Biomaterials (v.27, #23)

Calendar (I).

Development of a soft tissue seal around bone-anchored transcutaneous amputation prostheses by Catherine J. Pendegrass; Allen E. Goodship; Gordon W. Blunn (4183-4191).
Conventional amputation prosthetics are problematic because they rely on the stump–socket interface for attachment. Intraosseous transcutaneous amputation prostheses (ITAP) could solve these problems; however they rely on the integrity of the soft tissue–implant interface as a barrier to exogenous agents, and in the prevention of downgrowth and marsupilisation. We have used an in vivo animal model to study the soft tissue interfaces around bone-anchored transcutaneous implants. We hypothesise that by facilitating and increasing the area of dermal attachment to the implant epithelial down-growth will be reduced. A flange with a series of 24, 0.7 mm holes positioned immediately below the epithelium was used to increase dermal attachment. This significantly reduced downgrowth and optimised the integrity of the collagenous tissue-implant interface at the dermal level. We postulate that the flange reduces relative interfacial movement at the epithelium–implant interface by providing increased surface area for dermal tissue attachment. A tight seal at the dermal tissue level reduces the degree of downgrowth around ITAP, eliminating marsupilisation as a potential failure modality. Surface topography and coatings did not affect the degree of downgrowth or dermal attachment to straight or flanged implants. A significant negative correlation was observed between downgrowth and both epithelial and dermal attachment.This study shows that a soft tissue–implant interface capable of preventing downgrowth and marsupilisation can develop around a bone-anchored transcutaneous implant, given the incorporation of a porous flange positioned in the dermal tissues immediately below the epithelium. This will benefit applications where bone-anchored transcutaneous implants are used.
Keywords: Intraosseous; Transcutaneous; Amputation; Prostheses;

Early bone apposition in vivo on plasma-sprayed and electrochemically deposited hydroxyapatite coatings on titanium alloy by Hao Wang; Noam Eliaz; Zhou Xiang; Hu-Ping Hsu; Myron Spector; Linn W. Hobbs (4192-4203).
Three different implants, bare Ti–6Al–4V alloy, Ti–6Al–4V alloy coated with plasma-sprayed hydroxyapatite (PSHA), and Ti–6Al–4V alloy coated with electrochemically deposited hydroxyapatite (EDHA), were implanted into canine trabecular bone for 6 h, 7, and 14 days, respectively. Environmental scanning electron microscopy study showed that PSHA coatings had higher bone apposition ratios than those exhibited by bare Ti–6Al–4V and EDHA coatings after 7 days; however, at 14 days after implantation, EDHA and PSHA coatings exhibited similar bone apposition ratios, much higher than that for bare Ti–6Al–4V. The ultrastructure of the bone/implant interface observed by transmission electron microscope showed that the earliest mineralization (6 h—7 days) was in the form of nano-ribbon cluster mineral deposits with a Ca/P atomic ratio lower than that of hydroxyapatite. Later-stage mineralization (7–14 days) resulted in bone-like tissue with the characteristic templating of self-assembled collagen fibrils by HA platelets. Though adhesion of EDHA coatings to Ti–6Al–4V substrate proved problematical and clearly needs to be addressed through appropriate manipulation of electrodepositon parameters, the finely textured microstructure of EDHA coatings appears to provide significant advantage for the integration of mineralized bone tissue into the coatings.
Keywords: Bone; Hydroxyapatite coatings; Plasma spraying; Titanium alloy; TEM;

The influence of processes for the purification of human bone allografts on the matrix surface and cytocompatibility by Aline Dumas; Christine Gaudin-Audrain; Guillaume Mabilleau; Phillipe Massin; Laurent Hubert; Michel F. Baslé; Daniel Chappard (4204-4211).
Different industrial processes exist to purify allogenic bone, providing safe and cleaned blocks for bone allografting. However, they often make use of chemical reagents that can be aggressive for the bone matrix. Bone samples were processed with several soaking techniques used in industry: NaHCO3, H2O2, NaOH and H2O2+NaOH combined; the consequences on the bone matrix and cytocompoatibility were evaluated on femoral heads from osteoarthritic patients. Alterations of matrix were searched by histochemistry, atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cytocompatibility was evaluated by coculturing human osteoblast-like cells (SaOS-2) on bone slices. Collagen fibers were dramatically altered at the surface of bone treated with H2O2, NaOH (and their association), but not with NaHCO3. A marked reduction in the number of hydroxyapatite crystals was observed on the trabecular surfaces by TEM and morphological changes were evidenced in SEM and AFM. Argyrophilic proteins of the bone matrix were removed by H2O2 and NaOH (and their association), but not by NaHCO3. As a consequence, attachment, spreading, proliferation and alkaline phosphatase activity of SaOS-2 were reduced by H2O2 and NaOH treatments. Strong oxidizing reagents altered matrix integrity by modifying collagenous and non-collagenous proteins. Whether these changes have clinical consequences on the bone bonding and osseointegration in human necessitate further investigations.
Keywords: Bone allograft; Bone bank; Purification process; Collagen; Cytocompatibility;

