Biomaterials (v.27, #7)

Calendar (I).

Design of modular non-viral gene therapy vectors by Laura De Laporte; Jennifer Cruz Rea; Lonnie D. Shea (947-954).
Gene delivery has numerous potential applications both clinically and for basic science research. Non-viral vectors represent the long-term future of gene therapy and biomaterials are a critical component for the development of efficient delivery systems. Biomaterial development combined with fundamental studies of virus function and cellular processes will enable the molecular level design of modular vectors. Vectors are being developed based on cationic polymers or lipids that contain functional groups to mediate appropriate interactions with the extracellular environment or to interface with specific cellular processes. This review describes recent progress on the development of biomaterials for non-viral vectors and highlights opportunities for future development. Ultimately, efficient vectors will expand the traditional applications of gene therapy within the clinic and may enable numerous other opportunities within diagnostics, biotechnology, and basic science research.
Keywords: Gene therapy; Vector; Lipoplex; Polyplex; Cell trafficking;

Structural, mechanical and in vitro characterization of individually structured Ti–6Al–4V produced by direct laser forming by Dirk A. Hollander; Matthias von Walter; Tobias Wirtz; Richard Sellei; Bernhard Schmidt-Rohlfing; Othmar Paar; Hans-Josef Erli (955-963).
Direct laser forming (DLF) is a rapid prototyping technique which enables prompt modelling of metal parts with high bulk density on the base of individual three-dimensional data, including computer tomography models of anatomical structures. In our project, we tested DLF-produced material on the basis of the titanium alloy Ti–6Al–4V for its applicability as hard tissue biomaterial. To this end, we investigated mechanical and structural properties of DLF-Ti–6Al–4V. While the tensile and yield strengths of untreated DLF alloy ranged beyond 1000 MPa, a breaking elongation of 6.5±0.6% was determined for this material. After an additional post-DLF annealing treatment, this parameter was increased two-fold to 13.0±0.6%, while tensile and yield strengths were reduced by approx. 8%. A Young's modulus of 118.000±2.300 MPa was determined for post-DLF annealed Ti–6Al–4V. All data gained from tensile testing of post-DLF annealed Ti–6Al–4 V matched American Society of Testing and Materials (ASTM) specifications for the usage of this alloy as medical material. Rotating bending tests revealed that the fatigue profile of post-DLF annealed Ti–6Al–4 V was comparable to casted/hot isostatic pressed alloy. We characterized the structure of non-finished DLF-Ti–6Al–4V by scanning electron microscopy and observed a surface-associated layer of particles, which was removable by sandblasting as a finishing step. We manufactured porous specimens with nominal pore diameters of 500, 700 and 1000 μm. The diameters were reduced by the used DLF processing by approx. 300 μm. In an in vitro investigation, we cultured human osteoblasts on non-porous and porous blasted DLF-Ti–6Al–4V specimens to study morphology, vitality, proliferation and differentiation of the cells. The cells spreaded and proliferated on DLF-Ti–6Al–4V over a culture time of 14 days. On porous specimens, osteoblasts grew along the rims of the pores and formed circle-shaped structures, as visualized by live/dead staining as well as scanning electron microscopy. Overall, the DLF-Ti–6Al–4V approach proved to be efficient and could be further advanced in the field of hard tissue biomaterials.
Keywords: Titanium alloy; Rapid prototyping; Structure; Mechanical properties; Osteoblast; Cell spreading;

Optimising bioactive glass scaffolds for bone tissue engineering by Julian R. Jones; Lisa M. Ehrenfried; Larry L. Hench (964-973).
A 3D scaffold has been developed that has the potential to fulfil the criteria for an ideal scaffold for bone tissue engineering. Sol–gel derived bioactive glasses of the 70S30C (70 mol% SiO2, 30 mol% CaO) composition have been foamed to produce 3D bioactive scaffolds with hierarchical interconnected pore morphologies similar to trabecular bone. The scaffolds consist of a hierarchical pore network with macropores in excess of 500 μm connected by pore windows with diameters in excess of 100 μm, which is thought to be the minimum pore diameter required for tissue ingrowth and vasularisation in the human body. The scaffolds also have textural porosity in the mesopore range (10–20 nm). The scaffolds were sintered at 600, 700, 800 and 1000 °C. As sintering temperature was increased to 800 °C the compressive strength increased from 0.34 to 2.26 MPa due to a thickening of the pore walls and a reduction in the textural porosity. The compressive strength is in the range of that of trabecular bone (2–12 MPa). Importantly, the modal interconnected pore diameter (98 μm) was still suitable for tissue engineering applications and bioactivity is maintained. Bioactive glass foam scaffolds sintered at 800 °C for 2 h fulfill the criteria for an ideal scaffold for tissue engineering applications.
Keywords: Scaffold; Porosity; Bioactive glass; Mechanical properties; In vitro test;

