Biomaterials (v.28, #18)

Enzyme-mediated fast in situ formation of hydrogels from dextran–tyramine conjugates by Rong Jin; Christine Hiemstra; Zhiyuan Zhong; Jan Feijen (2791-2800).
Dextran hydrogels were formed in situ by enzymatic crosslinking of dextran-tyramine conjugates and their mechanical, swelling and degradation properties were evaluated. Two types of dextran–tyramine conjugates (M n,dextran=14 k, M w/M n=1.45), i.e. dextran–tyramine linked by a urethane bond (denoted as Dex–TA) or by an ester-containing diglycolic group (denoted as Dex–DG–TA), with different degrees of substitution (DS) were prepared. Hydrogels were rapidly formed under physiological conditions from Dex–TA DS 10 or 15 and Dex–DG–TA DS 10 at or above a concentration of 2.5 wt% in the presence of H2O2 and horseradish peroxidase (HRP). The gelation time ranged from 5 s to 9 min depending on the polymer concentration and HRP/TA and H2O2/TA ratios. Rheological analysis showed that these hydrogels are highly elastic. The storage modulus (G′), which varied from 3 to 41 kPa, increased with increasing polymer concentration, increasing HRP/TA ratio and decreasing H2O2/TA ratio. The swelling/degradation studies showed that under physiological conditions, Dex–TA hydrogels are rather stable with less than 25% loss of gel weight in 5 months, whereas Dex–DG–TA hydrogels are completely degraded within 4–10 d. These results demonstrate that enzymatic crosslinking is an efficient way to obtain fast in situ formation of hydrogels. These dextran-based hydrogels are promising for use as injectable systems for biomedical applications including tissue engineering and protein delivery.
Keywords: In situ forming; Hydrogel; Degradable; Enzymatic crosslinking; Dextran;

Effects of polymer amount and processing conditions on the in vitro behaviour of hybrid titanium dioxide/polycaprolactone composites by Roberto De Santis; Michelina Catauro; Lucy Di Silvio; Luigi Manto; Maria G. Raucci; Luigi Ambrosio; Luigi Nicolais (2801-2809).
Titanium dioxide (TiO2) and TiO2 glasses containing poly(ε-caprolactone) (PCL) up to 24% by weight were obtained by the sol–gel process. Powder compaction was achieved providing heat and pressure. Properties were evaluated through compression and bending tests assisted by X-ray micro-computed tomography imaging. The effects of compaction conditions (i.e. temperature, pressure and duration) on mechanical properties of inorganic/organic composites were investigated. Biocompatibility tests on organic/inorganic composites were carried out using human cells and the MTT assay to determine viability. Results indicated that the mechanical properties (i.e. Young's modulus and maximum strength), in both compression and bending, were a function of the compression moulding conditions. Highest mechanical properties were measured using a compaction pressure of 1500 MPa acting for 90 min at a die temperature of 100 °C. The results, however, also suggest that mechanical properties can be tailored by varying the amount of PCL to TiO2. Strength and stiffness spanned between the properties of spongy and cortical bone. Young's modulus in both compression and bending were higher for PCL amounts of 6%. Instead, higher bending strength values were measured for PCL amounts of 12%. These weight amounts of PCL also provide higher average density values, thus suggesting that the polymeric phase is effective in toughening TiO2-based materials. The investigated materials also showed a very good cytocompatibility as indicated by the MTT assay results.
Keywords: Titanium dioxide; Polycaprolactone; Mechanical properties; Microtomography; MTT assay; Bone;