Previously we demonstrated that high throughput gene expression experiments can yield novel information about how cells respond to a collagen–glycosaminoglycan (GAG) three-dimensional culture environment. The goal of the current study was to determine which of these differences result from culture in a three-dimensional construct versus those caused simply by the presence of the collagen–GAG biomaterial. To make this distinction, cells were cultured both in collagen–GAG scaffolds fabricated using a phase separation method and on thin two-dimensional coatings of the same material. Control cells were grown on standard tissue culture polystyrene (TCPS). Cell response was measured using histology and microarray analysis and select results were verified with real time polymerase chain reaction (RT-PCR) assays. Genes involved in matrix remodeling (matrix components, matrix metalloproteinases and growth factors) and angiogenesis (VEGF, HGF and HMOX) were shown to be differentially expressed between the treatment conditions. Several matrix metalloproteinases (MMPs) were up regulated in mesh grown cell while some of their inhibitors (TIMPs) were down regulated. These results suggest that the three-dimensional presentation of the collagen–GAG material to the cells is required to stimulate the observed increase in fibroblast remodeling behavior.
Keywords: Collagen; Biocompatibility; PCR (polymerase chain reaction); mRNA; metalloproteinases; Glycosaminoglycan;

In vitro re-endothelialization of detergent decellularized heart valves under simulated physiological dynamic conditions by Artur Lichtenberg; Igor Tudorache; Serghei Cebotari; Stefanie Ringes-Lichtenberg; Gerrit Sturz; Klaus Hoeffler; Christof Hurscheler; Gudrun Brandes; Andres Hilfiker; Axel Haverich (4221-4229).
The production of viable biological heart valves is of central interest in tissue engineering (TE). The aim of this study was to generate decellularized heart valves with an intact ultra-structure and to repopulate these with endothelial cells (EC) under simulated physiological conditions. Decellularization of ovine pulmonary valve conduits was performed under agitation in detergents followed by six wash cycles. Viability of EC cultures exposed to washing solution served to prove efficiency of washing. Resulting scaffolds were free of cells with preserved extracellular matrix. Biomechanical standard tension tests demonstrated comparable parameters to native tissue. Luminal surfaces of decellularized valvular grafts were seeded with ovine jugular vein EC in dynamic bioreactors. After rolling culture for 48 h, pulsatile medium circulation with a flow of 0.1 L/min was started. The flow was incremented 0.3 L/min/day up to 2.0 L/min (cycle rate: 60 beats/min), while pH, pO2, pCO2, lactate and glucose were maintained at constant physiological levels. After 7 days, a monolayer of cells covered the inner valve surface, which expressed vWF, indicating an endothelial origin. A complete endothelialization of detergent decellularized scaffold can be achieved under simulated physiological circulation conditions using a dynamic bioreactor system, which allows continuous control of the culture environment.
Keywords: Cardiac tissue engineering; Extracellular matrix; Endothelialization;

The in vitro inhibition of multidrug resistance by combined nanoparticulate titanium dioxide and UV irradition by Min Song; Renyun Zhang; Yongyuan Dai; Feng Gao; Huimei Chi; Gang Lv; Baoan Chen; Xuemei Wang (4230-4238).
The appearance of drug-resistant (especially, multidrug-resistant (MDR)) tumor cells is a major obstacle to the success of chemotherapy; thus, the development of effective anti-MDR agents plays an important role in the tumor therapy. In this report, the considerable effect of nano-TiO2 and UV illumination on the drug resistance of target cancer cells has been explored, and the fresh evidence from the fluorescence spectroscopy and microscopy as well as electrochemical studies demonstrates the significant enhancement effect of nano-TiO2 to the drug uptake by drug-resistant leukemia cells. Besides, it is also observed that the combination of the nano-TiO2 and UV irradiation with the accompanying anticancer drug daunorubicin could provoke some considerable changes of the cell membrane of the target leukemia cells, which indicates that nano-TiO2 could not only increase the drug accumulation in target cancer cells, but also act as an effective anti-MDR agent to inhibit the relative drug resistance.
Keywords: Drug delivery; Electrochemistry; Confocal fluorescence; Titanium oxide; Drug resistance; Leukemia;

Controlled release of gentamicin from calcium phosphate—poly(lactic acid-co-glycolic acid) composite bone cement by Julia Schnieders; Uwe Gbureck; Roger Thull; Thomas Kissel (4239-4249).
Modification of a self setting bone cement with biodegradable microspheres to achieve controlled local release of antibiotics without compromising mechanical properties was investigated. Different biodegradable microsphere batches were prepared from poly(lactic-co-glycolic acid) (PLGA) using a spray-drying technique to encapsulate gentamicin crobefate varying PLGA composition and drug loading. Microsphere properties such as surface morphology, particle size and antibiotic drug release profiles were characterized. Microspheres were mixed with an apatitic calcium phosphate bone cement to generate an antibiotic drug delivery system for treatment of bone defects. All batches of cement/microsphere composites showed an unchanged compressive strength of 60 MPa and no increase in setting time. Antibiotic release increased with increasing drug loading of the microspheres up to 30% (w/w). Drug burst of gentamicin crobefate in the microspheres was abolished in cement/microsphere composites yielding nearly zero order release profiles. Modification of calcium phosphate cements using biodegradable microspheres proved to be an efficient drug delivery system allowing a broad range of 10–30% drug loading with uncompromised mechanical properties.
Keywords: Bone cement; Hydroxyapatite; Biodegradation; Microspheres; Antimicrobial; Drug delivery;