One of the main issues in tissue engineering is the fabrication of scaffolds that closely mimic the biomechanical properties of the tissues to be regenerated. Conventional fabrication techniques are not sufficiently suitable to control scaffold structure to modulate mechanical properties. Within novel scaffold fabrication processes 3D fiber deposition (3DF) showed great potential for tissue engineering applications because of the precision in making reproducible 3D scaffolds, characterized by 100% interconnected pores with different shapes and sizes. Evidently, these features also affect mechanical properties. Therefore, in this study we considered the influence of different structures on dynamic mechanical properties of 3DF scaffolds. Pores were varied in size and shape, by changing fibre diameter, spacing and orientation, and layer thickness. With increasing porosity, dynamic mechanical analysis (DMA) revealed a decrease in elastic properties such as dynamic stiffness and equilibrium modulus, and an increase of the viscous parameters like damping factor and creep unrecovered strain. Furthermore, the Poisson's ratio was measured, and the shear modulus computed from it. Scaffolds showed an adaptable degree of compressibility between sponges and incompressible materials. As comparison, bovine cartilage was tested and its properties fell in the fabricated scaffolds range. This investigation showed that viscoelastic properties of 3DF scaffolds could be modulated to accomplish mechanical requirements for tailored tissue engineered applications.
Keywords: Rapid prototyping; Dynamic mechanical analysis; Scaffolds; Tissue engineering;

A series of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM)–poly(ethylene glycol) (PEG) block copolymers with various PNIPAM contents and copolymer architectures, such as linear, four-armed and eight-armed configurations, were prepared by iniferter-based photopolymerization of dithiocarbamylated PEGs (DC-PEGs) under ultraviolet (UV)-light irradiation. The increase in monomer/DC-PEG feed ratio resulted in an increase in both the molecular weight and PNIPAM content of copolymers. The measurement of the optical transmittances of aqueous solutions of PNIPAM–PEG block copolymers determined the lower critical solution temperatures (LCSTs) of block copolymers, which ranged from 31.3 to 34.0 °C. LCST decreased with increasing block length of PNIPAM and with the formation of a branched architecture. Rabbit chondrocytes were immobilized and cultured in a three-dimensional (3D) gel composed of PNIPAM–PEG block copolymer at 37 °C. Gels prepared from copolymers with higher PNIPAM contents at higher concentrations appeared to exhibit a minimal decrease in both cell number and cell viability during a 7-day culture. Cell viability dependencies on material and formulation parameters and the potential use of PNIPAM–PEG block copolymers as an in situ formable scaffold for an engineered cartilage tissue were discussed.
Keywords: Chondrocyte; Thermally responsive material;

Ability of zirconia double coated with titanium and hydroxyapatite to bond to bone under load-bearing conditions by Takashi Suzuki; Shunsuke Fujibayashi; Yasuaki Nakagawa; Iwao Noda; Takashi Nakamura (996-1002).
As a preclinical study, we evaluated the ability of hydroxyapatite and titanium on zirconia (HTOZ) to bond to bone under load-bearing conditions in animal experiments. HTOZ, HA, and Ti on Co–Cr alloy (HTOC) and Ti on Co–Cr alloy (TOC) were implanted into the weight-bearing portion of the femoral condyles of nine beagle dogs. Femurs were extracted 4, 12, and 52 weeks after implantation and examined mechanically by pullout testing, and histologically by toluidine blue staining, SEM, and calculation of the affinity index. The interfacial shear strengths (mean±SD) of the HTOZ, HTOC, and TOC groups were 4.42±0.453, 3.90±0.903, and 4.08±0.790 MPa at 4 weeks; 6.82±2.64, 6.00±1.88, and 6.63±1.63 MPa at 12 weeks; and 13.98±1.94, 11.95±1.51, and 10.78±0.83 MPa at 52 weeks. There were no significant differences in the interfacial shear strengths between the three groups at any time. Affinity indices (mean±SD) obtained from SEM images of the HTOZ, HTOC, and TOC groups were 49.6±6.52%, 43.3±10.43%, and 23.7±3.95% at 4 weeks; 55.0±6.72%, 51.5±3.07%, and 28.6±4.09% at 12 weeks; and 59.1±6.73%, 63.0±6.40%, and 34.3±6.72% at 52 weeks. HA-coated implants (HTOZ, HTOC) had significantly higher affinity indices than non-HA-coated implants (TOC) at all times. HTOZ has the ability to bond to bone equivalent to HTOC and TOC. HTOZ is an excellent material for components of cementless joint prostheses.
Keywords: Zirconia; Cementless; Load-bearing; Total knee arthroplasty;

Response of human chondrocytes to a non-uniform distribution of hydrophilic domains on poly (ethyl acrylate-co-hydroxyethyl methacrylate) copolymers by Marcos Pérez Olmedilla; Natalia Garcia-Giralt; Manuel Monleón Pradas; Pere Benito Ruiz; José Luis Gómez Ribelles; Enric Cáceres Palou; Joan Carles Monllau García (1003-1012).
A series of polymer and copolymer networks with varying hydrophilicity and distribution of the hydrophilic groups was synthesized and biologically tested with monolayer culture of human chondrocytes in vitro. Cell viability (MTT), proliferation (BrdU incorporation) and aggrecan expression (PG ELISA) were quantified at 7 and 14 days from seeding. Both assays (MTT and BrdU) showed complementary results that are consistent with positive cellular adhesion on the material. When human chondrocytes were cultured on polymer substrates in which the hydrophilic groups were homogeneously distributed, hydrophobic substrates showed higher values in all the biological parameters analysed. Adhesion, proliferation and viability decreased monotonously with the content of hydrophilic groups in the polymer chain. However poly(ethyl acrylate-co-hydroxyethyl methacrylate) copolymers, in which hydrophilic and hydrophobic nano-domains alternate as phase-separated domains, showed better results than the corresponding homopolymers.
Keywords: Monolayer cell culture; Polymer network; PolyHEMA; PolyHEA; PolyEA; PolyEMA;