Bone ingrowth in porous titanium implants produced by 3D fiber deposition by Jia Ping Li; Pamela Habibovic; Mirella van den Doel; Clayton E. Wilson; Joost R. de Wijn; Clemens A. van Blitterswijk; Klaas de Groot (2810-2820).
3D fiber deposition is a technique that allows the development of metallic scaffolds with accurately controlled pore size, porosity and interconnecting pore size, which in turn permits a more precise investigation of the effect of structural properties on the in vivo behavior of biomaterials.This study analyzed the in vivo performance of titanium alloy scaffolds fabricated using 3D fiber deposition. The titanium alloy scaffolds with different structural properties, such as pore size, porosity and interconnecting pore size were implanted on the decorticated transverse processes of the posterior lumbar spine of 10 goats. Prior to implantation, implant structure and permeability were characterized. To monitor the bone formation over time, fluorochrome markers were administered at 3, 6 and 9 weeks and the animals were sacrificed at 12 weeks after implantation. Bone formation in the scaffolds was investigated by histology and histomorphometry of non-decalcified sections using traditional light- and epifluorescent microscopy. In vivo results showed that increase of porosity and pore size, and thus increase of permeability of titanium alloy implants positively influenced their osteoconductive properties.
Keywords: Osteoconduction; Porous Ti6Al4V; Scaffold; 3D fiber deposition;

Surface roughness and surface free energy are two important factors that regulate cell responses to biomaterials. Previous studies established that titanium (Ti) substrates with micron-scale and submicron scale topographies promote osteoblast differentiation and osteogenic local factor production and that there is a synergistic response to micro-rough Ti surfaces that have retained their high surface energy via processing that limits hydrocarbon contamination. This study tested the hypothesis that the synergistic response of osteoblasts to these modified surfaces depends on both surface micro-structure and surface energy.Ti disks were manufactured to present three different surface structures: smooth pretreatment (PT) surfaces with R a of 0.2 μm; acid-etched surfaces (A) with a submicron roughness R a of 0.83 μm; and sandblasted/acid-etched surfaces (SLA) with R a of 3–4 μm. Modified acid-etched (modA) and modified sandblasted/acid-etched (modSLA) Ti substrates, which have low contamination and present a hydroxylated/hydrated surface layer to retain high surface energy, were compared with regular low surface energy A and SLA surfaces. Human osteoblast-like MG63 cells were cultured on these substrates and their responses, including cell shape, growth, differentiation (alkaline phosphatase, osteocalcin), and local factor production (TGF-β1, PGE2, osteoprotegerin (OPG)) were analyzed (N=6 per variable). Data were normalized to cell number.There were no significant differences between smooth PT and A surfaces except for a small increase in OPG. Compared to A surfaces, MG63 cells produced 30% more osteocalcin on modA, and 70% more on SLA. However, growth on modSLA increased osteocalcin by more than 250%, which exceeded the sum of independent effects of surface energy and topography. Similar effects were noted when levels of latent TGF-β1, PGE2 and OPG were measured in the conditioned media.The results demonstrate a synergistic effect between high surface energy and topography of Ti substrates and show that both micron-scale and submicron scale structural features are necessary.
Keywords: Titanium; Surface energy; Micro-structure; Submicron roughness; Osteoblast differentiation;

In vivo cellular repopulation of tubular elastin scaffolds mediated by basic fibroblast growth factor by Aditee Kurane; Dan T. Simionescu; Narendra R. Vyavahare (2830-2838).
In vivo tissue engineering has been explored as a method to repopulate scaffolds with autologous cells to create a functional, living, and non-immunogenic tissue substitute. In this study, we describe an approach to in vivo cellular repopulation of a tissue-derived tubular elastin scaffold. Pure elastin scaffolds were prepared from porcine carotid arteries (elastin tubes). Elastin tubes were filled with agarose gel containing basic fibroblast growth factor (bFGF) to allow sustained release of growth factor. These tubes were implanted in subdermal pouches in adult rats. The elastin tubes with growth factor had significantly more cell infiltration at 28 days than those without growth factor. Immunohistochemical staining indicated that most of these cells were fibroblasts, of which a few were activated fibroblasts (myofibroblasts). Microvasculature was also observed within the scaffolds. Macrophage infiltration was seen at 7 days, which diminished by 28 days of implantation. None of the elastin tubes with bFGF calcified. These results demonstrated that the sustained release of bFGF brings about repopulation of elastin scaffolds in vivo while inhibiting calcification. Results showing myofibroblast infiltration and vascularization are encouraging since such an in vivo implantation technique could be used for autologous cell repopulation of elastin scaffolds for vascular graft applications.
Keywords: Elastin based scaffold; Basic fibroblast growth factor; Subdermal implant; Calcification; Tissue engineering;

