Biomaterials (v.27, #2)

Calendar (I).

Microbial cellulose—the natural power to heal wounds by Wojciech Czaja; Alina Krystynowicz; Stanislaw Bielecki; R. Malcolm Brown (145-151).
Microbial cellulose (MC) synthesized in abundance by Acetobacter xylinum shows vast potential as a novel wound healing system. The high mechanical strength and remarkable physical properties result from the unique nanostructure of the never-dried membrane. This article attempts to briefly summarize the recent developments and applications of MC in the emerging field of novel wound dressings and skin substitutes. It considers the properties of the synthesized material, its clinical performance, as well as progress in the commercialization of MC for wound care products. Efficient and inexpensive fermentation techniques, not presently available, will be necessary to produce large quantities of the polymer.
Keywords: Microbial cellulose; Acetobacter; Wound healing; Wound dressing;

Gas foamed open porous biodegradable polymeric microspheres by Taek Kyoung Kim; Jun Jin Yoon; Doo Sung Lee; Tae Gwan Park (152-159).
Highly open porous biodegradable polymeric microspheres were fabricated for use as injectable scaffold microcarriers for cell delivery. A modified water-in-oil-in-water (W1/O/W2) double emulsion solvent evaporation method was employed for producing the microspheres. The incorporation of an effervescent salt, ammonium bicarbonate, in the primary W1 droplets spontaneously produced carbon dioxide and ammonia gas bubbles during the solvent evaporation process, which not only stabilized the primary emulsion, but also created well inter-connected pores in the resultant microspheres. The porous microspheres fabricated under various gas foaming conditions were characterized. The surface pores became as large as 20 μm in diameter with increasing the concentration of ammonium bicarbonate, being sufficient enough for cell infiltration and seeding. These porous scaffold microspheres could be potentially utilized for cultivating cells in a suspension manner and for delivering the seeded cells to the tissue defect site in an injectable manner.
Keywords: Biodegradable polymer; PLGA; Porous; Microspheres; Gas foaming; Polylactic acid; Scaffold; Biodegradation; Microcarrier;

Polyurethanes with radiopaque properties by Nirmala R. James; Juby Philip; A. Jayakrishnan (160-166).
An aliphatic, commercially available, medical grade polyurethane, Tecoflex 80 A was made radiopaque by coupling a 5-iodine-containing molecule, N-(2,6- diiodocarboxyphenyl)-3,4,5-triiodo benzamide (DCPTB) onto the polymer backbone. DCPTB was synthesized by coupling 4-amino-3,5-diiodobenzoic acid and 3,4,5-triiodobenzoic acid using dicyclohexyl carbodiimide. Radiopaque polyurethane thus obtained was characterized by IR, TGA, DSC and X-radiography. By optimizing the reaction conditions, it was possible to incorporate about 8% iodine in the polymer (wt/wt) to achieve radiopacity almost equivalent to that of a 2 mm thick aluminium wedge. However, the products differed from the starting polymer in thermal characteristics. The starting polymer showed two endothermic transitions, the first one due to glass transition of the soft segment and the second one due to disruption of the hard segments. After modification, the second transition shifted to a lower temperature, while the first transition remained unaltered. Also, the modified polymers showed reduced thermal stability compared to the starting polymer. These observations could be explained on the basis of the reduced extent of intermolecular hydrogen bonding among the hard segments of the end product. Radiopaque polyurethanes are expected to have significant advantage over their non-radiopaque counterparts in many medical and related applications.
Keywords: Polyurethane; Radiopacity; Molecular weight; DSC; Glass transition temperature;