In vitro and in vivo corrosion measurements of magnesium alloys by Frank Witte; Jens Fischer; Jens Nellesen; Horst-Artur Crostack; Volker Kaese; Alexander Pisch; Felix Beckmann; Henning Windhagen (1013-1018).
The in vivo corrosion of magnesium alloys might provide a new mechanism which would allow degradable metal implants to be applied in musculo-skeletal surgery. This would particularly be true if magnesium alloys with controlled in vivo corrosion rates could be developed. Since the magnesium corrosion process depends on its corrosive environment, the corrosion rates of magnesium alloys under standard in vitro environmental conditions were compared to corrosion rates in an in vivo animal model. Two gravity-cast magnesium alloys (AZ91D, LAE442) were used in these investigations. Standardized immersion and electrochemical tests according to ASTM norms were performed. The in vivo corrosion tests were carried out by intramedullar implantation of sample rods of the magnesium alloys in guinea pig femura. The reduction in implant volume was determined by synchrotron-radiation-based microtomography. We found that in vivo corrosion was about four orders of magnitude lower than in vitro corrosion of the tested alloys. Furthermore, the tendency of the corrosion rates obtained from in vitro corrosion tests were in the opposite direction as those obtained from the in vivo study. The results of this study suggest, that the conclusions drawn from current ASTM standard in vitro corrosion tests cannot be used to predict in vivo corrosion rates of magnesium alloys.
Keywords: Biodegradation; Implant; Magnesium; Corrosion; Bone;

Poly(ε-caprolactone) (PCL) fibres were produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. As-spun PCL fibres exhibited a mean strength and stiffness of 7.9 MPa and 0.1 GPa, respectively and a rough, porous surface morphology. Cold drawing to an extension of 500% resulted in increases in fibre strength (43 MPa) and stiffness (0.3 GPa) and development of an oriented, fibrillar surface texture. The proliferation rate of Swiss 3T3 mouse fibroblasts and C2C12 mouse myoblasts on as-spun, 500% cold-drawn and gelatin-modified PCL fibres was determined in cell culture to provide a basic measure of the biocompatibility of the fibres. Proliferation of both cell types was consistently higher on gelatin-coated fibres relative to as-spun fibres at time points below 7 days. Fibroblast growth rates on cold-drawn PCL fibres exceeded those on as-spun fibres but myoblast proliferation was similar on both substrates. After 1 day in culture, both cell types had spread and coalesced on the fibres to form a cell layer, which conformed closely to the underlying topography. The high fibre compliance combined with a potential for modifying the fibre surface chemistry with cell adhesion molecules and the surface architecture by cold drawing to enhance proliferation of fibroblasts and myoblasts, recommends further investigation of gravity-spun PCL fibres for 3-D scaffold production in soft tissue engineering.
Keywords: Polycaprolactone; Fibres; Tissue engineering; Fibroblasts; Myoblasts;

Biodegradable and photocrosslinkable polyphosphoester hydrogel by Qiang Li; Jun Wang; Shilpa Shahani; Danny D.N. Sun; Blanka Sharma; Jennifer H. Elisseeff; Kam W. Leong (1027-1034).
A new biodegradable, photocrosslinkable and multifunctional macromer, poly(6-aminohexyl propylene phosphate) (PPE-HA)-ACRL, was synthesized by conjugation of acrylate groups to the side chains of PPE-HA. By controlling the synthetic conditions, different weight fractions of acrylate in the macromers were achieved as confirmed by 1H NMR. The hydrogels obtained from PPE-HA-ACRL through photocrosslinking were dominantly elastic. With increasing acrylate contents in the macromers, the hydrogels exhibited a lower swelling ratio and higher mechanical strength. The hydrogels with different crosslinking densities lost between 4.3% and 37.4% of their mass in 84 days when incubated in phosphate-buffered saline at 37 °C. No significant cytotoxicity of the macromers against bone marrow-derived mesenchymal stem cells from goat (GMSC) was observed at a concentration up to 10 mg/ml. Finally, GMSCs encapsulated in the photopolymerized gel maintained their viability when cultured in osteogenic medium for three weeks. Clear mineralization in the hydrogel scaffold was revealed by Von Kossa staining. This study suggests the potential of these biodegradable and photocrosslinkable as injectable tissue engineering scaffolds.
Keywords: Polyphosphoester; Photopolymerization; Hydrogel; Tissue engineering scaffold; Osteogenesis;

