Biomaterials (v.29, #26)

Do we need synthetic osteotomy augmentation materials for opening-wedge high tibial osteotomy by Sebastian Aryee; Andreas B. Imhoff; Tim Rose; Thomas Tischer (3497-3502).
High tibial osteotomy (HTO) is an increasing popular method to treat unicompartimental osteoarthritis of the knee in younger, active patients. In so doing one tries to delay the need for total or unicompartimental joint replacement. The augmentation of HTO opening gaps with supporting material is discussed controversially, especially after the introduction of locking plates, which contribute to the decline of the non-union rate. Currently, we do not recommend synthetic augmentation, when using locking plates in HTO with opening angles less than 10°. In our recent randomized study we could histologically and radiologically demonstrate the complete rebuilding of lamelliform bone in patients without synthetic augmentation, whilst bony ingrowth into the hydroxyapatite/tricalcium phosphate (HA/TCP) wedge of augmented osteotomies just slowly progressed. In contrast to unaugmented osteotomies, there was no advantage in using HA/TCP wedges or the combination of HA/TCP wedges and platelet rich plasma (PRP) as supporting material after 12 months. In osteotomies where an opening angle bigger than 7.5° is chosen, rigid locking plates should be used. In our opinion, autologous iliac crest graft should be used in the high-risk patients (obese, smoker, opening angle bigger than 10°). Whether synthetic augmentation combined with PRP is equal or even superior to autologous iliac crest graft in openings bigger than 10° has not been proven yet.
Keywords: Osteoarthritis of the knee; High tibial osteotomy; Open wedge osteotomy; Supporting material; Hydroxyapatite; Synthetic augment;

We demonstrated the effect of synthesized sulfated hyaluronan (SHya), which is composed of a sulfated group and hyaluronan, and basic fibroblast growth factor 2 (FGF-2) on normal human astrocytes (NHA) activity and its morphological transformation in vitro study. Astrocyte is a kind of glial cell and stellated astrocyte (activating astrocyte) supports axons network, neurons survival and synaptic plasticity. Treatment of SHya hardly affected NHA proliferation. However combination treatment of SHya and FGF-2 increased NHA proliferation. Treatment of SHya promoted transformation of normal astrocyte into a stella morphology (stellation) and combination treatment of SHya and FGF-2 promoted stellation than that of SHya only. Treatment of SHya increased glial fibrillary acidic protein (GFAP), nestin mRNA and GFAP protein expression in the stellated NHA. The cell–cell adhesion of NHA increased by treatment of SHya. Treatment of SHya increased heparin-binding trophic factors FGF-2, midkine, and some other trophic factors mRNA level in the NHA. These results suggested that the treatment of SHya promoted NHA activity due to enhancing neurotrophins production and the morphological transformation of NHA and the effect of SHya on astrocytes partly involved FGF-2 activity. These findings indicate that SHya may be involved in the astrocyte activity and support neurons survivals.
Keywords: Hyaluronan; Sulfated groups; Astrocyte; FGF-2; Neurotrophic factors;

Immunoproteomic identification of bovine pericardium xenoantigens by Leigh G. Griffiths; Leila H. Choe; Kenneth F. Reardon; Steven W. Dow; E. Christopher Orton (3514-3520).
Bovine pericardium is an important biomaterial with current application in glutaraldehyde-fixed bioprosthetic heart valves and possible future application as an unfixed biological scaffold for tissue engineering. The importance of both humoral and cell-mediated rejection responses toward fixed and unfixed xenogeneic tissues has become increasingly apparent. However, the full scope and specific identities of bovine pericardium proteins that can elicit an immune response remain largely unknown. In this study, an immunoproteomic approach was used to survey bovine pericardium proteins for their ability to elicit a humoral immune response in rabbits. A two-stage protein extraction protocol was used to separate bovine pericardium proteins into water- and lipid-soluble fractions. Two-dimensional (2-D) gel electrophoresis was performed to separate the proteins from each fraction. Western blots were generated from 2-D gels of both bovine pericardium protein fractions. These blots were probed with serum from rabbits immunized with bovine pericardium and a secondary antibody was used to assess for IgG positivity. Western blots were compared to duplicate 2-D gels and proteins in matched spots were identified by tandem mass spectrometry. Thirty-one putative protein antigens were identified, eight of which are known to be antigenic from previous studies. All of the putative antigens demonstrated progressive staining intensity with increasing days of post-exposure serum. Identified antigenic proteins represented a variety of functional and structural protein types, and included both cellular and matrix proteins. The results of this study have implications for the use of bovine pericardium as a biomaterial in bioprostheses and tissue engineering applications, as well as xenotransplantation in general.
Keywords: Immunoproteomics; Scaffold; Tissue engineering; Xenoantigens; Heart valve;