Spontaneous growth of a laminin-apatite nano-composite in a metastable calcium phosphate solution by Ayako Oyane; Masaki Uchida; Kazuo Onuma; Atsuo Ito (167-175).
We have previously reported that a laminin-apatite composite layer is formed on an ethylene-vinyl alcohol copolymer (EVOH) in a laminin-containing calcium phosphate (LCP) solution. In this work, the stability of the LCP solution and growth process of the laminin-apatite composite layer have been investigated. Dynamic light scattering technique revealed that the LCP solution was stable for periods as long as 24 h; it did not induce homogeneous precipitation of laminin or calcium phosphates in the solution. Analysis of the EVOH surface and the LCP solution showed that the laminin-apatite composite layer was formed via coprecipitation of laminin and apatite on the EVOH plate, i.e., spontaneous growing of apatite and simultaneous immobilization of laminin molecules or laminin–calcium phosphate nano-complexes onto its surface. Transmission electron microscopy also revealed that the laminin molecules in the resulting composite layer were not localized or aggregated, but were dispersed on a nano-scale in the entire layer. Because of this nano-composite structure, a large number of laminin molecules were stably immobilized on the EVOH plate. This may be responsible for the excellent cell adhesion properties of this type of composite material.
Keywords: Laminin; Apatite; Composite; Ethylene-vinyl alcohol copolymer (EVOH); Calcium phosphate;

Functional compressive mechanics of a PVA/PVP nucleus pulposus replacement by Abhijeet Joshi; Garland Fussell; Jonathan Thomas; Andrew Hsuan; Anthony Lowman; Andrew Karduna; Ed Vresilovic; Michele Marcolongo (176-184).
Emerging techniques as an alternative to the current treatments of lower back pain include nucleus replacement by an artificial material, which aims to relieve pain and restore the normal spinal motion. The compressive mechanical behavior of the PVA/PVP hydrogel nucleus implant was assessed in the present study.PVA/PVP hydrogels were made with various PVP concentrations. The hydrogels were loaded statically under unconfined and confined conditions. Hydrogels were tested dynamically up to 10 million cycles for a compression fatigue. Also, hydrogel nucleus implants with a line-to-line fit, were implanted in the human cadaveric intervertebral discs (IVD) to determine the compressional behavior of the implanted discs.Hydrogel samples exhibited typical non-linear response under both unconfined and confined compressions. Properties of the confinement ring dictated the observed response. Hydrogel moduli and polymer content were not different pre- and post-fatigues. Slight geometrical changes (mostly recoverable) were observed post-fatigue. In cadavers, hydrogels restored the compressive stiffness of the denucleated disc when compared with equivalent condition of the IVD.The results of this study demonstrate that PVA/PVP hydrogels may be viable as nucleus pulposus implants. Further studies under complex loading conditions are warranted to better assess its potential as a replacement to the degenerated nucleus pulposus.
Keywords: Intervertebral disc; Hydrogel; Mechanical test; Spinal implant; Nucleus pulposus;

A “room-temperature” injection molding approach combined with particulate leaching (RTIM/PL) has been, for the first time, developed in this work to fabricate three-dimensional porous scaffolds composed of biodegradable polyesters for tissue engineering. In this approach, a “wet” composite of particulate/polymer/solvent was used in processing, and thus the injection was not performed at melting state. Appropriate viscosity and flowability were facilely obtained at a certain solvent content so that the composite was able to be injected into a mould under low pressure at room temperature, which was very beneficial for avoiding thermal degradation of polyesters. As a demonstration, tubular and ear-shaped porous scaffolds were fabricated from biodegradable poly(d,l-lactide-co-glycolide) (PLGA) by this technology. Porosities of the resulting scaffolds were as high as 94%. The pores were well interconnected. Besides the well-known characteristics of injection molding to be suitable for automatization of a fabrication process with high repeatability and precision, this RTIM/PL approach is much suitable for tailoring highly porous foams with its advantages flexible for shaping complicated scaffolds, free of thermal degradation and high-pressure machine, etc.
Keywords: Porous scaffold; Fabrication method; Injection molding; Poly(d,l-lactide-co-glycolide) (PLGA); Biodegradable polymer; Tubular scaffold;