An ex vivo model for chondrogenesis and osteogenesis by Jodie C. Pound; David W. Green; Helmtrud I. Roach; Stephen Mann; Richard O.C. Oreffo (2839-2849).
Loss of bone and cartilage are major healthcare issues. At present, there is a paucity of therapies for effectively repairing these tissues sustainably in the long term. A tissue engineering approach using advanced functional scaffolds may provide a clinically acceptable alternative. In this study, an innovative mineralized alginate/chitosan scaffold was used to provide tailored microenvironments for driving chondrogenesis and osteogenesis from single and mixed populations of human articular chondrocytes and human bone marrow stromal cells. Polysaccharide capsules were prepared with combinations of these cell types with the addition of type I or type II collagen to augment cell–matrix interactions and promote the formation of phenotypically distinct tissues and placed in a rotating (Synthecon) bioreactor or held in static 2D culture conditions for up to 28 days. Significant cell-generated matrix synthesis was observed in human bone marrow bioreactor samples containing type I collagen after 21–28 days, with increased cell proliferation, cell activity and osteocalcin synthesis. The cell-generated matrix was immuno-positive for types I and II collagen, bone sialoprotein and type X collagen, a marker of chondrogenic hypertrophy, demonstrating the formation of a mature chondrogenic phenotype with areas of new osteoid tissue formation. We present a unique approach using alginate/collagen capsules encapsulated in chitosan to promote chondrogenic and osteogenic differentiation and extracellular matrix formation and the potential for tissue-specific differentiation. This has significant implications for skeletal regeneration and application.
Keywords: Cartilage; Osteogenesis; Alginate; Polysaccharide; Bioreactor;

Production of a new tissue-engineered adipose substitute from human adipose-derived stromal cells by Mélanie Vermette; Valérie Trottier; Vincent Ménard; Lucie Saint-Pierre; Alphonse Roy; Julie Fradette (2850-2860).
Adipose tissue is an accessible and abundant source of mesenchymal stem cells for soft-tissue reconstruction. In an attempt to create a novel, entirely autologous tissue-engineered adipose substitute, we extracted human stromal cells from either lipoaspirated or resected fat, and assessed their capacity to produce a three-dimensional adipose tissue using an adapted “self-assembly” culture methodology. This strategy involved a concomitant induction of adipogenic differentiation whilst ascorbic acid supplementation stimulated the stromal cells to produce and organize their own “biomaterial” in the form of extracellular matrix, forming manipulatable sheets that are then assembled into thicker reconstructed adipose tissues. When compared to resected fat, lipoaspiration-derived cells featured an increased adipogenic potential and the enhanced ability to recreate an adipose substitute in vitro. When viewed by scanning electron microscopy, the appearance of these reconstructed adipose tissues was strikingly similar to subcutaneous fat. Furthermore, these substitutes secreted adipokines and mediated β-adrenergic receptor-stimulated lipolysis, hence reproducing known major biological functions of white adipose tissue. Therefore, our cell-based tissue engineering strategy led to the production of a functional and entirely natural reconstructed adipose tissue, which offers the potential to be used for specific in vitro applications as well as for autologous soft-tissue reconstruction.
Keywords: Adipose tissue engineering; Extracellular matrix (ECM); Mesenchymal stem cell; Cell culture; Scanning electron microscopy (SEM); Autologous cell;

