Biomaterials (v.28, #8)

The effect of cholecyst-derived extracellular matrix on the phenotypic behaviour of valvular endothelial and valvular interstitial cells by Sarah Brody; Jillian McMahon; Li Yao; Margret O’Brien; Peter Dockery; Abhay Pandit (1461-1469).
Cholecyst-derived extracellular matrix (CEM) is a novel, proteinaceous biomaterial, derived from the porcine cholecyst, which may have potential applications as a scaffold in the area of heart valve tissue engineering. In this study the potential of CEM to support the proliferation of valvular endothelial cells (VECs) and valvular interstitial cells (VICs), while maintaining their phenotypic mRNA synthesis, protein expression and morphology was assessed by biochemical assays, electron microscopy, immunostaining and reverse-transcriptase polymerase chain reaction. VICs and VECs were isolated from the porcine aortic valve and techniques were developed for the isolation of CEM for cell culture. VECs and VICs cultured on CEM adhered and proliferated, maintaining their phenotypic morphology. VECs synthesised von Willebrand factor mRNA and endothelial nitric oxide synthase (eNOS) mRNA and expressed eNOS and VICs synthesised alpha-smooth muscle actin (αSMA) mRNA and expressed αSMA. Cellular area fraction of VICs expressing αSMA was 87.7±6.8% and cellular area fraction of VECs expressing eNOS was 93.8±9.3%. Findings of this study support the hypothesis that CEM is a potential biomaterial for tissue engineered heart valve scaffold design.
Keywords: Cardiac tissue engineering; Heart valve; Biomimetic material; Endothelialisation; Scaffold;

Efficient differentiation of CD14+ monocytic cells into endothelial cells on degradable biomaterials by Guido Krenning; Patricia Y.W. Dankers; Danijela Jovanovic; Marja J.A. van Luyn; Martin C. Harmsen (1470-1479).
Vascular tissue engineering aims at creating self-renewing, anti-thrombogenic, vascular grafts, which can be based on endothelial progenitor cells (EPC). EPC harbor essential features such as plasticity and longevity. Unfortunately, the archetype CD34+ EPC is rare in peripheral blood. Monocytes, i.e. CD14+ cells also have the ability to differentiate into endothelial-like cells and are by far more abundant in peripheral blood than are CD34+ EPC. Therefore, CD14+ cells would seem appropriate candidates for tissue engineering of small-diameter blood vessels. In this study, we investigated the differentiation of CD14+ cells on three biodegradable biomaterials under angiogenic conditions. Morphological analyses, gene transcript analyses, endothelial marker (i.e. VE-Cadherin and eNOS) and macrophage marker (i.e. CD68 and CD163) expression analyses, revealed that a small fraction (15–25%) of cultured CD14+ cells differentiated into macrophages after 21 days of culture. The majority of CD14+ cells (>75%) differentiated into endothelial-like cells (ELC) on all biomaterials used. The expression of endothelial markers was similar to their expression on HUVEC. Since CD14+ cells are present in high numbers in adult peripheral blood, easy to isolate and because they easily differentiate into ELC on biomaterials, we conclude that CD14+ cells are a suitable cell source for progenitor-based vascular tissue engineering.
Keywords: Cell culture; Endothelial cell; Monotype; Polycaprolactone; Polyurethane; RGD peptide;

Electrodeposition of anchored polypyrrole film on microelectrodes and stimulation of cultured cardiac myocytes by Matsuhiko Nishizawa; Hyuma Nozaki; Hirokazu Kaji; Takahiro Kitazume; Noriyuki Kobayashi; Takeshi Ishibashi; Takashi Abe (1480-1485).
The electrically conducting polymer polypyrrole (PPy) was electrochemically deposited onto Pt microelectrodes on a polyimide (PI) substrate. Pre-modification of the PI surface with a self-assembled monolayer of octadecyltrichlorosilane-induced anisotropic lateral growth of PPy along the PI surface and enhanced adhesive strength of the PPy film. The lateral growth of PPy film around the electrode anchored the whole film to the substrate. External stimulation of cultured cardiac myocytes was carried out using the PPy-coated microelectrode. The myocytes on the microelectrode substrate were electrically conjugated to form a sheet, and showed synchronized beating upon stimulation. The threshold charge for effective stimulation of a 0.8 cm2 sheet of myocytes was around 0.2 μC, roughly corresponding to a membrane depolarization of 250 mV.
Keywords: Cardiomyocyte; Cell culture; Electrical stimulation; Electroactive polymer; Electrochemistry;