In vitro and in vivo studies on blends of isotactic and atactic poly (3-hydroxybutyrate) for development of a dura substitute material by Carmen Kunze; Hans Edgar Bernd; René Androsch; Claudia Nischan; Thomas Freier; Sven Kramer; Burkhard Kramp; Klaus-Peter Schmitz (192-201).
Blends of semicrystalline isotactic poly(3-hydroxybutyrate) (PHB) with amorphous atactic PHB (at-PHB) were prepared by solution-casting using 30%, 50% and 70% at-PHB, and were studied for medical applications. The mechanical properties of the blends including the elastic modulus and elongation at break are strongly affected by the blend composition. The elastic modulus decreases with increasing fraction of at-PHB in the blend from 3350 MPa in the case of PHB to 170 MPa of PHB/at-PHB (30/70). In contrast, the elongation at break increases from 2% in pure PHB up to 50% in the case of the blend with 70% at-PHB. The in vitro degradation is changed as well. The molecular weight of PHB/at-PHB (30/70) is reduced to 5% after 2 years storage in phosphate buffer compared to 35% for pure PHB stored at identical conditions. The in vitro cell vitality is slightly reduced depending on the composition.PHB/at-PHB blends with 30% and 50% at-PHB were selected as dura substitute in minipigs based on the results of the in vitro investigation and the mechanical testing. Patch films with a structured surface on one side were fabricated by a dipping-leaching method. Dura defects were clinically and histologically examined 3, 6, and 9 months after implantation, confirming defect closure, prevention of adhesions to brain tissue, and no signs of inflammation or malignant degeneration. The PHB-based patch materials fulfill the requirements which are necessary for a dura substitute.
Keywords: Polyhydroxybutyric acid (PHB); Dura; Biocompatibility; Degradation;

Influence of copolymer composition of polylactide implants on cranial bone regeneration by Christoph Sebastian Leiggener; Raymond Curtis; Andreas A. Müller; Dominik Pfluger; Sylwester Gogolewski; Berton A. Rahn (202-207).
Biodegradable polymers have become useful auxiliary materials for the functional and structural restoration of bone deficiencies. Commercial implants from poly(l/dl-lactide) 70:30 are used clinically for fracture fixation in regions of low load. Implants manufactured from poly(l/dl-lactide) 80:20 are currently being investigated experimentally. The higher degree of crystallinity results in a higher chemical strength and loading capacity which promises advantages for long-term implantation. In this study implants from these two copolymers were applied to promote bone regeneration of bilateral, full thickness, circular cranial defects in 16 adult New Zealand white rabbits. The defects were covered with melt extruded and laser cut polylactide burr hole covers epicranially and endocranially in direct contact to the dura. The defect spaces were kept open with a spacer which created a hollow chamber. Both materials were implanted in each animal. Bone seeking fluorochromes were used to assess the pattern of bone growth. After eight weeks bone regeneration in the defects was assessed radiologically, histologically and by fluorescence microscopy. During the eight weeks observation period the application of a hollow chamber design resulted in almost complete cranial defect healing, whereby the copolymer composition had no effect on the amount or the morphology of the regenerate. The dura mater showed no adverse tissue reactions during these early stages of implantation. Eight weeks is only a short period in the lifetime of the tested polymers and complete bone regeneration can only be expected after complete polymer degradation. Long-term studies or accelerated degradation studies are required to confirm the expected advantages of poly(l/dl-lactide) 80:20.
Keywords: Bone regeneration; Copolymer; Dura; Osteoconduction; Polylactic acid;