In vivo evaluation of an oral delivery system for P-gp substrates based on thiolated chitosan by Florian Föger; Thierry Schmitz; Andreas Bernkop-Schnürch (4250-4255).
Recently, thiolated polymers, so called thiomers, have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a proof-of-principle for a delivery system based on thiolated chitosan in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. In vitro, the permeation enhancing effect of unmodified chitosan, chitosan-4 thiobutylamidine (Ch-TBA) and the combination of Ch-TBA with reduced glutathione (GSH) was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to buffer only, Rho-123 transport in presence of 0.5% (w/v) chitosan, 0.5% (w/v) Ch-TBA and the combination of 0.5% (w/v) Ch-TBA/0.5% (w/v) GSH, was 1.8-fold, 2.6-fold, 3.8-fold improved, respectively. Furthermore, enteric-coated tablets based on unmodified chitosan or Ch-TBA/GSH, were investigated in vivo. In rats, the Ch-TBA/GSH tablets increased the area under the plasma concentration time curve (AUC0–12) of Rho-123 by 217% in comparison to buffer control and by 58% in comparison to unmodified chitosan. This in vivo study showed that a delivery system based on thiolated chitosan significantly increased the oral bioavailability of P-gp substrate Rho-123.
Keywords: Thiolated chitosan; P-glycoprotein; Rhodamine-123; P-gp inhibition; Oral drug delivery;

Nanoparticles of cationic amphiphilic cyclodextrins entangling anionic porphyrins as carrier-sensitizer system in photodynamic cancer therapy by Salvatore Sortino; Antonino Mazzaglia; Luigi Monsù Scolaro; Francesca Marino Merlo; Vincenza Valveri; Maria Teresa Sciortino (4256-4265).
The photodynamic activity of a carrier-sensitizer system consisting of heterotopic colloidal nanoparticles (diameter 100–1000 nm) of a cationic amphiphilic cyclodextrin, heptakis(2-ω-amino-O-oligo(ethylene oxide)-6-hexylthio)-β-CD (SC6CDNH2) encapsulating the anionic 5,10,15,20-tetrakis(4-sulfonatophenyl)-21H,23H-porphyrin (TPPS) is investigated by an interdisciplinary approach involving the combination of time-resolved absorption and emission techniques with in vitro studies on cultured tumor cells. In a range of TPPS:SC6CDNH2 molar ratios between 1:10 and 1:50 these nanoparticles preserve the photodynamic properties of the entrapped photoactive agent. In fact, the triplet state of TPPS is efficiently populated, very long-lived and, as a consequence, able to produce singlet oxygen (the essential species for the photodynamic action) with quantum yield comparable to the free TPPS. Photodynamic efficacy of the carrier/sensitizer system is proven by in vitro studies on tumor Hela cells treated with TPPS:SC6CDNH2 at different molar ratio, showing significant cells death upon illumination with visible light.
Keywords: Biocompatibility; Cyclodextrins; Drug delivery; Photoexcitation; Nanostructures; Singlet oxygen;

Combination of 3D tissue engineered scaffold and non-viral gene carrier enhance in vitro DNA expression of mesenchymal stem cells by Hossein Hosseinkhani; Tony Azzam; Hisatoshi Kobayashi; Yosuke Hiraoka; Hitoyata Shimokawa; Abraham J. Domb; Yasuhiko Tabata (4269-4278).
The objective of this study is to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSC) by combination of 3-dimensional (3D) tissue engineered scaffolds and non-viral gene carrier. As a carrier of plasmid DNA, dextran-spermine cationic polysaccharide was prepared by means of reductive-amination between oxidized dextran and the natural oligoamine, spermine. As the MSC scaffold, collagen sponges reinforced by incorporation of poly(glycolic acid) (PGA) fibers were used. A complex of the cationized dextran and plasmid DNA of BMP-2 was impregnated into the scaffolds. MCS were seeded into each scaffold and cultured by a 3D culture method. When MSC were cultured in the PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by the cationized dextran-plasmid DNA complex impregnated into the scaffold than by the cationized dextran-plasmid DNA complex in 2-dimensional (2D) (tissue culture plate) culture method. The alkaline phosphatase activity and osteocalcin content of transfected MSC cultured in the PGA-reinforced sponge were significantly higher compared with 2D culture method. We conclude that combination of cationized dextran plasmid DNA complex and 3D tissue engineered scaffold was promising to promote the in vitro gene expression for MSC.
Keywords: Plasmid DNA; In vitro transfection; Enhanced gene expression; Cationization; Scaffold; Osteoinduction;