Self-aggregation is key to hair follicle (HF) induction ability of dermal papilla (DP) cells and neogenesis of HF can be achieved by transplanting DP microtissues. However, there is currently lack of a suitable system that allows efficient production of DP microtissues and analysis of DP self-aggregation in vitro. We demonstrate that, at a higher seeding cell density, poly(ethylene-co-vinyl alcohol) (EVAL) membranes facilitate DP self-assembly into many compact spheroidal microtissues that are able to induce new HFs. This self-assembling process is associated with an enhanced cell movement and a declined cell–substrate adhesivity on EVAL. A compromised cell growth is also revealed on EVAL. On the contrary, a more adherent surface allows faster cell expansion but maintains DP cells in a flat morphology. Dynamically, cell migration, intercellular collision and intercellular adhesion contribute to DP microtissue formation on EVAL. Our results suggest that, for large-scale production of DP microtissues for HF regeneration, an adhesive surface is needed for quick cell expansion and a biomaterial with a lower adhesivity is required for self-aggregation. In addition, this system can be a model for investigation of DP self-aggregation in vitro.
Keywords: Aggregation; Dermal papilla; Spheroid; Tissue engineering; Adhesivity; Biomaterial;

The dosage dependence of VEGF stimulation on scaffold neovascularisation by Neil Davies; Stephan Dobner; Deon Bezuidenhout; Christian Schmidt; Michael Beck; Andreas H. Zisch; Peter Zilla (3531-3538).
Growth factors are often used in tissue regeneration to stimulate vascularisation of polymeric scaffolds, with vascular endothelial growth factor (VEGF) having been extensively studied for short-term vessel ingrowth. We have therefore evaluated the effect of different concentrations of VEGF on the vascularisation of a porous scaffold in the short-, intermediate- and long-term, by delivering 15, 150 and 1500 ng VEGF/day to polyurethane scaffolds by osmotic pumps for up to 6 weeks. An increased vascularisation months after termination of VEGF delivery was only achieved with 150 ng/day (46%, p  < 0.05). This dosage consistently showed elevated levels of vascularisation (144, 125, 160 and 60% above PBS controls at 10, 20, 30 and 42 days, respectively, p  < 0.05), whilst the vessels induced by the highest dosage, though initially maximally elevated (265 and 270% at 10 and 20 days, p  < 0.05) tended to regress after 20 days of VEGF delivery. Pericyte coverage was decreased at 20 days for the highest dosage (30%, p  < 0.05). Lectin perfusion demonstrated that vessels within the scaffold were connected to the host vasculature at all time points and perfusion was substantially raised by VEGF delivery at day 20. These results suggest concentration of VEGF plays a critical role in the nature and persistence of vasculature formed in a tissue regenerative scaffold.
Keywords: Vascular endothelial growth factor; Angiogenesis; Tissue regeneration; Scaffolds; Vessel regression; Polyurethane;

Design of graded biomimetic osteochondral composite scaffolds by Anna Tampieri; Monica Sandri; Elena Landi; Daniele Pressato; Silvia Francioli; Rodolfo Quarto; Ivan Martin (3539-3546).
With the ultimate goal to generate suitable materials for the repair of osteochondral defects, in this work we aimed at developing composite osteochondral scaffolds organized in different integrated layers, with features which are biomimetic for articular cartilage and subchondral bone and can differentially support formation of such tissues. A biologically inspired mineralization process was first developed to nucleate Mg-doped hydroxyapatite crystals on type I collagen fibers during their self-assembling. The resulting mineral phase was non-stoichiometric and amorphous, resembling chemico-physical features of newly deposited, natural bone matrix. A graded material was then generated, consisting of (i) a lower layer of the developed biomineralized collagen, corresponding to the subchondral bone, (ii) an upper layer of hyaluronic acid-charged collagen, mimicking the cartilaginous region, and (iii) an intermediate layer of the same nature as the biomineralized collagen, but with a lower extent of mineral, resembling the tidemark. The layers were stacked and freeze-dried to obtain an integrated monolithic composite. Culture of the material for 2 weeks after loading with articular chondrocytes yielded cartilaginous tissue formation selectively in the upper layer. Conversely, ectopic implantation in nude mice of the material after loading with bone marrow stromal cells resulted in bone formation which remained confined within the lower layer. In conclusion, we developed a composite material with cues which are biomimetic of an osteochondral tissue and with the capacity to differentially support cartilage and bone tissue generation. The results warrant testing of the material as a substitute for the repair of osteochondral lesions in orthotopic animal models.
Keywords: Biomimetic material; Biomineralization; Chondrocyte; Collagen; Composite; Magnesium;