This study attempted to accelerate the peripheral nerve regeneration, using the high outflow rate of asymmetric poly(dl-lactic acid-co-glycolic acid) (PLGA) nerve conduits. Asymmetric PLGA nerve conduits of monomer ratio 85/15 were prepared by immersion–precipitation method to serve as possible materials. In this study, mandrels were immersed into a 20% (wt/wt) of PLGA/1,4-dioxane solution and precipitated in a non-solvent bath followed by freeze-drying. Different concentrations of isopropyl alcohol (95%, 40% and 20%) were used as precipitation baths where non-asymmetric (95%) and asymmetric (40% and 20%) conduits could easily form. The asymmetric nerve conduits that consisted of macrovoids on the outer layer, and interconnected micropores in the inner sublayer, possessed characters of larger outflow rate than inflow rate. The asymmetric conduits were implanted to 10 mm right sciatic nerve defects in rats. Autografts, silicone and non-asymmetric PLGA conduits were performed as the control and the contrast groups. Implanted graft specimens of all groups were harvested for histological analysis at 4 and 6 weeks following surgery. The asymmetric PLGA conduits maintained a stable supporting structure and inhibited exogenous cells invasion during entire regeneration process. Asymmetric PLGA conduits were found to have statistically greater number of regenerated axons at the midconduit and distal nerve site of implanted grafts, as compared to the silicone and non-asymmetric groups at 4 and 6 weeks. Of interest was that the results of 4 weeks in asymmetric groups were better than the non-asymmetric groups at 6 weeks in number of axons. According to the results of permeability, the asymmetric structure in the conduit wall seemed to enhance the removal of the blockage of the waste drain from the inner inflamed wound in the early stage, which may have improved the efficacy of the peripheral nerve regeneration. The asymmetric structure could be adequately employed in the future as optimal nerve conduits in peripheral nerve regeneration.
Keywords: Poly(dl-lactic acid-co-glycolic acid); Asymmetric conduit; Nerve regeneration; Immersion–precipitation; Phase inversion;

The regulation of expanded human nasal chondrocyte re-differentiation capacity by substrate composition and gas plasma surface modification by Tim B.F. Woodfield; Sylvie Miot; Ivan Martin; Clemens A. van Blitterswijk; Jens Riesle (1043-1053).
Optimizing re-differentiation of clinically relevant cell sources on biomaterial substrates in serum containing (S+) and serum-free (SF) media is a key consideration in scaffold-based articular cartilage repair strategies. We investigated whether the adhesion and post-expansion re-differentiation of human chondrocytes could be regulated by controlled changes in substrate surface chemistry and composition in S+ and SF media following gas plasma (GP) treatment. Expanded human nasal chondrocytes were plated on gas plasma treated (GP+) or untreated (GP−) poly(ethylene glycol)-terephthalate–poly(butylene terephthalate) (PEGT/PBT) block co-polymer films with two compositions (low or high PEG content). Total cellularity, cell morphology and immunofluorescent staining of vitronectin (VN) and fibronectin (FN) integrin receptors were evaluated, while post-expansion chondrogenic phenotype was assessed by collagen types I and II mRNA expression.We observed a direct relationship between cellularity, cell morphology and re-differentiation potential. Substrates supporting high cell adhesion and a spread morphology (i.e. GP+ and low PEG content films), resulted in a significantly greater number of cells expressing α 5 β 1 FN to αVβ 3 VN integrin receptors, concomitant with reduced collagen type II/I mRNA gene expression. Substrates supporting low cell adhesion and a spherical morphology (GP− and high PEG content films) promoted chondrocyte re-differentiation indicated by high collagen type II/I gene expression and a low percentage of α 5 β 1 FN integrin expressing cells.This study demonstrates that cell–substrate interactions via α 5 β 1 FN integrin mediated receptors negatively impacts expanded human nasal chondrocyte re-differentiation capacity. GP treatment promotes cell adhesion in S+ media but reverses the ability of low PEG content PEGT/PBT substrates to maintain chondrocyte phenotype. We suggest alternative cell immobilization techniques to GP are necessary for clinical application in articular cartilage repair.
Keywords: Chondrocyte; Gene expression; Surface modification; PCR; Fibronectin; Vitronectin; Cartilage tissue engineering;

Modified collagen fleece, a scaffold for transplantation of human bladder smooth muscle cells by Carina Danielsson; Sylvie Ruault; Aurelia Basset-Dardare; Peter Frey (1054-1060).
Several congenital and acquired diseases of the human genito-urinary tract may need, due to lack or destruction of functional tissues, mechanically stable biomaterials as cell carriers for the engineering of these tissues.When using collagen scaffolds, both their capacity to induce tissue regeneration and their biocompatibility are advantageous characteristics to render them apt for tissue engineering. The attachment of extracellular matrix or serum proteins to their surfaces does further improve these characteristics, mimicking a close to natural cell environment. In this study, equine collagen scaffolds (TissueFleece®) were modified by coating fetal bovine serum proteins, before human bladder smooth muscle cells were seeded. Cell growth was evaluated by WST-1 proliferation assay and improved when using modified collagen scaffolds. However, cell penetration assessed by histology showed similar results on modified and native scaffolds. These cell-scaffold constructs were further implanted in the dorsal subcutaneous space of athymic mice. In vivo studies showed the presence of the fluorescent-labeled transplanted smooth muscle cells until day 3 and thereafter angiogenesis was induced and infiltration of mouse fibroblasts and polymorphonuclear cells were observed. The latter had completely disappeared after 3 weeks.
Keywords: Tissue engineering; Collagen scaffolds; Human bladder smooth muscle cells; Transplantation; Athymic mice;