Plasma coagulation response to surfaces with nanoscale chemical heterogeneity by Rachel Miller; Zhe Guo; Erwin A. Vogler; Christopher A. Siedlecki (208-215).
A mixed film bearing nanoscale domains of one chemical functionality surrounded by another chemical functionality is shown to prolong material-induced coagulation of whole human blood plasma. In comparison, surfaces with uniform silane chemistry or physical mixtures of control surfaces bearing two different, uniform silane chemistries are found to be much more efficient activators of plasma coagulation on a per-unit-area basis. Binary mixed films are deposited on glass substrates by the sequential adsorption of 0.0001% 3-aminopropyltriethoxysilane (APS) followed by 0.1% n-butyltrichlorosilane (BTS). Creation of APS islands in a sea of BTS is confirmed by atomic force microscopy in friction mode. Results suggest that some yet-to-be-determined interfacial phenomena, perhaps associated with protein adsorption near the interface, may be altered by this nanoscale spatially distributed chemical heterogeneity, causing a decrease in contact activation.
Keywords: Blood compatibility; Haemocompatibility; Coagulation; Self-assembly; Silane;

Porcine-derived xenogeneic bone (PDXB) was derived from cancellous bone of adult porcine. Its morphology and structure were characterized by SEM, FTIR and XRD. A series of composite films consisting of PDXB and poly(glycolide-co-lactide-co-caprolactone) (PGLC) polymer were prepared. Because of the introduction of PGLC polymer, the PDXB/PGLC composites especially PDXB/PGLC(30/70) and PDXB/PGLC(50/50) showed good processability and mechanical properties. In addition, the hydrophilicity of the composites was enhanced as well since the PDXB component was hydrophilic. Osteoblast-like cells (OCT-1) were used as an in-vitro model to assess the affinity of the PDXB/PGLC composites. It was found that compared with the pure PGLC film, PDXB/PGLC(30/70) and PDXB/PGLC(50/50) composite films promoted cell attachment, proliferation and ALP (alkaline phosphatase) activity obviously. In addition, the cells preferred growing on the areas of exposed PDXB. It was considered that the hydrophilicity, osteoconductivity and appropriate surface roughness ( S a = 3.30 , 4.00 μm) induced by PDXB facilitate cell growth. However, the introduction of too much PDXB, such as PDXB/PGLC(70/30) film, would obtain an adverse effect on the cell growth since the value of S a was up to 7.33 μm. It indicated that only the composites with appropriate surface topography could favor cell growth. Surface topography probably has a more important effect on cell growth process than surface chemistry.
Keywords: Porcine-derived xenogeneic bone; PGLC; Composite; Osteoblast-like cell; Cell affinity; Surface topography;

Characterization of the Flexcell™ Uniflex™ cyclic strain culture system with U937 macrophage-like cells by Loren A. Matheson; N. Jack Fairbank; Geoffrey N. Maksym; J. Paul Santerre; Rosalind S. Labow (226-233).
Mechanical forces alter many cell functions in a variety of cell types. It has been recognized that stimulation of cells in culture may be more representative of some physiologic conditions. Although there are commercially available systems for the study of cells cultured in a mechanical environment, very little has been documented on the validation techniques for these devices. In this study, Flexcell's™ recently introduced Uniflex™ cyclic strain system was programmed to apply 10% longitudinal sinusoidal strain (0.25 Hz) for 48 h to U937 cells cultured on Uniflex™ plates. Image analysis was employed to characterize the actual strain field. For a chosen amplitude of 10% the applied strain was highly reproducible and relatively uniform (10.6±0.2%) in a central rectangular region of the membrane (dimensions of 9.2±2×13.6±0.8 mm2). The strain increased the release of IL-6, esterase and acid phosphatase activity (p<0.05) from adherent U937 cells. Cells also displayed altered morphology, aligning and lengthening with the direction of strain, whereas static cells maintained a round appearance showing no preferred orientation. These data indicate that cyclic mechanical strain applied by the Uniflex™ strain system modulates U937 cell function leading to selective increases in enzymatic activities as well as orientation in a favored direction.
Keywords: Macrophage; Mechanical test; Esterase; Uniaxial strain; Siloxane;

