Biomaterials (v.26, #5)

Calendar (I).

In vivo evaluation of tetrahedral amorphous carbon by David A. LaVan; Robert F. Padera; Thomas A. Friedmann; John P. Sullivan; Robert Langer; Daniel S. Kohane (465-473).
The in vivo behavior and tissue reaction to tetrahedral amorphous carbon (ta-C) has been evaluated for periods of up to 6 months in SV129 mice. Two sample types were tested—silicon die coated with ta-C (n=53) and micromachined particles (n=40). The coated samples were compared to uncoated silicon die (n=22). Die samples were implanted subcutaneously, and tissue reaction and capsule formation were evaluated at various time points. Micromachined particles of 1, 3, 10, and 30 μm were injected adjacent to the sciatic nerve, and tissue samples were examined histologically at various time points (4 days–6 months).Tissue reaction to ta-C was mild and was localized to the area of the injection or implantation. Samples with a higher ratio of 3-fold bonding appeared to shed material during the experiments; this was not observed on samples with a higher level of 4-fold bonding, nor on uncoated silicon die. The results strongly suggest that films with greater 4-fold bonding character (more diamond-like) are more resistant to in vivo fragmentation than films with higher 3-fold character (more graphitic).
Keywords: Biocompatibility; Amorphous diamond; Amorphous carbon; Thin film; In vivo testing;

The influence of the phosphorus content on the bioactivity of sol–gel glass ceramics by S Padilla; J Román; A Carenas; M Vallet-Regı́ (475-483).
The aim of this work was to study the influence of the phosphorus on the crystallization and bioactivity of glass-ceramics obtained from sol–gel glasses. For this purpose two sol–gel glasses with a similar composition but one of them containing P2O5 (70% SiO2; 30% CaO and 70% SiO2; 26% CaO; 4% P2O5, mol%) were prepared. Pieces of these glasses were treated at temperatures ranging between 700°C and 1400°C for 3 h. The obtained materials were characterized by XRD, FTIR, SEM-EDS and the biaxial flexural strength was determined in samples heated at 1100°C. In addition, an in vitro bioactivity study in simulated body fluid (SBF) was carried out.The results showed that phosphorus plays an important role in the crystallization of the glasses: it induced the crystallization of calcium phosphate phases, the stabilization of the wollastonite phase at high temperature as well as the crystallization of SiO2 phases at low temperatures. Moreover, the presence of phosphorus produced a heterogeneous distribution of defects in the pieces and, therefore, the flexural strength of samples containing this element decreased. Finally, glass-ceramics obtained from glasses containing phosphorus showed the fastest formation rate of the apatite layer when soaked in SBF.
Keywords: Glass-ceramics; Sol–gel glasses; Bioactivity; In vitro test; Calcium silicate;

In vitro cytocompatibility of MG63 cells on chitosan-organosiloxane hybrid membranes by Yuki Shirosaki; Kanji Tsuru; Satoshi Hayakawa; Akiyoshi Osaka; Maria Ascensão Lopes; José Domingos Santos; Maria Helena Fernandes (485-493).
Chitosan-silicate hybrids were synthesized using γ-glycidoxypropyltrimethoxysilane (GPSM) as the agent for cross-linking the chitosan chains. CaCl2 was introduced in the hybrids in expectation that it would improve cell adhesion and differentiation of the hybrid surfaces. Fourier-transform infrared (FT–IR) spectroscopy and 29Si CP-MAS NMR spectroscopy were used to analyze the structures of the hybrids. Cytocompatibility of the hybrids was investigated in terms of proliferation of an osteoblastic cell line, MG63. The adhesion and proliferation of the osteoblastic cells cultured on the surface of a chitosan-GPSM hybrid without calcium were similar to those on a control culture plate, and were better than those on a chitosan membrane. The ALP activity of the cells cultured on this hybrid was higher than that on the chitosan membrane. Contrary to expectations, the incorporation of calcium ions into the hybrids did not improve cell attachment and proliferation on their surfaces.
Keywords: Chitosan; γ-Glycidoxypropyltrimethoxysilane (GPSM); Organic–inorganic hybrid; Cytocompatibility; MG63 osteoblastic cells;

