Biomaterials (v.26, #35)

Calendar (I).

Special Issue of Biomaterials Dedicated to Canadian Biomaterials Research by Hasan Uludag; J. Paul Santerre; Gaetan Laroche; Mark Filiaggi (7207-7208).

Biomaterials in Canada: The first four decades by John L. Brash (7209-7220).
Biomaterials research in Canada began in the 1960s. Over the past four decades significant contributions have been made across a broad spectrum covering dental, orthopaedic, cardiovascular, neuro, and ocular biomaterials. Canadians have also been active in the derivative area of tissue engineering. Biomaterials laboratories are now established in universities and research institutes from coast to coast, supported mainly by funding from the Federal and Provincial Governments. The Canadian Biomaterials Society was formed in 1971 and has played an important role in the development of the field. The Society played host to the 5th World Biomaterials Congress in Toronto in 1996. The work of Canadian researchers over the past four decades is summarized briefly. It is concluded that biomaterials and tissue engineering is a mature, strong area of research in Canada and appears set to continue as such into the future.
Keywords: Canada; Dental biomaterials; Orthopaedic biomaterials; Cardiovascular biomaterials; Neuro biomaterials; Ocular biomaterials; Research funding in Canada; Canadian Biomaterials Society;

Impact of heat on nanocrystalline silver dressings by P.L. Taylor; A.L. Ussher; R.E. Burrell (7221-7229).
Thermal stability of heat-treated nanocrystalline silver dressings was investigated using chemical techniques and biological assays. Dressings were heat-treated for 24 h at temperatures from 23 to 110 °C. Bactericidal efficacy of heat-treated dressings was measured using a log reduction assay, while antibacterial longevity was determined via plate-to-plate transfer corrected zone of inhibition assays. Over the temperature range tested, biological activity dropped from excellent to negligible. Biological longevity results showed that controlled release properties of the dressings were significantly reduced by heat treatments above 75 °C. These data illustrate nanocrystalline silver sensitivity to heat. Further, it was clear that dressing efficacy is determined by total available soluble silver, not total silver in the dressing. It was determined that the quantity of soluble silver decreased significantly with increased heat treatment temperatures. These results should be considered in developing new nanocrystalline drug delivery systems.
Keywords: Antibacterial; Antimicrobial; Bioactivity; In vitro test; Nanocrystalline; Silver;

Impact of heat on nanocrystalline silver dressings by P.L. Taylor; O. Omotoso; J.B. Wiskel; D. Mitlin; R.E. Burrell (7230-7240).
Keywords: SEM (scanning electron microscopy); XPS (X-ray photoelectron spectroscopy); XRD (X-ray diffraction); Crystal growth; Nanocrystalline; Silver;

Chitosan, the deacetylated derivative of chitin, is a promising scaffold material for skin tissue engineering applications. It is biocompatible and biodegradable, and the degradation products are resorbable. However, the rapid degradation of chitosan and its low mechanical strength are concerns that may limit its use. In this study, chitosan with 80%, 90% and 100% degree of deacetylation (DDA) was crosslinked with dimethyl 3-3, dithio bis’ propionimidate (DTBP) and compared to uncrosslinked scaffolds. The scaffolds were characterized with respect to important tissue engineering properties.The tensile strength of scaffolds made from 100% DDA chitosan was significantly higher than for scaffolds made from 80% and 90% DDA chitosan. Crosslinking of scaffolds with DTBP increased the tensile strength. Crosslinking with DTBP had no significant effect on water vapour transmission rate (WVTR) or water absorption but had significant effect on the pore size and porosity of the samples. All samples showed a WVTR and pore size distribution suitable for skin tissue engineering; however, the water absorption and porosity were lower than the optimal values for skin tissue engineering. The biodegradation rate of scaffolds crosslinked with DTBP and glutaraldehyde (GTA) were reduced while no significant effect was observed in biodegradation of the samples made from 100% DDA chitosan whether crosslinked or uncrosslinked after 24 days of degradation.
Keywords: Chitin/chitosan; Scaffold; Wound dressing;

Polymeric micelles for the solubilization and delivery of cyclosporine A: pharmacokinetics and biodistribution by Hamidreza Montazeri Aliabadi; Dion R. Brocks; Afsaneh Lavasanifar (7251-7259).
The aim of this study was to assess the potential of polymeric micelles to modify the pharmacokinetics and tissue distribution of cyclosporine A (CsA). Drug-loaded methoxy poly(ethylene oxide)-b-poly( ε -caprolactone) (PEO-b-PCL) micellar solutions in isotonic medium were prepared and administered intravenously to healthy Sprague-Dawley rats. Blood and tissues were harvested and assayed for CsA, and resultant pharmacokinetic parameters and tissue distribution of CsA in its polymeric micellar formulation were compared to its commercially available intravenous formulation (Sandimmune®). In the pharmacokinetic assessment, a 6.1 fold increase in the area under the blood concentration versus time curve (AUC) was observed for CsA when given as polymeric micellar formulation as compared to Sandimmune®. The volume of distribution and clearance of CsA as PEO-b-PCL formulation were observed to be 10.0 and 7.6 fold lower, respectively, compared to the commercial formulation. No significant differences in t 1/2 or MRT could be detected. In the biodistribution study, analysis of tissue samples indicated that the mean AUC of CsA in polymeric micelles was lower in liver, spleen and kidney (1.5, 2.1 and 1.4-fold, respectively). Similar to the pharmacokinetic study in these rats, the polymeric micellar formulation gave rise to 5.7 and 4.9-fold increase in the AUC of CsA in blood and plasma, respectively. Our results show that PEO-b-PCL micelles can effectively solubilize CsA, at the same time confining CsA to the blood circulation and restricting its access to tissues such as kidney, perhaps limiting the onset of toxicity.
Keywords: Cyclosporine A; Block copolymer micelles; Biodistribution; Drug delivery; Polyethylene oxide; Polycaprolactone;