Thin collagen film scaffolds for retinal epithelial cell culture by James T. Lu; Christina J. Lee; Stacey F. Bent; Harvey A. Fishman; Eric E. Sabelman (1486-1494).
Collagen films have been used in biological implantation and surgical grafts. The development of thin collagen films on the order of 10 μm thick that ensure a planar distribution of implanted cells is a necessary step towards surgical grafts for treatment of age-related macular degeneration (AMD). Here, collagen films were manufactured on a Teflon support to a thickness of 2.4±0.2 μm, comparable to that of native Bruch's membrane. Because one important function of Bruch's membrane is allowing the flow of nutrients and waste to and from the retinal pigment epithelium the diffusion properties of the collagen films were studied using blind-well chambers. The diffusion coefficient of the collagen film was determined to be 4.1×10−10  cm2/s for 71,200 Da dextran molecules. Viability studies utilizing the blind-well chambers also confirmed that nutrient transport through the films was sufficient to sustain retinal pigment epithelial (RPE) cells. The films were bioassayed in a RPE cell culture model to confirm cell attachment and viability. RPE cells were shown to form an epithelial phenotype and were able to phagocytize photoreceptor outer segments.
Keywords: Retina; Collagen; Epithelial cell; Cell viability;

Bone tissue engineering strategies are fundamentally based upon porous scaffold materials that serve as a support for ingrowth of host cells and/or provide a substrate for exogenously delivered cells. Here we report the application of a surface calcium phosphate (CaP) mineral layer to a macroporous polymeric/CaP composite biomaterial, with a macroporous interconnectivity, and its subsequent in vivo evaluation in a rodent femoral defect. The application of the mineral layer eliminates the fibrous tissue encapsulation and foreign body giant cell response commonly seen at the interface of polymeric materials, yet retains the unique characteristics of the parent material as being macroporous, completely biodegradable and possessing a high degree of interconnectivity. This represents the third generation of this scaffold material, incorporating iterative changes to the scaffold design in response to both materials and biological design criteria to produce a material with enhanced in vitro and in vivo performance.
Keywords: Bone tissue engineering; Scaffold; Calcium phosphate coating; Simulated body fluid; In vivo test; Foreign body response;

Dynamics of smooth muscle cell deadhesion from thermosensitive hydroxybutyl chitosan by Beiyi Chen; Jiyoung Dang; Tuan Lin Tan; Ning Fang; Wei Ning Chen; Kam W. Leong; Vincent Chan (1503-1514).
Thermoresponsive polymer (TRP) enables the enzyme-free harvesting of cells through an acute increase in surface hydrophilicity of TRP across its lower critical solution temperature (LCST), rendering feasible the generation of polymer-free cell sheets for regenerative medicine applications. To date, the intricate mechanisms of cell deadhesion/detachment on TRP surface remain obscure. Elucidation of such biophysical responses would be valuable for the cell sheet technology. In this study, integrative biophysical techniques are applied to probe the thermal-induced deadhesion kinetics of smooth muscle cell (SMC) on thermoresponsive hydroxybutyl chitosan (HBC29) against different periods of pre-culture time at 37 °C. Atomic force microscopy demonstrates that both the surface topography and mechanical property of HBC29 film in water are acutely modulated across its LCST. Firstly, cells show negligible changes in adhesion contact area during low-temperature incubation on unmodified tissue culture polystyrene (TCPS). Secondly, the recession of adhesion contact and retraction of cell body for cells with different pre-culture times are triggered by HBC29 coating on TCPS. Interestingly, the initial rate of reduction in the normalized adhesion contact area of SMC is negatively correlated with the pre-culture time. Thirdly, the degree of cell deformation and average adhesion energy are reducing functions of time only for SMCs with the lowest pre-culture time. In contrast, adhesion energy per cell is a reducing function of time irrespective of the change of pre-culture time. Lastly, the temporal dynamics of cytoskeleton organization and β-actin/smoothelin-B mRNA expression for SMCs is strongly dependent on the pre-culture time. Overall, this study demonstrates that the thermal-induced deadhesion of SMC on TRP is characterized by the evolution of its contractile phenotypes.
Keywords: Deadhesion kinetics; Chitosan derivative; Cell biophysics; Cytoskeleton dynamics;

In recent years, functional biomaterial research has been directed towards the development of improved scaffolds and new drug delivery systems. The objective of this study was to develop growth-factor gene releasing coral composites as a regenerative material for periodontal regeneration. In this study, porous chitosan/coral composites combined with plasmid encoding platelet-derived growth factor B (PDGFB) gene were prepared through a freeze-drying process. These scaffolds were evaluated in vitro by analysis of microscopic structure and cytocompatibility. The expression of PDGFB and type-I collagen were detected with RT-PCR after human periodontal ligament cells (HPLCs) were seeded in this scaffold. Then these scaffolds were implanted subcutaneously into athymic mice. Results indicated that HPLCs showed much better proliferation properties on the gene-activated scaffolds than on the pure coral scaffolds, and the expression of PDGFB and type-I collagen up-regulated in gene-activated scaffold. After implanted in vivo, HPLCs not only proliferate but also increased the expression of PDGFB. This study demonstrated the potential of coral scaffold combined PDGFB gene as a good substrate candidate in periodontal tissue regeneration.
Keywords: Chitosan; Coral; Scaffold; Platelet-derived growth factor B; Gene transfer; Periodontium;