The better understanding of cell biology and cell communication requires novel culture systems that better represent the natural cell environment in tissues and organs. We developed a spherical organoid (spheroid) microarray culture system using a combination of microfabrication and microcontact printing. The system consisted of a chip that had cylindrical cavities of 300 μm diameter at a density of 700 cavities/cm2. The bottom faces of these cavities were defined as two different regions that either supported or inhibited cell adhesion. In the cell adhesion region, the center of the bottom face of a 100 μm diameter in a cavity was modified with collagen (Col), and in the non-adhesion region, the entire region around the cavity, except the Col spots, was modified with polyethylene glycol. Primary hepatocytes spontaneously formed spheroids with a uniform diameter at the center of each cavity on the chip. Hepatocytes forming spheroids had a cuboidal cell shape, similar to hepatocytes in vivo, and stably maintained liver-specific phenotypes, such as liver-enriched transcriptional factors, albumin secretion, urea cycle enzymes, and intercellular adhesion molecules. This novel culture system may be applicable as a cellular platform for fundamental studies in cell biology and tissue engineering applications.
Keywords: Rat hepatocyte; Spheroid; Microfabrication; Microcontact printing; Polyethylene glycol; Cellular platform;

Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits by Xin Xin Shao; Dietmar W. Hutmacher; Saey Tuan Ho; James C.H. Goh; Eng Hin Lee (1071-1080).
The objective of this study was to evaluate the feasibility and potential of a hybrid scaffold system in large- and high-load-bearing osteochondral defects repair. The implants were made of medical-grade PCL (mPCL) for the bone compartment whereas fibrin glue was used for the cartilage part. Both matrices were seeded with allogenic bone marrow-derived mesenchymal cells (BMSC) and implanted in the defect (4 mm diameter×5 mm depth) on medial femoral condyle of adult New Zealand White rabbits. Empty scaffolds were used at the control side. Cell survival was tracked via fluorescent labeling. The regeneration process was evaluated by several techniques at 3 and 6 months post-implantation. Mature trabecular bone regularly formed in the mPCL scaffold at both 3 and 6 months post-operation. Micro-Computed Tomography showed progression of mineralization from the host–tissue interface towards the inner region of the grafts. At 3 months time point, the specimens showed good cartilage repair. In contrast, the majority of 6 months specimens revealed poor remodeling and fissured integration with host cartilage while other samples could maintain good cartilage appearance. In vivo viability of the transplanted cells was demonstrated for the duration of 5 weeks. The results demonstrated that mPCL scaffold is a potential matrix for osteochondral bone regeneration and that fibrin glue does not inherit the physical properties to allow for cartilage regeneration in a large and high-load-bearing defect site.
Keywords: Osteochondral tissue engineering; Scaffold; Bone marrow-derived precursor cells; Fibrin glue;

The mechanical integrity of in vivo engineered heterotopic bone by Patrick H. Warnke; Ingo N. Springer; Yahya Acil; Gerrit Julga; Jörg Wiltfang; Klaus Ludwig; Paul A.J. Russo; Eugene Sherry; Sureshan Sivananthan; Jürgen Hedderich; Hendrik Terheyden (1081-1087).
Recent advances in tissue engineering have aroused interest in growth of heterotopic bone for the repair of skeletal defects. This study demonstrates an in vivo method in minipigs of engineering individual human-sized mandible replacements of heterotopic bone with a mechanical integrity similar to natural bone.Ten individualized mandible replacement scaffolds were created using computer-aided design (CAD) techniques. Five had a resorbable external scaffold made of polylactite mesh (test group 1) and five had had a non-resorbable external scaffold of titanium mesh (test group 2). The mesh scaffolds were loaded each with five BioOss® blocks serving as internal scaffolds and 3.5 mg recombinant human Bone Morphogenetic Protein-7. The loaded mesh scaffolds were implanted into the latissimus dorsi muscles of five infant minipigs.After 6 weeks the mandible replacements were harvested. Core biopsy cylinders were taken from the replacements of both test groups and from the natural pig mandibles (control 1). Also, core biopsies from plain BioOss Blocks were gained (control 2). The core biopsy cylinders were loaded axially into a compression test device to evaluate the mechanical compression resistance. Additional specimen underwent histological examination.Both test groups resulted in successful bone induction with degrees of compression resistance [Test 1: 1.62 MPa (SD±0.73); Test 2: 1.51 MPa (SD±0.56)] statistically insignificant when compared to natural porcine mandibular bone [1.75 MPa (SD±0.69)]. This differed significantly from the much lower compression resistance seen in the unadulterated BioOss [0.92 MPa (SD±0.04)]. Following this, the in vivo engineered bone has a similar mechanical compression stability as natural bone.
Keywords: In vivo tissue engineering; BioOss; BMP 7; Bone Morphogenetic Proteins; Heterotopic bone; Mechanical stability;