Therapeutic potential of nanoparticulate systems for macrophage targeting by Fatiha Chellat; Yahye Merhi; Alain Moreau; L’Hocine Yahia (7260-7275).
The use of non-viral nanoparticulate systems for the delivery of therapeutic agents is receiving considerable attention for medical and pharmaceutical applications. This increasing interest results from the fact that these systems can be designed to meet specific physicochemical requirements, and they display low toxic and immunogenic effects. Among potential cellular targets by drug-loaded nanoparticles, macrophages are considered because they play a central role in inflammation and they act as reservoirs for microorganisms that are involved with deadly infectious diseases. The most common and potent drugs used in macrophage-mediated diseases treatment often induce unwanted side effects, when applied as a free form, due to the necessity of high doses to induce a satisfactory effect. This could result in their systemic spreading, a lack of bioavailability at the desired sites, and a short half-life. Therefore, the use of drug-loaded nanoparticles represents a good alternative to avoid, or at least decrease, side effects and increase efficacy. In this manuscript, we present an overview of the usefulness of nanoparticles for macrophage-mediated therapies in particular. We discuss, though not exhaustively, the potential of therapeutic agent-loaded nanoparticles for some macrophage-mediated diseases. We also underline the most important parameters that affect the interaction mechanisms of the macrophages and the physicochemical aspects of the particulate systems that may influence their performance in macrophage-targeted therapies.
Keywords: Review; Macrophage targeting; Nanoparticles; Inflammatory diseases;

Calcium polyphosphate (CPP) antibiotic delivery matrices were prepared using a unique processing technique involving the exposure of antibiotic-loaded CPP pastes to high humidity for 0, 5, or 24 h. After the designated gelling period, samples were dried for a minimum of 24 h. At several time points out to 130 h, the elution medium was monitored for vancomycin, Ca2+ ion and ortho and poly phosphate release levels. Vancomycin activity was also assessed after 1, 24 and 130 h, while solution 31P-NMR was used to monitor changes in chain length within a 24 hr gelled VCM disc throughout the elution process. The gelling and drying process significantly reduced the rate of vancomycin release during the initial 2–4 h of elution, while extending the effective antibiotic release period by an additional 80 h. The mild conditions associated with matrix fabrication readily allowed for vancomycin incorporation within an environment that did not disrupt antibiotic activity. Throughout the elution process, all sample groups experienced considerable swelling followed by some apparent bulk erosion. Phosphate chain lysis was clearly observed by the end of the elution period. Generally, no strong or consistent correlation existed between matrix degradation and antibiotic release for the treatment groups investigated. An ability to delay antibiotic release using CPPs in conjunction with this protocol supports further investigations into the potential of this matrix as a localized drug delivery system.
Keywords: Calcium phosphate; Drug delivery; Degradation; Bone repair;

EGF-grafted PDMS surfaces in artificial cornea applications by B.J. Klenkler; M. Griffith; C. Becerril; J.A. West-Mays; H. Sheardown (7286-7296).
Lack of epithelial cell coverage has remained a persistent problem in the design of an artificial cornea. In this work, polydimethylsiloxane (PDMS) surfaces were modified with epidermal growth factor (EGF) to improve the growth of corneal epithelial cells. The EGF was covalently tethered to PDMS substrates aminated by plasma polymerization of allylamine via a homobifunctional polyethylene glycol (PEG) spacer. Surface modification was confirmed by contact angle and X-ray photoelectron spectroscopy measurements. By varying the ratio of EGF to PEG from 1:50 to 1:5, EGF amounts from 40 to 90 ng/cm2 could be bound, as determined by surface plasmon resonance (SPR) and 125I radiolabelling. Human corneal epithelial cells on the various modified surfaces were cultured both in the presence and absence of EGF in the culture medium to determine the effect of covalently bound EGF on the cells. The results demonstrated that covalently bound EGF on the surfaces is active with respect to promoting epithelial cell coverage. This was significant when compared to unmodified controls.
Keywords: Artificial cornea; Epidermal growth factor; PDMS; PEO; Corneal epithelial cells;

In-situ preparation of poly(propylene fumarate)—hydroxyapatite composite by Dorna Hakimimehr; Dean-Mo Liu; Tom Troczynski (7297-7303).
In-situ precipitation of hydroxyapatite (HAp) in the presence of poly(propylene fumarate) (PPF) is investigated. Amorphous calcium phosphate (ACP) precipitates in the presence of the polymer and remains in the amorphous form for a relatively long time, e.g. even after 24 h of coexistence with the mother solution. Our observations suggest that PPF interacts with the surface of the ACP particles and prevents them from transformation to crystalline hydroxyapatite. The PPF polymer seems to be more efficient in hindering the ACP to HAp transformation at higher pH conditions. From spectroscopic observations we hypothesize that the C=O bond of the PPF molecules interact with the calcium ion of the ACP particles. In case of low molecular weight PPF this interaction may lead to the incorporation of the polymer within the growing ACP particles.
Keywords: In-situ synthesis; Amorphous calcium phosphate; Poly(propylene fumarate); Composite;

