle fate and enhance the efficacy of nanoscale therapeutics in cells. Simultaneous use of high-efficacy polymers along with mediators of intracellular trafficking is an attractive synergistic strategy for enhancing polymer-mediated transgene expression.

Keywords: Non-viral gene delivery; Histone deacetylase inhibitors; HDAC; Tubacin; Microtubule; Trichostatin A

Chitosan–alginate 3D scaffolds as a mimic of the glioma tumor microenvironment by Forrest M. Kievit; Stephen J. Florczyk; Matthew C. Leung; Omid Veiseh; James O. Park; Mary L. Disis; Miqin Zhang (pp. 5903-5910).
Despite recent advances in the understanding of its cell biology, glioma remains highly lethal. Development of effective therapies requires a cost-effective in vitro tumor model that more accurately resembles the in vivo tumor microenvironment as standard two-dimensional (2D) tissue culture conditions do so poorly. Here we report on the use of a three-dimensional (3D) chitosan–alginate (CA) scaffold to serve as an extracellular matrix that promotes the conversion of cultured cancer cells to a more malignant in vivo-like phenotype. Human U-87 MG and U-118 MG glioma cells and rat C6 glioma cells were chosen for the study. In vitro tumor cell proliferation and secretion of factors that promote tumor malignancy, including VEGF, MMP-2, fibronectin, and laminin, were assessed. The scaffolds pre-cultured with U-87 MG and C6 cells were then implanted into nude mice to evaluate tumor growth and blood vessel recruitment compared to the standard 2D cell culture and 3D Matrigel matrix xenograft controls. Our results indicate that while the behavior of C6 cells showed minimal differences due to their highly malignant and invasive nature, U-87 MG and U-118 MG cells exhibited notably higher malignancy when cultured in CA scaffolds. CA scaffolds provide a 3D microenvironment for glioma cells that is more representative of the in vivo tumor, thus can serve as a more effective platform for development and study of anticancer therapeutics. This unique CA scaffold platform may offer a valuable alternative strategy to the time-consuming and costly animal studies for a wide variety of experimental designs.

Keywords: Chitosan; Alginate; Natural polymer; Scaffold; Tumor microenvironment; Glioma

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