Controlled fabrication of a biological vascular substitute by Joel Stitzel; Jie Liu; Sang Jin Lee; Makoto Komura; Joel Berry; Shay Soker; Grace Lim; Mark Van Dyke; Richard Czerw; James J. Yoo; Anthony Atala (1088-1094).
Autologous and synthetic vessel grafts have been used as a vascular substitute for cardiovascular bypass procedures. However, these materials are limited by the availability of appropriate caliber autologous vessels, increased susceptibility to thrombosis and intimal hyperplasia following surgery. Electrospinning technology offers the potential for controlling composition, structure and mechanical properties of biomaterials. Vascular graft scaffolds have been fabricated using electrospun polymer blends of Type I collagen, elastin from ligamentum nuchae, and poly (d,l-lactide-co-glycolide). This study demonstrates improved electrospinning characteristics versus previous studies by increasing polymer concentration and adding PLGA to the polymer blend. Additionally, new in vitro biocompatibility and mechanical testing data is presented. The scaffolds possess tissue composition and mechanical properties similar to native vessels. The electrospun vessel matrix is biocompatible and does not elicit local or systemic toxic effects when implanted in vivo. This study demonstrates the promise of electrospinning as a fabrication process for a functional vascular graft for clinical use.
Keywords: Collagen; Elastin; Electrospinning; Mechanical properties; Scaffold; Vascular grafts;

Porous chitosan-gelatin scaffold containing plasmid DNA encoding transforming growth factor-β1 for chondrocytes proliferation by Ting Guo; Jianning Zhao; Jianbin Chang; Zhi Ding; Hao Hong; Jiangning Chen; Junfeng Zhang (1095-1103).
Cartilage defects as a result of disease or injury have a very limited ability to heal spontaneously. Recently, tissue engineering and local therapeutic gene delivery systems have been paid much attention in the cartilage natural healing process. Gene-activated matrix (GAM) blends these two strategies, serving as local bioreactor with therapeutic agents expression and also providing a structural template to fill the lesion defects for cell adhesion, proliferation and synthesis of extracellular matrix (ECM). In the current study, we used chitosan-gelatin complex as biomaterials to fabricate three-dimensional scaffolds and plasmid DNA were entrapped in the scaffolds encoding transforming growth factor-β1 (TGF-β1), which has been proposed as a promoter of cartilage regeneration for its effect on the synthesis of matrix molecules and cell proliferation. The plasmid DNA incorporated in the scaffolds showed a burst release in the first week and a sustained release for the other 2 weeks. The gene transfectd into chondrocytes expresses TGF-β1 protein stably in 3 weeks. The histological and immunohistochemical results confirmed that the primary chondrocytes cultured into the chitosan-gelatin scaffold maintained round and owned characters of high secretion of specific ECM. From this study, it can be concluded that this gene-activated chitosan-gelatins matrix has a potential in the application of cartilage defects regeneration.
Keywords: Cartilage defects; Gene-activated matrix; Transforming growth factor-β1; Chitosan; Gelatin;

Collagen composite hydrogels for vocal fold lamina propria restoration by Mariah S. Hahn; Benjamin A. Teply; Molly M. Stevens; Steven M. Zeitels; Robert Langer (1104-1109).
Chronic voice impairment due to scarring of the vocal fold (VF) lamina propria (LP) can be debilitating in terms of quality of life. Due to the dependence of normal VF vibration on proper VF geometry, an implant inserted to restore appropriate shape and pliability to scarred LP should ideally maintain its insertion-dimensions while being replaced by newly synthesized extracellular matrix (ECM). In the present study, collagen–alginate and collagen–hyaluronan (HA) composite hydrogels were investigated for their ability to support ECM synthesis by VF fibroblasts with limited hydrogel compaction and/or resorption. Collagen–HA composites showed significant mass loss over 28 days of culture, with little evidence of new matrix production. Collagen–alginate composites, in contrast, resisted scaffold compaction and mass loss for at least 42 days in culture while allowing for ECM synthesis. Collagen–alginate hydrogels appear to be promising materials for VF restoration, warranting further investigation.
Keywords: Vocal fold; Tissue engineering; Augmentation; Hydrogel; Collagen; Hyaluronan;

The use of a bioresorbable nano-crystalline hydroxyapatite paste in acetabular bone impaction grafting by J.J. Chris Arts; Nico Verdonschot; Berend W. Schreurs; Pieter Buma (1110-1118).
Calcium phosphates such as TCP-HA granules are considered promising bone graft substitutes. In the future, they may completely replace allograft bone for impaction grafting procedures. Mechanically, acetabular reconstructions with TCP-HA granules show high stability, however this is partly caused by excessive cement penetration, which is unfavourable from a biological perspective.It has been hypothesised that mixtures of morselised cancellous bone grafts (MCB) and/or TCP-HA granules with a nano-crystalline hydroxyapatite paste (Ostim®) may reduce cement penetration while maintaining adequate implant stability and biocompatibility of the graft mixture. To investigate this hypothesis, destructive lever-out tests and in vivo animal test were performed with various combinations of materials.Mechanically, the addition of 10% Ostim to mixtures of MCB and/or TCP-HA granules reduced cement penetration and resulted in a mechanical stability comparable to pure allograft (the current gold standard). Biologically, the application of Ostim with MCB or TCP-HA granules did not hamper the biocompatibility of the materials. Ostim was mostly osseous-integrated with MCB or TCP-HA granules after 8 weeks. Also, non-osseous-integrated Ostim remnants were observed. In tartrate resistant acid phosphatase stained sections, these few non-osseous integrated Ostim remnants were actively being resorbed by osteoclasts.In conclusion, Ostim HA-paste could be a valuable addition when TCP-HA ceramic granules are being used for acetabular bone impaction grafting procedures.
Keywords: Biocompatibility; Bone graft; Hydroxyapatite; Joint replacement; Nano-composite; Osseointegration;