Helical rosette nanotubes: A biomimetic coating for orthopedics? by Ai Lin Chun; Jesus G. Moralez; Thomas J. Webster; Hicham Fenniri (7304-7309).
Helical rosette nanotubes (HRN) are obtained through an entropically driven self-assembly process of low-molecular-weight synthetic modules under physiological conditions. Counter-intuitively, these materials undergo extensive self-assembly under the effect of temperature, resulting in networks of very long nanotubes. We have previously shown, using an in vitro model, that titanium (Ti) coated with HRN containing a lysine side chain (HRN-K1) displayed enhanced osteoblast (OB) adhesion when compared to uncoated Ti (p<0.01). Because it has been widely known that proteins play a critical role in OB adhesion on nanophase materials, here we examine OB adhesion on heated (+T) and unheated (−T) HRN-K1-coated Ti under serum (+S, presence of proteins) and serum-free (–S, absence of proteins) conditions. The results demonstrated that (a) while proteins enhanced OB adhesion on +T HRN-K1-coated Ti, they had no effect on –T HRN-K1-coated Ti, suggesting an active role played by the rosette nanotubes in promoting OB adhesion, and (b) under –S conditions, +T HRN-K1 induced the same level of OB adhesion as uncoated Ti under +S conditions, suggesting that +T HRN-K1 acts as a protein substitute. Finally, transmission electron microscopy and atomic force microscopy studies of +T and –T HRN-K1-coated Ti revealed a significant change in surface coverage, density and hierarchical organization of the nanotubes upon heating, which was correlated with their ability to promote cell adhesion.
Keywords: Biomimetic material; Bone tissue engineering; Cell adhesion; Osteoblast; Protein; Self-assembly; Surface treatment;

Calcium phosphate cement composites in revision hip arthroplasty by Andrew D. Speirs; Thomas R. Oxland; Bassam A. Masri; Anoush Poursartip; Clive P. Duncan (7310-7318).
Loosening of the femoral component in a total hip arthroplasty with concomitant bone loss can pose a problem for revision surgery due to inadequate structure in the remaining femur. While impaction allografting has shown promise, it has also shown serious complications, especially with moderate to severe bone loss. It may be possible to stabilize the graft layer with a bioresorbable cement to improve clinical results. This study examines the mechanical properties of a potential morsellized bone-bioresorbable composite. Morsellized bone was mixed with a commercially available bioresorbable cement (α-BSM, Etex Corp.) in compositions of 0%, 25%, 50% and 75% bone. Unconfined compression and diametral tensile and confined compression tests were performed to determine the composite mechanical properties.The composition containing 50% bone tended to exhibit the highest uniaxial strengths, as well as the highest confined compression modulus. The uniaxial compressive strength and stiffness of this composition was in the range of cancellous bone. Uniaxial compressive modulus decreased with increasing bone fraction whereas elongation exhibited the opposite trend. Bone fraction had a significant effect on compressive strength (p<0.0001), compressive modulus (p<0.0001), elongation (p<0.01), tensile strength (p<0.0001) and confined compressive modulus (p=0.04).The addition of a bioresorbable cement to the allograft layer may improve the properties of the layer, preventing early subsidence seen in some clinical studies of impaction allografting, and therefore improving the clinical results. Further testing is required to evaluate the in vitro mechanical performance, as well as in vivo remodelling characteristics.
Keywords: Impaction allografting; Composite; Strength; Stiffness; Calcium phosphate;

Three-dimensional growth of differentiating MC3T3-E1 pre-osteoblasts on porous titanium scaffolds by Jean-Philippe St-Pierre; Maxime Gauthier; Louis-Philippe Lefebvre; Maryam Tabrizian (7319-7328).
The present work assesses the potential of three-dimensional porous titanium scaffolds produced by a novel powder metallurgy process for applications in bone engineering through in vitro experimentation. Mouse MC3T3-E1 pre-osteoblasts were used to investigate the proliferation (DNA content), differentiation (alkaline phosphatase activity and osteocalcin release) and mineralisation (calcium content) processes of cells on titanium scaffolds with average pore sizes ranging from 336 to 557 μm, using mirror-polished titanium as reference material. Scanning electron microscopy was employed to qualitatively corroborate the results. Cells proliferate on all materials before reaching a plateau at day 9, with proliferation rates being significantly higher on foams (ranging from 123 to 163 percent per day) than on the reference material (80% per day). Alkaline phosphatase activity is also significantly elevated on porous scaffolds following the proliferation stage. However, cells on polished titanium exhibit greater osteocalcin release toward the end of the differentiation process, resulting in earlier mineralisation of the extracellular matrix. Nevertheless, the calcium content is similar on all materials at the end of the experimental period. Average pore size of the porous structures does not have a major effect on cells as determined by the various analyses, affecting only the proliferation stage. Thus, the microstructured titanium scaffolds direct the behaviour of pre-osteoblasts toward a mature state capable of mineralising the extracellular matrix.
Keywords: Osteoblasts; Differentiation; Mineralisation; Three-dimensional scaffolds; Titanium;