Neomycin prevents enzyme-mediated glycosaminoglycan degradation in bioprosthetic heart valves by Devanathan Raghavan; Dan T. Simionescu; Naren R. Vyavahare (2861-2868).
Bioprosthetic heart valves (BHVs) derived from glutaraldehyde crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, BHVs have limited durability and fail either due to degeneration or calcification. Glycosaminoglycans (GAGs), one of the integral components of heart valve cuspal tissue, are not stabilized by conventional glutaraldehyde crosslinking. Previously we have shown that valvular GAGs could be chemically fixed with GAG-targeted chemistry. However, chemically stabilized GAGs were only partially stable to enzymatic degradation. In the present study an enzyme inhibitor was incorporated in the cusps to effectively prevent enzymatic degradation. Thus, neomycin trisulfate, a known hyaluronidase inhibitor, was incorporated in cusps via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry followed by glutaraldehyde crosslinking (NEG). Controls included cusps crosslinked with either EDC/NHS followed by glutaraldehyde (ENG) or only with glutaraldehyde (GLUT). NEG group showed improved resistance to in vitro enzymatic degradation as compared to GLUT and ENG groups. All groups showed similar collagen stability, measured as a thermal denaturation temperature by differential scanning calorimetry (DSC). The cusps were implanted subdermally in rats to study in vivo degradation of GAGs. NEG group preserved significantly more GAGs than ENG and GLUT. NEG and ENG groups showed reduced calcification than GLUT.
Keywords: Neomycin; Hexosamine; GAGs; Porcine aortic valves; Valve fixation;

Lack of toxicological side-effects in silver-coated megaprostheses in humans by Jendrik Hardes; Helmut Ahrens; Carsten Gebert; Arne Streitbuerger; Horst Buerger; Michael Erren; Andreas Gunsel; Christian Wedemeyer; Guido Saxler; Winfried Winkelmann; Georg Gosheger (2869-2875).
Deep infection of megaprostheses remains a serious complication in orthopedic tumor surgery. Furthermore, reinfection gets a raising problem in revision surgery of patients suffering from infections associated with primary endoprosthetic replacement of the knee and hip joint. These patients will need many revision surgeries and in some cases even an amputation is inevitable. Silver-coated medical devices proved their effectiveness on reducing infections, but toxic side-effects concerning some silver applications have been described as well. Our study reports about a silver-coated megaprosthesis for the first time and can exclude side-effects of silver-coated orthopedic implants in humans. The silver-levels in the blood did not exceed 56.4 parts per billion (ppb) and can be considered as non-toxic. Additionally we could exclude significant changes in liver and kidney functions measured by laboratory values. Histopathologic examination of the periprosthetic environment in two patients showed no signs of foreign body granulomas or chronic inflammation, despite distant effective silver concentrations up to 1626 ppb directly related to the prosthetic surface. In conclusion the silver-coated megaprosthesis allowed a release of silver without showing any local or systemic side-effects.
Keywords: Antimicrobial; Antibacterial; Bone; Metal ion release; Metal ion toxicity; Metal surface treatment;

Privileged delivery of polymer nanoparticles to the perinuclear region of live cells via a non-clathrin, non-degradative pathway by Samuel K. Lai; Kaoru Hida; Stan T. Man; Clive Chen; Carolyn Machamer; Trina A. Schroer; Justin Hanes (2876-2884).
The efficacy of many therapeutic molecules could be greatly enhanced by polymer-based nanoparticle systems capable of delivering them to the direct vicinity of the cell nucleus. However, degradation of the particles and encapsulated drugs within the enzyme-rich and low-pH environments of the endo/lysosomal pathway of cells has dramatically limited the efficacy of such systems. In this paper, we discovered that small polymeric particles (<25 nm) but not larger particles (>42 nm) enter live cells via a novel mechanism that leads to trafficking outside the endo/lysosomal pathway. Sub-25 nm particles rapidly transport to the perinuclear region of cells in vesicles that never acidify. The pathway is non-degradative, cholesterol independent, and non-clathrin and non-caveolae mediated. This privileged non-acidic pathway may be general since our results are surprisingly obtained with standard latex polymer beads without addition of ligands and may, therefore, provide a promising route for drug and gene delivery using biomaterial-based nanodevices.
Keywords: Intracellular delivery; Trafficking; Size; Gene delivery; Non-clathrin; Non-caveolae;