Osteointegration of femoral stem prostheses with a bilayered calcium phosphate coating by Eric Goyenvalle; Eric Aguado; Jean-Michel Nguyen; Norbert Passuti; Laurent Le Guehennec; Pierre Layrolle; Guy Daculsi (1119-1128).
Our purpose was to evaluate the osteointegration of bilayered calcium phosphate (CaP)-coated femoral hip stems in a canine model. A first layer of hydroxyapatite (HA) 20 μm thick and a superficial layer of Biphasic Calcium Phosphate (BCP) 30 μm thick were plasma-sprayed on to the proximal region of sandblasted Ti6Al4V prostheses. Bilayered CaP-coated and non-coated canine femoral stems were implanted bilaterally under general anesthesia in 6 adult female Beagle dogs. After 6 and 12 months, a significant degradation of the bilayered coating occurred with a remainder of 33.1±12.4 and 23.6±9.2 μm in thickness, respectively. Lamellar bone apposition was observed on bilayered coated implants while fibrous tissue encapsulation was observed on non-coated femoral stems. The bone-implant contacts (BIC) were 91±3% and 81±8% for coated and 7±8% and 8±12% for non-coated implants, at 6 and 12 months, respectively. Our study supports the concept of a direct relationship between the biodegradation of CaP coating and the enhanced osteointegration of titanium prostheses. A bilayered CaP coating might therefore enhance bone apposition in the early stages because of the superior bioactivity of the BCP layer while the more stable HA layer might sustain bone bonding over long periods.
Keywords: Canine hemiarthroplasty; Calcium phosphate coating; Bone apposition; Mineral apposition rate; Biodegradation;

Visualisation of bacterial sequestration and bactericidal activity within hydrating Hydrofiber® wound dressings by Geoffrey R. Newman; Michael Walker; Jan A. Hobot; Philip G. Bowler (1129-1139).
The fluid handling and microbiological properties of a non-antimicrobial Hydrofiber® (NAH) wound dressing have been compared with those of a silver salt-containing Hydrofiber® (SCH). Fluorescent dyes ( BacLight™, Live/Dead™ Kit) were added to fresh cultures of two wound pathogens (Pseudomonas aeruginosa and Staphylococcus aureus), and used to visualise their viability. Live bacteria stained green and dead/dying bacteria turned red. When inoculated into samples of the NAH and SCH dressings, the viability of the bacteria could be effectively monitored over time using a rapid form of confocal laser scanning microscopy (RCLSM—Leica® UK). When the NAH dressing was hydrated with stained bacterial culture, its fibres swelled quickly, reducing interstitial spaces between the fibres, resulting in the formation of a cohesive gel. Bacteria became immobilised in the gel, forming characteristic clumps, but remained largely green (viable) for more than 20 h with no apparent increase in numbers. The SCH initially behaved in a similar manner, however, using 3-D data from RCLSM time-lapse sequences P. aeruginosa was observed to turn progressively red (i.e. died) within 1.5–3 h and S. aureus similarly turned red within 5–7 h of contact with the SCH dressing. The ability of both Hydrofiber® dressings to sequester and immobilise potentially pathogenic wound micro-organisms has been demonstrated. Additionally the SCH dressing was shown to kill immobilised bacteria, as a consequence of the ionic silver bactericide. These properties of the Hydrofiber® dressings may contribute to providing an environment that is supportive to wound healing.
Keywords: Bacteria; Confocal microscopy; Silver; Hydrofiber® wound dressing; Wound healing;

Synthesis and characterization of novel aromatic azo bond-containing pH-sensitive and hydrolytically cleavable IPN hydrogels by Padmanabh Chivukula; Karel Dušek; Dong Wang; Mirka Dušková-Smrčková; Pavla Kopečková; Jindřich Kopeček (1140-1151).
Novel interpenetrating network (IPN) hydrogels, composed of pH-sensitive, aromatic azo group containing network as one component (Network A), and a hydrolyzable network as the other (Network B), were prepared by a sequential process. The first network was formed by crosslinking of a reactive polymer precursor (copolymer of N,N-dimethylacrylamide, acrylic acid, N-tert.butylacrylamide, and N-methacryloylglycylglycine p-nitrophenyl ester) with an aromatic azo group containing diamine ((N,N′-ε-aminocaproyl)-4,4′-diaminoazobenzene). The second network was formed by radical crosslinking copolymerization of N-(2-hydroxypropyl)methacrylamide with N,O-dimethacryloylhydroxylamine. The composition of the hydrogels was manipulated to determine the influence of hydrogel composition on the equilibrium degree of swelling, modulus of elasticity in compression, and on the rate of degradation of Network B. Modeling of network structure was accomplished using the statistical branching theory. The major advantage of IPN hydrogels, when compared to traditional pH-sensitive networks, is the linear swelling profile following abrupt change of pH from 2 to 7.4. This indicates the suitability of IPN as carriers for oral drug delivery.
Keywords: Hydrogel; Interpenetrating polymer network; Degradation; Mechanical properties;