RGD-grafted thermoreversible polymers to facilitate attachment of BMP-2 responsive C2C12 cells by Erin Smith; Jennifer Yang; Locksley McGann; Walter Sebald; Hasan Uludag (7329-7338).
The purpose of this study was to design thermoreversible biomaterials for enhanced adhesion of bone morphogenetic protein-2 (BMP-2)-responsive cells. Peptides containing the arginine–glycine–aspartic acid (RGD) sequence were conjugated to N-isopropylacrylamide (NiPAM) polymers via amine-reactive N-acryloxysuccinimide (NASI) groups. In monolayer cultures, the adhesion of BMP-2-responsive C2C12 cells to RGD-grafted NiPAM/NASI surfaces was significantly higher than adhesion on ungrafted NiPAM/NASI surfaces. Although the morphology of cells adhered to RGD-grafted NiPAM/NASI surfaces was comparable to cells adhered on tissue culture polystyrene (TCPS), long-term cell growth was limited on the NiPAM/NASI surfaces, even for RGD-grafted surfaces. Treatment of C2C12 cells with recombinant BMP-2 induced dose-dependent osteoblastic differentiation as assessed by alkaline phosphatase (ALP) activity. In the absence of BMP-2, cells cultured on NiPAM/NASI polymers (either grafted with RGD peptide or not) expressed significantly higher levels of ALP activity than the cells cultured on TCPS, indicating that the polymer surfaces induced some osteoblastic activity in C2C12 cells without the need for BMP-2. We conclude that NiPAM-based thermoreversible biomaterials, despite their limited ability to support cell growth, allowed an enhanced expression of the chosen osteogenic marker (ALP) by C2C12 cells in vitro.
Keywords: Thermoreversible polymers; NiPAM; RGD; Cell attachment; BMP-2;

In this study, porcine bone–anterior cruciate ligament–bone (B–ACL–B) grafts were decellularized using one of three protocols incorporating surfactants lauryl sulfate (SDS), Triton X-100, and/or an organic solvent (tributyl phosphate (TnBP)). The effectiveness of Triton–SDS, Triton–Triton or Triton–TnBP treatments in removing cellular materials was determined and possible changes in biochemical composition and mechanical properties due to each treatment were investigated. Treatment with Triton–SDS was most effective at removing cell nuclei and intracellular protein (vimentin) from the ACL but affected both the collagen and glycosaminoglycan (GAG) components of the extracellular matrix while increasing the tensile stiffness of the ligament. Triton–Triton was the least effective of the three treatments in terms of cellular extraction, but did not significantly change the mechanical and biochemical properties of the ACL. Triton–TnBP matched the level of decellularization achieved by Triton–SDS in terms of visible cell nuclei; however, the extraction of intracellular vimentin was less consistent. TnBP treatment also slightly decreased the collagen content of the ACL but did not alter its mechanical properties.Overall, all three decellularization treatments maintained adequate mechanical and biochemical properties of B–ACL–B grafts to justify the further investigation of all three decellularization protocols. The selection of a superior treatment will depend on future studies of the propensity of treated tissues for repopulation by host ACL fibroblasts and, ultimately, on any immunogenic and/or remodeling host response induced in vivo.
Keywords: Decellularized; Ligament; Anterior cruciate ligament; Allograft;

The effect of calcium phosphate surface deposit and the surface adsorption of the serum proteins, bovine serum albumin (BSA) and fibrinogen, on the corrosion resistance and electrochemical behavior of (cp)titanium in phosphate buffer saline solution (pH 7.4) was investigated at physiological temperature, 37 °C, using electrochemical impedance spectroscopy and dc electrochemical polarization techniques. The formation of calcium phosphate deposit on the Ti surface decreased both the corrosion rate at the open circuit potential (OCP) and the anodic reaction current in the high anodic potential range (>2.6 V). Addition of BSA significantly moved the OCP towards a more negative (cathodic) potential and inhibited the cathodic corrosion reaction, but did not significantly change the corrosion resistance at the OCP. Addition of fibrinogen showed a similar, but less pronounced effect than BSA. The possible mechanisms leading to these observed effects are discussed.
Keywords: Titanium; Calcium phosphate; Bovine serum albumin; Fibrinogen; Corrosion; Biocompatibility; Electrochemistry;