The dependence of fibrillar adhesions in human fibroblasts on substratum chemistry by Nathalie Faucheux; Rumiana Tzoneva; Marie-Daniele Nagel; Thomas Groth (234-245).
Little is known about the influence of substratum properties and composition on the ability of cells to translocate α5β1 integrins and to form fibrillar adhesion. We have examined the impact of self-assembled monolayers (SAMs) bearing different functional end groups (amines (NH2) or carboxylic acids (COOH)) on the presence of fibrillar adhesions in human fibroblasts attached to fibronectin-coated SAMs. Most of the fibroblasts incubated in serum-free medium for 2 h on COOH showed segregation of focal contact components (αv integrin subunits, phosphotyrosine proteins) and fibrillar adhesions (α5 integrin subunits, tensin) while the majority of cells plated on NH2 did not. Analysis of fibronectin fibril formation confirmed also that human fibroblasts plated on COOH formed matrix fibrils significantly better. The surface-associated α5 to αv integrin ratio was smaller in cells on COOH than on NH2 due to a decreased α5 integrin binding. In addition, human fibroblasts migrated more readily on COOH in comparison to NH2 which points to a different binding strength of integrins to the substratum. Overall, the results indicate that the molecular composition of substrata has a strong influence on FN matrix formation by promoting or inhibiting segregation of focal and fibrillar adhesions.
Keywords: Focal adhesions; Fibrillar adhesions; Integrin; Fibronectin; Cell migration;

Platinum nanoparticle-doped chitosan (CHIT) solution can be easily prepared by treating the CHIT solution with aqueous H2PtCl6 solution followed by chemical reduction of Pt(IV) with NaBH4. Multiwalled carbon nanotubes (MWCNT) are then dispersed in the nanoparticle-doped solution. The resulting Pt-CNT-CHIT material brings new capabilities for electrochemical devices by using the synergistic action of Pt nanaoparticles and CNT. Positively charged Pt-CNT-CHIT solution and negatively charged poly(sodium-p-styrenesulfonate) salt (PSS) have been employed to fabricate stable ultrathin multilayer films on gold electrode and quartz glass slides in a layer-by-layer fashion. Cyclic voltammetric and UV–vis adsorption spectroscopy confirms the consecutive growth of the multilayer films. The modified gold electrode allows low-potential detection of hydrogen peroxide with high sensitivity and fast response time. With the immobilization of cholesterol oxidase onto the electrode surface using glutaric dialdehyde, a biosensor that responds sensitively to cholesterol has been constructed. In pH 6.98 phosphate buffer, almost interference free determination of cholesterol has been realized at 0.1 V vs. SCE with a linear range from 0.01 to 3 mM and response time<30 s. With the immobilization of another cholesterol esterase enzyme layer, the biosensor was used to determine total cholesterol in serum samples with satisfactory results.
Keywords: Biosensor; Chitosan; Electroactive polymer; Enzyme; Nanoparticle;

In vivo behavior of acrylic bone cement in total hip arthroplasty by Michael D. Ries; Ernest Young; Laila Al-Marashi; Philip Goldstein; Alexander Hetherington; Timothy Petrie; Lisa Pruitt (256-261).
Polymethylmethacrylate (PMMA) bone cement serves as the primary fixation material between bone and the prosthetic component in cemented total hip arthroplasty. In vivo degradation of bone cement may lead to a decrease in mechanical properties of PMMA and result in aseptic loosening. However, other factors such as porosity and location of the cement relative to the bone implant interface may also contribute to mechanical behavior in vivo. This study investigated the mechanical properties of Simplex® cement retrieved from 43 patients undergoing revision total hip arthroplasty. The time in vivo was between 1 month and 27 years. The variables studied included fracture toughness (K IC), porosity, molecular weight, time in vivo of the cement, and relative in vivo location of the cement with respect to the implant and bone. K IC did not correlate with time in vivo of the samples or with molecular weight. This suggests that time in vivo may not be the limiting factor in the mechanical integrity of the bone cement, A significant and inverse relationship was found between porosity and K IC. This implies that porosity is the most important factor in the mechanical behavior of bone cement during in vivo use.
Keywords: Bone cement; In vivo degradation; Fracture toughness; Porosity;