Multiphoton autofluorescence imaging of intratissue elastic fibers by K. König; K. Schenke-Layland; I. Riemann; U.A. Stock (495-500).
Multiphoton induced blue/green autofluorescence by near infrared femtosecond laser pulses has been used to selectively image intratissue elastic fibers in native and tissue engineered (TE) viable heart valves without any invasive tissue removal, embedding, fixation, and staining. Elastic fibers could be clearly distinguished from collagenous structures which emit ultraviolet/violet radiation when excited with intense ultrashort pulses due to second harmonic generation. Deep-tissue three-dimensional imaging of elastic fibers with submicron spatial resolution was performed by optical sectioning of heart valves using a multiphoton laser scanning microscope in connection with a tunable 80 MHz femtosecond laser source. The technology was used to diagnose extracellular matrix structures and cell resettlement of TE heart valves prior implantation. This novel non-invasive method opens the general possibility of high-resolution in situ imaging of elastic fibers, collagen structures and intracellular organelles in living intact tissues without staining.
Keywords: Cardiac tissue engineering; Extracellular matrix (ECM); Elastin; Heart valve; Near infrared (NIR) multiphoton laser microscopy; Second harmonic generation (SHG);

An asymmetric viologen, N-hexyl-N′-(4-vinylbenzyl)-4,4′-bipyridinium bromide chloride (HVV), was synthesized and graft copolymerized with commercial PET films. The surface graft concentration of HVV on the PET film is easily controlled by varying the monomer concentration used in the UV-induced graft copolymerization process. The HVV surface functionalized PET film functions as a smart window whose transmittance is reduced upon exposure to light. Concomitantly, the film possesses antibacterial activity, as shown by its bactericidal effect on Escherichia coli (E. coli). The antibacterial activity depends on the concentration of pyridinium groups on the surface and a surface concentration of 25 nmol/cm2 on PET has been shown to be highly effective in killing the bacteria.
Keywords: Antibacterial; Surface modification; Polyethylene terephthalate; Graft copolymerization; Viologen;

Improvement in biocompatibility of ZrO2–Al2O3 nano-composite by addition of HA by Young-Min Kong; Chang-Jun Bae; Su-Hee Lee; Hae-Won Kim; Hyoun-Ee Kim (509-517).
The biocompatibility of zirconia–alumina (ZA) nano-composites in load-bearing applications such as dental/orthopedic implants was significantly enhanced by the addition of bioactive HA. The ZA matrix was composed of nano-composite powder obtained from the Pechini process and had higher flexural strength than conventionally mixed zirconia–alumina composite. Because the ZA nano-composite powder effectively decreased the contact area between HA and zirconia for their reaction during the sintering process, the HA-added ZA nano-composites contained biphasic calcium phosphates (BCP) of HA/TCP and had higher flexural strength than conventionally mixed ZA–HA composite. From the in vitro test with osteoblastic cell-lines, the proliferation and the differentiation (as expressed by the alkaline phosphatase activity) of the cellular response on the HA-added ZA nano-composites gradually increased as the amount of HA added increased. From the mechanical and biological evaluations of the HA-added ZA nano-composites, 30HA (30 vol% HA+70 vol% ZA) was found to be the optimal composition for load-bearing biological applications.
Keywords: Hydroxyapatite (HA); Zirconia–Alumina (ZA); Nano-composite; In vitro; Alkaline phosphatase;

In vivo cytokine secretion and NF-κB activation around titanium and copper implants by Felicia Suska; Christina Gretzer; Marco Esposito; Lena Emanuelsson; Ann Wennerberg; Pentti Tengvall; Peter Thomsen (519-527).
The early biological response at titanium (Ti), copper (Cu)-coated Ti and sham sites was evaluated in an in vivo rat model. Material surface chemical and topographical properties were characterized using Auger electron spectroscopy, energy dispersive X-ray spectroscopy and interferometry, respectively. The number of leukocytes, cell types and cell viability (release of lactate dehydrogenase) were determined in the implant-interface exudate. The contents of activated nuclear transcription factor NF-κB, interleukin-6 (IL-6) and interleukin-10 (IL-10) were determined by enzyme linked immunosorbent assay. An increase in the number of leukocytes, in particular, polymorphonuclear leukocytes, was observed between 12 and 48 h around Cu. A marked decrease of exudate cell viability was found around Cu after 48 h. The total amounts of activated NF-κB after 12 h was highest in Ti exudates whereas after 48 h the highest amount of NF-κB was detected around Cu. The levels of cytokine IL-6 were consistently high around Cu at both time periods. No differences in IL-10 contents were detected, irrespective of material/sham and time. The results show that materials with different toxicity grades (titanium with low and copper with high toxicity) exhibit early differences in the activation of NF-κB, extracellular expression and secretion of mediators, causing major differences in inflammatory cell accumulation and death in vivo.
Keywords: Titanium; Copper; Inflammation; Cytokine;