The human macrophage response during differentiation and biodegradation on polycarbonate-based polyurethanes: Dependence on hard segment chemistry by Rosalind S. Labow; Danne Sa; Loren A. Matheson; Donna Lee M. Dinnes; J. Paul Santerre (7357-7366).
Human monocytes, isolated from whole blood, were seeded onto tissue culture grade polystyrene (PS) and three polycarbonate-based polyurethanes (PCNUs) (synthesized with either 1,6-hexane diisocyanate (HDI) or 4,4′-methylene bis-phenyl diisocyanate (MDI), poly(1,6-hexyl 1,2-ethyl carbonate) diol (PCN) and 1,4-butanediol (BD) in different stoichiometric ratios (HDI:PCN:BD 4:3:1 or 3:2:1 and MDI:PCN:BD 3:2:1) (referred to as HDI431, HDI321 and MDI321, respectively). Following their differentiation to monocyte-derived macrophages (MDMs) the cells were trypsinized and reseeded onto each of the PCNUs synthesized with either 14C-HDI or 14C-BD and degradation was measured by radiolabel release (RR). When the differentiation surface was MDI321, there was more RR from 14C-HDI431 than from any other surface ( p < 0.0001 ) whereas the amount of esterase (identified by immunoblotting) as well as the esterase activity was the greatest in MDM differentiated on PS, reseeded on 14C-HDI431 ( p < 0.0001 ). The effect of potential degradation products (methylene dianiline (MDA) and BD) from the PCNUs was carried out to determine possible links between products and substrate-induced activation of MDM. MDA was found to inhibit RR 60% from MDM seeded on 14C-MDI321B ( p < 0.0001 ), ∼20% from 14C-HDI431 ( p = 0.002 ) and no effect from 14C-HDI321B. MDA inhibited esterase activity 30% from MDM only on 14C-MDI321B ( p = 0.003 ), but no effect on esterase activity was observed for the other two polymers. BD had no inhibitory effect on RR from any PCNU, but did inhibit esterase activity in MDM on 14C-HDI431 ( p = 0.025 ). This study indicates that the degradation of a specific material is a multi-factorial process, dictated by its susceptibility to hydrolysis, the effect of specific products generated during this course of action, and perhaps not as well appreciated, the material's inherent ability to influence enzyme synthesis and release.
Keywords: Macrophages; Polyurethanes; Enzymes; Hard segment; Biodegradation;

In previous work, it had been shown that platelet adhesion could be reduced by fluorinating surfaces with oligomeric fluoropolymers, referred to as surface-modifying macromolecules (SMMs). In the current study, two in vitro blood-contacting experiments were carried out on a polyetherurethane modified with three different SMMs in order to determine if altered platelet adhesion levels could be related to the pattern of adsorbed protein and more specifically to the manner in which fibrinogen (Fg) distribution occurs at the surface. In the first experiment, the materials were placed in whole human blood and the adherent platelets were viewed with high-resolution scanning electron microscopy (SEM). In a second experiment, the materials were incubated with human plasma with the absence of platelets. The plasma contained 5% fluorescent-Fg. The materials were then viewed with a fluorescence microscope and images were collected to define the distribution of high-density fluorescent-Fg areas. The SEM and fluorescent-Fg images were imported to Image Pro Plus™ imaging software to measure the area, length and circularity and a bivariate correlation test was conducted between the two sets of data. For area and length morphology parameters, there were high and significant correlations ( r > 0.9 , p < 0.05 ) between the platelets and Fg aggregates. The data suggest that the Fg distribution may serve as a predictor of platelet morphology/activation and provides insight into the non-thrombogenic character of biomaterials containing the fluorinated SMMs.
Keywords: Polyetherurethane; Fluoropolymers; Surface modification; Platelets morphology; Fibrinogen adsorption;

Cardiomyocytes are terminally differentiated cells and therefore unable to regenerate heart tissue after infarction. The successful engraftment of various cell types resulting in improved cardiac function has been reported, however methods for improving the delivery of donor cells to the infarct site still need to be developed. The use of bioengineered cardiac grafts has been suggested to replace infarcted myocardium and enhance cardiac function. In this study, we cultured embryonic stem (ES) cell-derived cardiomyocytes on thin polyurethane (PU) films. The films were coated with gelatin, laminin or collagen IV in order to encourage cell adhesion. Constructs were examined for 30 days after seeding. Cells cultured on laminin and collagen IV, exhibited preferential attachment, as assessed by cellular counts, and viability assays. These surfaces also resulted in a greater number of contracting films compared to controls. A degradable elastomer seeded with embryonic stem cell-derived cardiomyocytes may hold potential for the repair of damaged heart tissue.
Keywords: Embryonic stem cells; Cardiomyocytes; Biodegradable polyurethane; ECM; Cardiac tissue engineering;

Evaluation of biodegradable synthetic scaffold coated on arterial prostheses implanted in rat subcutaneous tissue by Zhaoxu Wang; Shengguo Wang; Yves Marois; Robert Guidoin; Ze Zhang (7387-7401).
Polyester arterial prostheses impregnated with various synthetic biodegradable materials and with gelatin were implanted subcutaneously in rats for 3–180 days. The inflammation was assessed by quantifying the activity of alkaline phosphatase and by histology. The degradation of the scaffold materials was determined by scanning electron microscopy (SEM), size exclusion chromatography (SEC), and differential scanning calorimetry (DSC). The alkaline phosphatase activity induced by the polymer-impregnated grafts was similar to that induced by the non-impregnated controls during most of the post-implantation periods. Histological studies revealed that the acute inflammatory response was moderate to mild and was similar for all types of specimens, except for the gelatin-impregnated grafts that induced a severe acute inflammation during the first 2 weeks post-implantation. At 4 and 6 months, significant disintegration of the scaffold was observed, accompanied by enhanced tissue infiltration and a reactivation of the acute inflammatory phase. Linear and exponential degradation rates of the synthetic polymers were described. The relative degradation rates of the biodegradable polymers were ranked as following: PLLACL>PDLLA>PLLA>PCEL. In conclusion, biodegradable polymers may provide an option as sealant/scaffolding materials for vascular prosthesis. It is suggested that the degradation rate of the polymer scaffolding materials should be higher to achieve early healing while without inducing strong inflammation.
Keywords: Biodegradable polymers; Vascular prostheses; Scaffold; Sealant; Implant;