Effects of trehalose click polymer length on pDNA complex stability and delivery efficacy by Sathya Srinivasachari; Yemin Liu; Lisa E. Prevette; Theresa M. Reineke (2885-2898).
Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA into cells. In this study, a series of trehalose-based glycopolymers containing four secondary amines in the repeat unit were synthesized via the ‘click reaction’ [degrees of polymerization (n w)=35, 53, 75, or 100] to elucidate how the polymer length affects the bioactivity. The four structures bound and charge-neutralized pDNA with similar affinity that was independent of the length, as determined through gel electrophoresis, heparin competitive displacement, and isothermal titration calorimetric assays. Dynamic light scattering measurements revealed that the polyplexes formed with the longer polymers (n w=53, 75, or 100) inhibited flocculation in media containing serum, whereas the polyplexes formed with the shorter polymer (n w=35) aggregated rapidly. Similar results were observed via transmission electron microscopy studies, where the nanoparticles formed with the polymers having longer degrees of polymerization showed discrete particles in media containing 10% serum. Transfection experiments revealed that the polymers exhibited low cytotoxicity at low N/P ratios and could facilitate high cellular uptake and gene expression in HeLa and H9c2(2-1) cells, and the results were dependent on the degrees of polymerization (longer polymers yielded higher transfection and toxicity).
Keywords: Bioactivity; Copolymer; DNA; Gene transfer; Polymerization; In vitro;

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG by Huayu Tian; Wei Xiong; Jizheng Wei; Yu Wang; Xuesi Chen; Xiabin Jing; Qingyu Zhu (2899-2907).
The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(γ-benzyl l-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase I. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI-25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.
Keywords: Hyperbranched polyethylenimine; Gene carriers; Cytotoxicity; Transfection;

2D mapping of texture and lattice parameters of dental enamel by Maisoon Al-Jawad; Axel Steuwer; Susan H. Kilcoyne; Roger C. Shore; Robert Cywinski; David J. Wood (2908-2914).
We have used synchrotron X-ray diffraction to study the texture and the change in lattice parameter as a function of position in a cross section of human dental enamel. Our study is the first to map changes in preferred orientation and lattice parameter as a function of position within enamel across a whole tooth section with such high resolution. Synchrotron X-ray diffraction with a micro-focused beam spot was used to collect two-dimensional (2D) diffraction images at 150 μm spatial resolution over the entire tooth crown. Contour maps of the texture and lattice parameter distribution of the hydroxyapatite phase were produced from Rietveld refinement of diffraction patterns generated by azimuthally sectioning and integrating the 2D images. The 002 Debye ring showed the largest variation in intensity. This variation is indicative of preferred orientation. Areas of high crystallite alignment on the tooth cusps match the expected biting surfaces. Additionally we found a large variation in lattice parameter when travelling from the enamel surface to the enamel-dentine junction. We believe this to be due to a change in the chemical composition within the tooth. The results provide a new insight on the texture and lattice parameter profiles within enamel.
Keywords: Enamel; Hydroxyapatite; Apatite structure; Synchrotron X-ray diffraction; Texture; Preferred orientation;

The role of specific and non-specific interactions in the receptor-mediated endocytosis of nanoparticles is analyzed. The characteristic time τ w , the threshold R th and optimal R opt radii for particle endocytosis are estimated as a function of the binding energy factor C, bond elasticity factor G, and non-specific attractive/repulsive factor F at the cell–particle interface. It is shown that the contribution of F is as important as that of C and G . General and ready to use formulas are presented that can be a guide in designing nanoparticles with controlled endocytic performances.
Keywords: Nanobiotechnology; Bioadhesion; Particle internalization; Surface properties; Drug delivery;