Bladder acellular matrix as a substrate for studying in vitro bladder smooth muscle–urothelial cell interactions by Allison L Brown; Tamara T Brook-Allred; Jennifer E Waddell; Jacinta White; Jerome A Werkmeister; John A.M Ramshaw; Dàrius J Bagli; Kimberly A Woodhouse (529-543).
The objective of this study was to evaluate the ability of bladder acellular matrix (BAM) to support the individual and combined growth of primary porcine bladder smooth muscle (SMC) and urothelial (UEC) cells. An in vitro co-culture system was devised to evaluate the effect of UEC on (i) SMC-mediated contraction of BAM discs, and (ii) SMC invasiveness into BAM. Cells were seeded onto BAM discs under 4 different culture conditions. Constructs were incubated for 1, 7, 14 and 28 days. Samples were then harvested for evaluation of matrix contraction.Immunohistochemistry (IHC) was utilized to examine cellular organization within the samples and conditioned media supernatants analyzed for net gelatinase activity. BAM contraction was significantly increased with co-culture. The same side co-culture configuration lead to a greater reduction in surface area than opposite side co-culture. IHC revealed enhanced SMC infiltration into BAM when co-culture was utilized. A significant increase in net gelatinase activity was also observed with the co-culture configuration. Enhanced infiltration and contractile ability of bladder SMCs with UEC co-culture may, in part, be due to an increase in gelatinase activity. The influence of bladder UECs on SMC behaviour in vitro indicates that BAM may contain some key inductive factors that serve to promote important bladder cell–cell and cell–matrix interactions.
Keywords: Bladder tissue engineering; Extracellular matrix; Smooth muscle cell; Co-culture; Matrix metalloproteinase;

Effect of functionalization of multilayered polyelectrolyte films on motoneuron growth by Constant Vodouhê; Matthieu Schmittbuhl; Fouzia Boulmedais; Dominique Bagnard; Dominique Vautier; Pierre Schaaf; Christophe Egles; Jean-Claude Voegel; Joelle Ogier (545-554).
We studied in vitro cell–substrate interaction of motoneurons with functionalized polylectrolyte films. Thin polylectrolyte films were built on glass by alternating polycations, poly(ethylene-imine) PEI, poly(l-lysine) PLL, or poly(allylamine hydrochloride) PAH, and polyanions, poly(sodium-4-styrenesulfonate) PSS or poly(l-glutamic acid) (PGA). These architectures were functionalized with Brain Derived Neurotrophic Factor (BDNF) or Semaphorin 3A (Sema3A). We used Optical Waveguide Lightmode Spectroscopy (OWLS) and Atomic Force Microscopy (AFM) to characterize the architectures. The viability of motoneurons was estimated by the acid phosphatase method, and morphometrical measures were performed to analyse the influence of different architectures on cell morphology. Motoneurons appeared to adhere and spread on all the architectures tested and preferentially on PSS ending films. The viability of motoneurons on polyelectrolyte multilayers was higher compared to polyelectrolyte monolayers. BDNF and Sema3A embedded in the films remained active and thereby create functionalized nanofilms.
Keywords: Surface modification; Bioactivity; Growth factors; Nerve regeneration; Cell spreading;