Improving arterial prosthesis neo-endothelialization: Application of a proactive VEGF construct onto PTFE surfaces by M. Crombez; P. Chevallier; R.C. -Gaudreault; E. Petitclerc; D. Mantovani; G. Laroche (7402-7409).
The formation of a confluent endothelium on expanded polytetrafluoroethylene (PTFE) vascular prostheses has never been observed. This lack of endothelialization is known to be one of the main reasons leading to the development of thromboses and/or intimal hyperplasia. In this context, several efforts were put forward to promote endothelial cell coverage on the internal surface of synthetic vascular prostheses. The goal of the present study was to immobilize the vascular endothelial growth factor (VEGF) onto Teflon® PTFE surfaces to generate a proactive polymer construct favoring interaction with endothelial cells. An ammonia plasma treatment was first used to graft amino groups on PTFE films. Subsequent reactions were performed to covalently bind human serum albumin (HSA) on the polymer surface and to load this protein with negative charges, which allows adsorbtion of VEGF onto HSA via strong electrostatic interactions. X-ray photoelectron spectroscopy (XPS) experiments along with surface derivatization strategies were performed between each synthesis step to ascertain the occurrence of the various molecules surface immobilization. Finally, the electrostatic binding of VEGF to the negatively charged HSA matrix was performed and validated by ELISA. Endothelial cell adhesion and migration experiments were carried out to validate the potential of this VEGF-containing biological construct to act as a proactive media toward the development of endothelial cells.
Keywords: Vascular endothelial growth factor; Surface modification; Endothelial cells; Plasma treatment;

Biological performances of collagen-based scaffolds for vascular tissue engineering by F. Boccafoschi; J. Habermehl; S. Vesentini; D. Mantovani (7410-7417).
 Collagen is widely used for biomedical applications and it could represent a valid alternative scaffold material for vascular tissue engineering. In this work, reconstituted collagen films were prepared from neutralized acid-soluble solutions for subsequent haemocompatibility and cell viability performance assays. First, haemoglobin-free, thrombelastography and platelet adhesion tests were performed in order to investigate the blood contact performance. Secondly, specimens were seeded with endothelial cells and smooth muscle cells, and cell viability tests were carried out by MTT and SEM. Results show that neutralized acid-soluble type I collagen films do not enhance blood coagulation, do not alter normal viscoelastic properties of blood and slightly activate platelet adhesion and aggregation. Cell culture shows that the samples are adequate substrates to support the adhesion and proliferation of endothelial and smooth muscle cells.
Keywords: Collagen; Blood compatibility; Endothelial cells; Smooth muscle cells; Platelet adhesion;

Immobilization of heparin on a silicone surface through a heterobifunctional PEG spacer by Hong Chen; Yang Chen; Heather Sheardown; Michael A. Brook (7418-7424).
A novel method of immobilizing heparin on a silicone surface through a heterobifunctional PEG spacer was used yield well defined surfaces with highly active surface immobilized heparin and low non-specific protein adsorption. The heparin surface density achieved using this technique was 0.68 μg/cm2. Sessile drop water contact angles showed increased hydrophilicity of the silicone surface after PEG modification and a further decrease in the contact angles following the grafting of heparin. High specificity for ATIII with little fibrinogen adsorption was noted in plasma adsorption studies. This ATIII adsorption was mediated by the heparin layer, since surfaces modified with PEG only did not adsorb significant quantities of AT. The thrombin resistance of the heparin modified surfaces was demonstrably greater as measured by a chromogenic thrombin generation assay. The results suggest that the heterbifunctional PEG linker results in a high density of active heparin on the surfaces.
Keywords: Silicone; PEG spacer; Heparin; Thrombogenic; Fibrinogen; Antithrombin III;

Semi-synthetic collagen/poloxamine matrices for tissue engineering by Alejandro Sosnik; Michael V. Sefton (7425-7435).
Collagen-containing poloxamine hydrogels were produced with the aim of overcoming the low stiffness displayed by collagen gels that are not otherwise chemically crosslinked. Matrices were obtained by functionalization of a four-arm PEO-PPO block copolymer (poloxamine, Tetronic™) with methcrylate groups and subsequent free radical polymerization of water solutions of the modified polymer in the presence of collagen. The resulting matrices had a sharp increase in stiffness, when compared to pure collagen gels. For example, whereas collagen had a storage modulus (G′) around 70 Pa and a loss modulus (G″) of 10 Pa, a crosslinked collagen/poloxamine system containing 8.3% crosslinked poloxamine had G′ and G″ values of 7400 and 1000 Pa, respectively. HepG2 cells were seeded within the gels before the crosslinking and the viability levels estimated by AlamarBlue™ assay were between 65% and 91% for systems containing 0.04–0.09 wt% photoinitiator. HepG2 and endothelial cells also adhered to and spread on the surface of the collagen-containing specimens, suggesting their potential utility in tissue engineering.
Keywords: Collagen; Crosslinked poloxamine hydrogels; Rheology; Cytocompatibility; HepG2 cells; Endothelial cells;