Construction and enzymatic degradation of multilayered poly-l-lysine/DNA films by Kefeng Ren; Jian Ji; Jiacong Shen (1152-1159).
The layer-by-layer (LbL) self-assembly of poly-l-lysine (PLL) and deoxyribonucleic acid (DNA) was used to construct the enzymatic biodegradable multilayered films. The LbL build up of DNA multilayers was monitored by UV–vis spectrometry, and atomic force microscopy (AFM). AFM, UV–vis spectrometry and fluorescence spectrometry measurements indicated that 90% of DNA within the films was released almost linearly under 5 U mL –1 α–chymotrypsin in PBS at 37 °C in 35 h. TEM and zeta potential experiments revealed that the released DNA molecules were condensed into the slight positive complexes with size from 20 to several hundred nanometers. The well-structured, easy processed enzymatic biodegradable multilayered film may have great potential for gene applications in tissue engineering, medical implants, etc.
Keywords: Biodegradable; DNA delivery; Layer-by-layer; Poly-l-lysine; Self-assembly;

Combined use of designed scaffolds and adenoviral gene therapy for skeletal tissue engineering by Rachel M. Schek; Erin N. Wilke; Scott J. Hollister; Paul H. Krebsbach (1160-1166).
While tissue engineering remains the most researched alternative to conventional therapies for repair and regeneration, how to optimally combine two of the most promising techniques, designed solid scaffolds and localized gene therapy, is largely unknown. We have conducted a systematic screening of several variables that may affect generation of bone via adenoviral gene therapy vector delivery, on image-based designed and solid freeform-fabricated scaffolds. These variables included: gene therapy type (ex vivo or in vivo); scaffold base material (sintered hydroxyapatite or a polypropylene fumarate/ tricalcium phosphate (PPF/TCP) composite), secondary carrier used to attach the biofactor to the scaffold (fibrin gel or a poly-lactic acid sponge), and scaffold pores size (300 or 800 μm). The in vivo formation of bone following implantation of these scaffolds was then analyzed. Gene therapy method had the largest effect, with ex vivo gene therapy yielding significant amounts of bone on nearly all the implants and in vivo gene therapy failing to produce any bone on most implants. Secondary carrier was the next most important variable, with fibrin gel consistently producing bone encompassing the implants and producing 2–4 times as much bone as the polymer sponge, which triggered only isolated bone growth. Though both scaffold base materials allowed bone growth, hydoxyapatite scaffolds generated twice as much bone as PPF/TCP scaffolds. The pore sizes tested had no significant effect on tissue generation.
Keywords: Adenovirus; Gene therapy; Hydroxyapatite; Bone; Free form fabrication; Poly-lactic acid;

Highly non-toxic and conductive ormosil composite film was prepared using (3-aminopropyl)triethoxysilane and 2-(3,4-epoxycyclohexyl)-ethyltrimethoxysilane by doping with ferrocenemonocarboxylic acid–bovine serum albumin (FMC–BSA) conjugate and multiwall carbon nanotubes (MWNTs). With glucose oxidase (GOD) as a model enzyme this film could be used to design an amperometric biosensor for glucose determination. The entrapped FMC–BSA conjugate performed excellent redox electrochemistry and the immobilized GOD was highly stable. Under optimal conditions this biosensor was able to detect glucose with a detection limit of 20 μm ( S / N = 3 ) in the linear range of 0.05–20.0 mm in flow system, which was wider than the batch amperometric mode, with an analysis time of 25 s for each sample. The value of K M app was 6.6 mm. The proximity of these three components FMC–BSA, MWNTs and GOD enhanced the electron transfer between the film and electrode. This film could be used efficiently for the entrapment of other redox bioactive compounds and biosensing/bioelectrochemical applications.
Keywords: Biosensors; Flow analysis; Composite; Multiwall carbon nanotubes; Ferrocenemonocarcoxylic acid–bovine serum albumin conjugate; Glucose oxidase;

Spatial control of protein within biomimetically nucleated mineral by Linh N. Luong; Sun Ig Hong; Rusha J. Patel; Mark E. Outslay; David H. Kohn (1175-1186).
An ideal approach for bone tissue engineering allows for osteoconductivity, osteoinductivity, and cell transplantation. In this study, we examined coprecipitation and surface adsorption schemes with respect to their abilities to control the spatial quantity and localization of a model protein, bovine serum albumin (BSA), that is incorporated into a biomimetic apatite layer nucleated onto polylactic-co-glycolic acid (PLGA) films. Protein incorporation was characterized by determining protein: presence, quantity loaded, retention, effects on mineral morphology, and localization. FT-IR confirmed the presence of protein in all coprecipitation samples with stronger peaks in the coprecipitated samples compared to the surface adsorbed samples. Coprecipitation resulted in higher loading capacities and higher protein retention versus adsorption. Protein incorporation via coprecipitation changed the mineral morphology from sharp plate-like structures to more rounded structures, whereas, surface adsorption did not change mineral structure. By using confocal microscopy to examine the incorporation of fluorescently labeled proteins, spatial control over protein localization was exhibited. By controlling the loading quantity and localization of the model protein through the mineral thickness, a desired release profile can be achieved. A desired and effective delivery system of biological agents utilizing coprecipitation for bone regeneration can therefore be designed.
Keywords: Coprecipitation; Biomineralization; Biomimetic coating; Protein localization; Simulated body fluid (SBF); Hydroxyapatite-drug;