A multifunctional bioreactor for three-dimensional cell (co)-culture by Artur Lichtenberg; Goekhan Dumlu; Thorsten Walles; Michael Maringka; Stefanie Ringes-Lichtenberg; Arjang Ruhparwar; Heike Mertsching; Axel Haverich (555-562).
Investigation of cell abilities to growth, proliferation and (de)-differentiation in a three-dimensional distribution is an important issue in biotechnological research. Here, we report the development of a new bioreactor for three-dimensional cell culture, which allows for co-cultivation of various cell types with different culture conditions in spatial separation. Preliminary results of neonatal rat cardiomyocyte cultivation are shown.Isolated neonatal rat cardiomyocytes were cultured in spatial separated bioreactor compartments in recirculating medium on a biodegradable fibrin matrix for 2 weeks. Glucose, lactate, and lactate dehydrogenase (LDH), pO2, pCO2, and pH levels were monitored in the recirculated medium, daily. Morphological characterization of matrix and cells was assessed by hematoxylin and eosin staining, and MF-20 co-immunostaining with 4′,6-diamidino-2-phenylindole (DAPI). Cell viability was determined by LIVE/DEAD staining before cultivation and on day 3, 7, and 14.The optimized seeding density in the matrix was 2.0×107 cells retaining cellular proportions over the cell culture period. The bioreactor allows the maintenance of physiologic culture conditions with aerobic cell metabolism (low release of lactate, LDH), a high oxygen tension (pO2–183.7±18.4 mmHg) and physiological pH values (7.4±0.02) and a constant level of pCO2 (43.1±2.9) throughout the experimental course. The cell viability was sufficient after 2 weeks with 82±6.7% living cells. No significant differences were found between spatial separated bioreactor compartments.Our novel multifunctional bioreactor allows for a three-dimensional culture of cells with spatial separation of the co-cultured cell groups. In preliminary experiments, it provided favorable conditions for the three-dimensional cultivation of cardiomyocytes.
Keywords: Bioreactor; Co-culture; Cardiomyocyte; Cell viability;

Osteoblast alignment, elongation and migration on grooved polystyrene surfaces patterned by Langmuir–Blodgett lithography by Steven Lenhert; Marie-Beatrice Meier; Ulrich Meyer; Lifeng Chi; Hans Peter Wiesmann (563-570).
Topographically patterned surfaces are known to influence cellular behavior in a controllable manner. However, the relatively large surface areas (several cm2) required for many biomaterial applications are beyond the practical limits of traditional lithography. Langmuir–Blodgett lithography, a recently developed method, was used to fabricate regularly spaced grooves of different depths (50 and 150 nm) with a periodicity of 500 nm over several square centimeter on silicon surfaces. These topographies were transferred into polystyrene surfaces by means of nanoimprinting. Primary osteoblasts were cultured on the patterned polymer surfaces. They were observed to align, elongate and migrate parallel to the grooves. The combination of Langmuir–Blodgett lithography with nanoimprinting enables the fabrication of large, nanostructured surface areas on a wide spectrum of different biomaterials. Osteoblasts show a significant anisotropic behavior to these surfaces, which can enhance cell settlement on the surface or be used to direct tissue generation on the biomaterial interface.
Keywords: Osteoblast; Bone tissue engineering; Interface; Langmuir–Blodgett lithography; Nanoimprinting; Nanotopography;

In this study the formulation and rheological characterisation of novel candidate ophthalmic viscosurgical devices (OVD) based on binary interactive polymer gels is described. Primary systems containing either hydroxyethylcellulose (HEC) or sodium carboxymethylcellulose (NaCMC) or binary interactive gels composed of HEC and NaCMC were manufactured. Rheological characterisation was performed using texture profile analysis and oscillatory rheometry. All formulations exhibited pseudoplastic flow. Systems composed of HEC or HEC and NaCMC behaved as gels (G′>G′′) over the range of oscillatory frequencies whereas systems composed of NaCMC were primarily elastoviscous. Increasing the polymer concentration in all systems increased the compressional rheological properties (hardness, compressibility), zero frequency viscosity (derived from the Cross model) and the viscoelastic properties (G′, G′′ and η′). Rheological synergy was observed in the binary gels and was indicative of interaction between the parent polymers. Importantly, the range of rheological properties offered by the binary mixtures was greater than those exhibited by the primary systems. The binary systems described in this study possessed viscoelastic properties and steady–state viscosities that were similar to commercially available systems and would therefore be appropriate for the maintenance of the ocular space. The acceptable compressional rheological and pseudoplastic properties of these systems would facilitate administration into the eye using a syringe. Additionally and uniquely, the excellent adhesive properties of the binary interactive gels would suggest an ability to interact with the corneal endothelium that would offer protection during phacoemulsification. Based on the described rheological properties it is suggested that binary gels composed of mass ratios of HEC to NaCMC of either 3.6: 2.4 or 2.4: 3.6 would be acceptable as OVD and would uniquely offer duality of function.
Keywords: Viscoelastic; Primary/binary gels; Bioadhesive; Pseudoplastic; Dynamic analysis; Texture profile analysis;