The short-term effects on restenosis and thrombosis of echinomycin-eluting stents topcoated with a hydrophobic heparin-containing polymer by Yong-Kyu Lee; Jae Hyung Park; Hyun Tae Moon; Dong Yun Lee; Jung Han Yun; Youngro Byun (1523-1530).
Although drug-eluting stents (DESs) have become the most effective means of treating coronary artery disease, safety concerns regarding their thrombogenicities remain to be surmounted. Here, we report on a novel type of DES capable of preventing restenosis and thrombosis. The DES was prepared by coating a bare metal stent with echinomycin (an anti-proliferative drug) in polyurethane by a spray drying method. Hydrophobic heparinized polymer was then topcoated onto stent over echinomycin/PU layer by dipping to improve hemocompatibility. The two-layered stent was characterized regarding surface and cross-sectional morphology, drug release pattern, platelet adhesion in vitro, and restenosis in vivo. It was found that the heparin topcoat acts as a diffusion barrier that allows the controlled release of drug in a sustained manner. Also, the heparin coated layer effectively reduced platelet adhesion, indicating excellent hemocompatibility. From the animal test using pigs, it was evident that the developed DESs can minimize neointimal proliferation and thrombus formation. The devised hydrophobic heparinized polymer-coated DES effectively reduced both restenosis and thrombosis, suggesting that they have potential as tools for the treatment of coronary artery diseases.
Keywords: Drug-eluting stent; Heparinized polymer; Echinomycin; Restenosis; Thrombosis;

The mechanically stable steam sterilization of bone grafts by G.F. Draenert; M. Delius (1531-1538).
Bone allografts are the standard material used in augmentative bone surgery. However, steam-sterilized bone has a low mechanical stability and limited ossification based on low strain-adapted bone remodelling. Here we describe a new technique which allows the bone to be autoclaved without losing its mechanical stability and osteoconductivity. The compression strength of the new material was compared with steam-sterilized and fresh bone based on mechanical testing using bone cylinders (n=30/group). Allogeneic new material and fresh bone were press-fit implanted into rabbit patellar grooves and examined under fluorescent light and conventional microscopy. Initial healing was assessed after 30 d (n=5/group). Osseous integration and remodelling was studied after 100 d (n=12/group). Steam-sterilized bone showed no mechanical stability, whereas the new material was stiff and had compression curves similar to fresh bone; both groups showed equal degrees of direct ossification after 30 d, advanced bony ingrowth and remodelling after 100 d, and similar ingrowth depths on histomorphometric analysis. The new method preserved the stiffness and osteoconductivity of bone after steam sterilization, and microstructure, mineralization, and composition were conserved. This technique could be useful for bone banking in Third World countries.
Keywords: Bone graft; Sterilization; Animal model; Transplantation; Osteoconduction; Osseointegration;

Polymerization of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) around living neural cells by Sarah M. Richardson-Burns; Jeffrey L. Hendricks; Brian Foster; Laura K. Povlich; Dong-Hwan Kim; David C. Martin (1539-1552).
In this paper, we describe interactions between neural cells and the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) toward development of electrically conductive biomaterials intended for direct, functional contact with electrically active tissues such as the nervous system, heart, and skeletal muscle. We introduce a process for polymerizing PEDOT around living cells and describe a neural cell-templated conducting polymer coating for microelectrodes and a hybrid conducting polymer-live neural cell electrode. We found that neural cells could be exposed to working concentrations (0.01  m) of the EDOT monomer for as long as 72 h while maintaining 80% cell viability. PEDOT could be electrochemically deposited around neurons cultured on electrodes using 0.5–1 μA/mm2 galvanostatic current. PEDOT polymerized on the electrode and surrounded the cells, covering cell processes. The polymerization was impeded in regions where cells were well adhered to the substrate. The cells could be removed from the PEDOT matrix to generate a neural cell-templated biomimetic conductive substrate with cell-shaped features that were cell attracting. Live cells embedded within the conductive polymer matrix remained viable for at least 120 h following polymerization. Dying cells primarily underwent apoptotic cell death. PEDOT, PEDOT+live neurons, and neuron-templated PEDOT coatings on electrodes significantly enhanced the electrical properties as compared to the bare electrode as indicated by decreased electrical impedance of 1–1.5 orders of magnitude at 0.01–1 kHz and significantly increased charge transfer capacity. PEDOT coatings showed a decrease of the phase angle of the impedance from roughly 80° for the bare electrode to 5–35° at frequencies >0.1 kHz. Equivalent circuit modeling indicated that PEDOT-coated electrodes were best described by R(C(RT)) circuit. We found that an RC parallel circuit must be added to the model for PEDOT+live neuron and neuron-templated PEDOT coatings.
Keywords: Electroactive polymer; Neural prosthesis; Interface; Electrode; Biomimetic material;