Paclitaxel is one of the best anticancer drugs, which has excellent therapeutic effects against a wide spectrum of cancers. The formulation of paclitaxel used in its currently clinical administration includes Cremophor EL, which has been found to cause serious side effects. Nanoparticle formulation of paclitaxel may provide an ideal solution for this problem and achieve a sustained chemotherapy. A novel copolymer, poly(lactide)-vitamin E TPGS (PLA-TPGS), was synthesized from lactide and d-α-tocopheryl polyethylene glycol 1000 succinate by bulk polymerization for nanoparticle formulation of anticancer drugs. 1H NMR, FTIR and GPC were used to detect molecular structure of the copolymer. Paclitaxel-loaded PLA-TPGS nanoparticles were fabricated by a modified solvent extraction/evaporation technique with or without emulsifier involved, which were characterized by laser light scattering for size and size distribution; field emission scanning electron microscopy for surface morphology; zeta potential for surface charge; X-ray photoelectron spectroscopy for surface chemistry. The drug encapsulation efficiency and the in vitro drug release kinetics were measured by high-performance liquid chromatography. Formulation optimization was pursued. The particles were found of around 300 nm in size and narrow size distribution. Of all, 89% drug encapsulation efficiency has been achieved for nanoparticles of 5% drug loading. The drug release from PLA-TPGS nanoparticles was found to be biphasic with an initial burst of 17% in the first day, followed by a sustained pattern with 51% release after 31 days.
Keywords: Biodegradable polymers; Cancer nanotechnology; Chemotherapy; Paclitaxel; Taxol®;

To increase topical delivery of low-molecular-weight heparin (LMWH), cationic, neutral, and anionic flexible liposomes (cFlexosome, nFlexosome, and aFlexosome) were prepared. The effects of surface charge of Flexosome on physicochemical properties and skin penetration of LMWH were also investigated. Among the different formulations of Flexosome, cFlexosome demonstrated three-times higher entrapment efficiency of LMWH, and better physicochemical stability than nFlexosome and aFlexosome. In vitro skin penetration and in vivo localization into the deeper skin layer of LMWH were significantly greater from cFlexosome compared to other formulations. Changes of skin surface charge after LMWH-cFlexosome application were investigated as a function of time. In the process of skin penetration, the Flexosomes act as drug carrier with the associated LMWH. Overall, macromolecular LMWH could be delivered deeply into the skin by topical application of cFlexosome for the treatment of superficial thrombosis, subcutaneous wounds, bruise, and burns.
Keywords: Low-molecular-weight heparin; Flexible liposome; Liposome surface charge; Topical delivery;

In vitro osteogenetic activity of pearl by Yutian Shen; Jing Zhu; Hongbiao Zhang; Fei Zhao (281-287).
In vivo and in vitro studies have shown that shell nacre and hydroxyapatite (HA) are promising bioactive materials for bone repair. In this work, the osteogenetic activity of pearl is evaluated by soaking it in simulated body fluid (SBF) and cell culture, taking shell nacre and HA as control materials at the same time. After soaking in SBF, HA particles were rapidly formed on the surface of pearl, the dissolution of CaCO3 and the binding between organic components and Ca2+ ions in pearl provide favorable conditions for the HA precipitation, and the whole process follows a dissolution−binding−precipitation mechanism. Calcium surplus, not conventional calcium deficiency, is found in HA crystal structure; it implies that type B-HA is formed on pearl surface in this study. HRTEM observation shows that HA is poorly crystallized with so many dislocations and shuttle-like amorphous areas. Cell culture reveals that pearl could stimulate osteoblast proliferation, which proceeded more quickly and smoothly than that on shell nacre and HA, and abundant extracellular matrix occupied the whole pearl surface by 5 days. It is concluded that pearl is a superior osteoinductive material with high osteogenetic activity.
Keywords: Pearl; Nacre; Hydroxyapatite; Simulated body fluids; OCT-1 osteoblast; Osteogenetic activity;