Macroporous interconnected dextran scaffolds of controlled porosity for tissue-engineering applications by Stéphane G. Lévesque; Ryan M. Lim; Molly S. Shoichet (7436-7446).
Dextran hydrogels have been studied as drug delivery vehicles but not as scaffolds for tissue-engineering likely because previously synthesized dextran hydrogels had pores too small for cell penetration. Our goal was to create macroporous, interconnected dextran scaffolds. To this end, we took advantage of the liquid–liquid immiscibility of poly(ethylene glycol) and methacrylated dextran during radical crosslinking of the methacrylated moieties. By controlling the degree of methacrylate substitution on dextran, dextran molar mass and PEG concentration, macroporous hydrogels were created. The presence of PEG in solution had a significant effect on the final morphology of the dextran hydrogel leading to the formation of different types of structures, from microporous gel to macroporous gel-wall to a macroporous interconnected-beaded structure. A series of formulation diagrams were prepared which allowed us to determine which conditions led to the formation of macroporous interconnected-beaded scaffolds. Dextran macroporous interconnected-beaded gels had a high water content, between 89% and 94%, a homogeneous morphology, determined by scanning electron microscopy, with interconnected macroporous pores, as determined by protein diffusivity where the effective diffusion coefficients of BSA were calculated to be 3.1×10−7  cm2/s for Dex-MA 40 kDa DS 5 and 1×10−7  cm2/s for Dex-MA 6 kDa DS10, which are similar to that of BSA in water, 5.9×10−7  cm2/s. Mercury intrusion porosimetry showed that the macroporous interconnected-beaded scaffolds had a bimodal distribution of macropores, with a median diameter of 41 μm, interconnected by throats, which had a median diameter of 11 μm. Together, these data suggest that the dextran scaffolds will be advantageous in applications that require an interconnected macroporous geometry, such as those of tissue engineering where cell penetration and nutrient diffusion are necessary for tissue regeneration.
Keywords: Hydrogel; Macroporous structure; Methacrylated dextran; Phase diagram; Biomaterials;

A PLGA membrane controlling cell behaviour for promoting tissue regeneration by G. Rh. Owen; J. Jackson; B. Chehroudi; H. Burt; D.M. Brunette (7447-7456).
Barrier membranes are used in periodontal applications with the aim of supporting bone regeneration by physically blocking migrating epithelial cells. We report a membrane design that has a surface topography that can inhibit epithelial cell migration and proliferation on one side and a topography that guides osteoblast migration to a desired area. A PLGA copolymer (85:15) blended with MePEG, was cast to have surfaces with smooth, grooved or sandblasted-acid-etched topographies. Epithelial cells spread on smooth surfaces after 24 h, and cell numbers increased after 5 days. Cells on the smooth surface exhibited no preferred direction of migration. On the sandblasted-acid-etched topography epithelial cells spread but the cell number did not significantly increase after 5 days. Cell migration was inhibited on this surface. Osteoblasts spread on both grooved and smooth surfaces and cell number increased after 5 days on all surfaces. The cells that adhered in the grooves migrated preferentially in the direction of the grooves. Positive alkaline phosphatase staining was seen on all surfaces within 4 weeks and positive Von Kossa nodule staining within 6 weeks. These results suggest that surface topographies replicated on opposite sides of a biodegradable polymers membrane can inhibit proliferation and migration of the epithelial cells, and promote proliferation and directional migration of osteoblasts. Addition of appropriate surface topographies to membranes used in guided tissue regeneration has the possibility of improving clinical performance in periodontal tissue regeneration procedures.
Keywords: Polglycolic acid; Polylactic acid; Epithelial cell; Osteoblast; Cell culture; Surface topography;

After almost half a century of use in the health field, polyurethanes (PUs) remain one of the most popular group of biomaterials applied for medical devices. Their popularity has been sustained as a direct result of their segmented block copolymeric character, which endows them with a wide range of versatility in terms of tailoring their physical properties, blood and tissue compatibility, and more recently their biodegradation character. While they became recognized in the 1970s and 1980s as the blood contacting material of choice in a wide range of cardiovascular devices their application in long-term implants fell under scrutiny with the failure of pacemaker leads and breast implant coatings containing PUs in the late 1980s. During the next decade PUs became extensively researched for their relative sensitivity to biodegradation and the desire to further understand the biological mechanisms for in vivo biodegradation. The advent of molecular biology into mainstream biomedical engineering permitted the probing of molecular pathways leading to the biodegradation of these materials. Knowledge gained throughout the 1990s has not only yielded novel PUs that contribute to the enhancement of biostability for in vivo long-term applications, but has also been translated to form a new class of bioresorbable materials with all the versatility of PUs in terms of physical properties but now with a more integrative nature in terms of biocompatibility. The current review will briefly survey the literature, which initially identified the problem of PU degradation in vivo and the subsequent studies that have led to the field's further understanding of the biological processes mediating the breakdown. An overview of research emerging on PUs sought for use in combination (drug+polymer) products and tissue regeneration applications will then be presented.
Keywords: Degradation; Biodegradation; Polyurethanes; Oxidation; Hydrolysis; Enzymes; Implants; Polymers; Macrophages; Bioresorbable; Biostability; Tissue regeneration; Scaffolds;