Ocular drug delivery by liposome–chitosan nanoparticle complexes (LCS-NP) by Yolanda Diebold; Miguel Jarrín; Victoria Sáez; Edison L.S. Carvalho; María Orea; Margarita Calonge; Begoña Seijo; María J. Alonso (1553-1564).
This study evaluated in vitro and in vivo a colloidal nanosystem with the potential to deliver drugs to the ocular surface. This nanosystem, liposome–chitosan nanoparticle complexes (LCS-NP), was created as a complex between liposomes and chitosan nanoparticles (CS-NP). The conjunctival epithelial cell line IOBA-NHC was exposed to several concentrations of three different LCS-NP complex to determine the cytotoxicity. The uptake of LCS-NP by the IOBA-NHC conjunctival cell line and by primary cultured conjunctival epithelial cells was examined by confocal microscopy. Eyeball and lid tissues from LCS-NP-treated rabbits were evaluated for the in vivo uptake and acute tolerance of the nanosystems. The in vitro toxicity of LCS-NP in the IOBA-NHC cells was very low. LCS-NPs were identified inside IOBA-NHC cells after 15 min and inside primary cultures of conjunctival epithelial cells after 30 min. Distribution within the cells had different patterns depending on the LCS-NP formulation. Fluorescence microscopy of the conjunctiva revealed strong cellular uptake of LCS-NP in vivo and less intensive uptake by the corneal epithelium. No alteration was macroscopically observed in vivo after ocular surface exposure to LCS-NP. Taken together, these data demonstrate that LCS-NPs are potentially useful as drug carriers for the ocular surface.
Keywords: Cell viability; Confocal microscopy; Controlled drug release; Epithelial cell; Mucosa;

Gene silencing using short interfering RNA (siRNA) is fast becoming an attractive approach to probe gene function in mammalian cells. Although there have been some success in the delivery of siRNA using various methods, tracking their delivery and monitoring their transfection efficiency prove to be hard without a suitable tracking agent. Therefore, a challenge lies with the design of an efficient and at the same time, self-tracking, transfection agent for RNA interference. In this paper, chitosan nanoparticles (NPs) with encapsulated quantum dots (QDs) were synthesized and used to deliver HER2/neu siRNA. Using such a construct, the delivery and transfection of the siRNA can be monitored by the presence of fluorescent QDs in the chitosan NPs. Targeted delivery of HER2 siRNA to HER2-overexpressing SKBR3 breast cancer cells was shown to be specific with chitosan/QD NP surface labeled with HER2 antibody targeting the HER2 receptors on SKBR3 cells. Gene-silencing effects of the conjugated siRNA was also established using the luciferase and HER2 ELISA assays. These self-tracking siRNA delivery NPs will also aid in the monitoring of future gene silencing studies in vivo.
Keywords: Chitosan; Nanoparticle; Fluorescence; Gene therapy;

Influence of glass and polymer coatings on CHO cell morphology and adhesion by Thomas Sordel; Frederique Kermarec-Marcel; Stephanie Garnier-Raveaud; Nicolas Glade; Fabien Sauter-Starace; Catherine Pudda; Mathias Borella; Marc Plissonnier; Francois Chatelain; Franz Bruckert; Nathalie Picollet-D’hahan (1572-1584).
Successful development of cell-on-chip microsystems where living cells are deposited and grown in microfabricated structures is highly dependent on the control of cell/substrate interactions. In this study, several materials of interest were tested for CHO cell growth and morphology: (i) glass, fibronectin-, poly-l-lysine- and 3-aminopropyltriethoxysilane (APTES)—treated glass and UV/O3-modified PDMS coating on glass as well as (ii) silicon, poly-l-lysine-, APTES-, O2 plasma-treated and oxide-coated silicon. In addition, we quantitatively characterized cell adhesion to these substrates using a radial flow detachment assay. Lack of correlation between cell adhesion and cell morphology was systematically observed for all substrates. In particular, we show that PDMS coatings on glass can be finely tuned by UV/O3 treatment to enhance cell adhesion and induce elongated morphology. Moreover, we observed a low shear stress cell detachment mechanism on silicon oxide coatings on silicon wafers. It is therefore possible with these coatings to selectively influence either cell adhesion or morphology.
Keywords: Cell adhesion; Cell morphology; Surface energy; Surface coatings; Biochips;