Characterization and evaluation of whey protein-based biofilms as substrates for in vitro cell cultures by Vanessa Gilbert; Mahmoud Rouabhia; Hongxum Wang; Anne-Lise Arnould; Gabriel Remondetto; Muriel Subirade (7471-7480).
Whey proteins-based biofilms were prepared using different plasticizers in order to obtain a biomaterial for the human keratinocytes and fibroblasts in vitro culture. The film properties were evaluated by Fourier Transform Infrared Spectroscopy (FTIR) technique and mechanical tests. A relationship was found between the decrease of intermolecular hydrogen bond strength and film mechanical behavior changes, expressed by a breaking stress and Young modulus values diminishing. These results allow stating that the film molecular configuration could induce dissimilarities in its mechanical properties. The films toxicity was assessed by evaluating the cutaneous cells adherence, growth, proliferation and structural stratification. Microscopic observation demonstrated that both keratinocytes and fibroblasts adhered to the biofilms. The trypan blue exclusion test showed that keratinocytes grew at a significantly high rate on all the biofilms. Structural analysis demonstrated that keratinocytes stratified when cultured on the whey protein-based biofilms and gave rise to multi-layered epidermal structures. The most organized epidermis was obtained with whey protein isolate/DEG biofilm. This structure had a well-organized basal layer under supra-basal and corneous layers. This study demonstrated that whey proteins, an inexpensive renewable resource which can be obtained readily, were non-toxic to cutaneous cells and thus they could be useful substrates for a variety of biomedical applications, including tissue engineering.
Keywords: Whey proteins; Biofilms; Cell culture;

This paper reviews reports on three-dimensional mammalian tissue growth in bioreactors and the corresponding mammalian tissue growth requirements. The needs for nutrient and waste removal of several mammalian tissues are reviewed and compared with the environment of many reactors currently in use such as the continuous stirred tank, the hollow fiber, the Couette–Taylor, the airlift, and the rotating-wall reactors developed by NASA. Many studies conclude that oxygen supply appears to be one of the most important factors limiting tissue growth. Various correlations to describe oxygen mass transfer are presented and discussed with the aim to provide some guidance to design, construct, and test reactors for tissue mass culture. To obtain tissue thickness clinically valuable, dimensionless and other types of analysis tend to point out that diffusive transport will have to be matched with an important convection to bring sufficient oxygen molecular flux to the growing cells located within a tissue mass. As learned from solid-state fermentation and hairy root culture, during the growth of large biomass, heterogeneity (i.e., channeling, temperature gradients, non-uniform cell growth, transfer gradients, etc.) can cause some important problems and these should be addressed in tissue engineering as well. Reactors (along with the scaffolds) should be designed to minimize these issues. The role of the uterus, the reactor built by Nature, is examined, and the environment provided to a growing embryo is reported, yielding possible paths for further reactor developments. Finally, the importance of cell seeding methods is also addressed.
Keywords: Bioreactors; Tissue engineering; Oxygenation; Organ culture; 3-D growth; Cell culture;

Mechanical testing methodologies developed for engineering materials may result in artifactual material properties if applied to soft planar connective tissues. The use of uniaxial tissue samples with high aspect ratios or biaxial samples with slender cruciform arms could lead to preferential loading of only the discrete subset of extracellular fibres that fully extend between the grips. To test this hypothesis, cruciform biaxial connective tissue samples that display distinctly different material properties (bovine pericardium, fish skin), as well as model textile laminates with predefined fibrous orientations, were repeatedly tested with decreasing sample arm lengths. With mechanical properties determined at the sample centre, results demonstrated that the materials appeared to become stiffer and less extensible with less slender sample geometries, suggesting that fibre recruitment increases with decreasing sample arm length. Alterations in the observed shear behaviour and rigid body rotation were also noted. The only truly reliable method to determine material properties is through in vivo testing, but this is not always convenient and is typically experimentally demanding. For the in vitro determination of the biaxial material properties, appropriate sample geometry should be employed in which all of the fibres contribute to the mechanical response.
Keywords: Biaxial; Mechanical testing; Sample geometry; Pericardium; Skin; Artifacts;

Rapid three-dimensional biointerfaces for real-time immunoassay using hIL-18BPa as a model antigen by Shawn D. Carrigan; George Scott; Maryam Tabrizian (7514-7523).
With the goal of designing a rapid and affordable system of real-time immune monitoring for future diagnostic applications in sepsis, we have developed a biointerface composed of polyethyleneimine (PEI) and carboxymethylcellulose (CMC) to provide a means of prompt and facile immunoassay. Biointerface assembly is complete within 30 min, with all preparation performed and monitored within the measurement chamber of a quartz crystal microgravimetry with dissipation (QCM-D) sensor. Optimised biointerface composition, as determined by the mass of antibody immobilised, the level of antigen detection, and the amount of non-specific binding of human serum albumin, was determined to consist of a 4.0 mg/mL CMC hydrogel layer cross-linked to a 0.5 mg/mL PEI sub-layer. Tapping mode atomic force microscopy (AFM) in liquid demonstrates highly uniform and smooth surfaces using these hydrogels. Sensitivity of the biointerface for rhIL-18BPa is 400 ng/mL, with detection of 1 μg/mL achievable following 25 surface regenerations. Performance of the biointerface is verified using surface plasmon resonance (SPR), demonstrating the ability of the biointerface to be applied across platforms.
Keywords: QCM; SPR; AFM; Immunoassay; Real-time; Sepsis;