Bioengineered cardiac patch constructed from multilayered mesenchymal stem cells for myocardial repair by Hao-Ji Wei; Chun-Hung Chen; Wen-Yu Lee; Iwen Chiu; Shiaw-Min Hwang; Wei-Wen Lin; Chieh-Cheng Huang; Yi-Chun Yeh; Yen Chang; Hsing-Wen Sung (3547-3556).
Growing three-dimensional scaffolds that contain more than a few layers of seeded cells is crucial for the creation of thick and viable cardiac tissues. To achieve this goal, a bioengineered cardiac patch (the MSC patch) composed of a sliced porous biological scaffold inserted with multilayered mesenchymal stem cells (MSCs) was developed for myocardial repair in a syngeneic rat model. After culture, sliced layers of the scaffold were stuck together and seeded MSCs were redistributed throughout the scaffold. Immunofluorescence analyses indicated that cells were viable and tightly adhered to a robust fibronectin meshwork within the scaffold. Results of echocardiography and heart catheterization revealed that the MSC-patch group had a superior heart function to the infarct group. Cells together with neo-muscle fibers and neo-microvessels were clearly observed in the entire MSC patch to fill its original pores, indicating that the implanted patch became well integrated into the host. The thickness of the retrieved MSC patch increased significantly as compared to that before implantation. When compared with the infarct group, expressions of angiogenic cytokines (bFGF, vWF and PDGF-B) and cardioprotective factors (IGF-1 and HGF) were significantly increased in the MSC-patch group. The aforementioned results indicated that transplantation of the MSC patch could restore the dilated LV and preserve cardiac functions after infarction.
Keywords: Myocardial infarction; Bioengineered scaffold; Cell sheet; Tissue engineering;

The effects of high dose irradiation on the cross-linking of vitamin E-blended ultrahigh molecular weight polyethylene by Ebru Oral; Christine Godleski Beckos; Arnaz S. Malhi; Orhun K. Muratoglu (3557-3560).
Vitamin E-stabilized, highly cross-linked ultrahigh molecular weight polyethylene (UHMWPE) is a promising oxidation and wear resistant UHMWPE with improved mechanical strength in comparison with the first generation, irradiated and melted UHMWPE. One approach of incorporating vitamin E in UHMWPE is through blending of vitamin E in UHMWPE powder followed by consolidation and radiation cross-linking. However, radiation cross-linking efficiency of UHMWPE decreases in the presence of vitamin E. Therefore an optimum vitamin E concentration and radiation dose level need to be determined to achieve a cross-link density comparable to 100-kGy irradiated and melted UHMWPE, which has shown excellent wear properties in vivo. We investigated the cross-link density and mechanical properties of vitamin E-blended UHMWPEs as a function of vitamin E concentration in the blend and gamma irradiation doses up to 200 kGy. We found that 0.3 wt% vitamin E-blended UHMWPE could not be cross-linked above a cross-link density achieved at a radiation dose of 65 kGy for virgin UHMWPE and 1.0 wt% vitamin E-blended UHMWPE could not be cross-linked above a cross-link density achieved at a radiation dose of 25 kGy for virgin UHMWPE even when the these UHMWPEs were irradiated to a radiation dose of 200 kGy. In addition, higher plasticity at vitamin E concentrations at and above 0.3 wt% indicated that increased chain scissioning may be prevalent. Since the wear resistance of this irradiated UHMWPE would be expected to be low, vitamin E concentrations equal to or above 0.3 wt% are not recommended for subsequent irradiation to achieve a wear resistant cross-linked UHMWPE. The long-term oxidative stability of irradiated blends with low vitamin E concentrations has yet to be studied to determine an optimum between cross-link density and long-term oxidative stability.
Keywords: Antioxidant; Arthroplasty; Cross-linking; Gamma irradiation; Joint replacement; Polyethylene;

Fullerene (C60), a third carbon allotrope, is a classical engineered material with the potential application in biomedicine. One of the biologically most relevant features of C60 is the ability to quench various free radicals, behaving as a “free radical sponge”. Conversely, photosensitization of C60 leads to its transition to a long-lived triplet excited state and the subsequent energy or electron transfer to molecular oxygen, yielding highly reactive singlet oxygen (1O2) or superoxide anion (O2 •−), respectively. These reactive oxygen species (ROS) react with a wide range of biological targets and are known to be involved in both cellular signaling and cell damage. Therefore, the dual property of fullerenes to either quench or generate cell-damaging ROS could be potentially exploited for their development as cytoprotective or cytotoxic anticancer/antimicrobial agents. However, the attempts to that effect have been hampered by the extremely low water solubility of C60, and by the fact that solubilization procedures profoundly influence the ROS-generating/quenching properties of C60, either through chemical modification or through formation of complex nanoscale particles with different photophysical properties. We here analyze the mechanisms and biological consequences of ROS generation/quenching by C60, focusing on the influence that different physico-chemical alterations exert on its ROS-related biological behavior.
Keywords: Carbon; Nanoparticle; Antioxidant; Cytotoxicity;

Enhancement of neurite outgrowth using nano-structured scaffolds coupled with laminin by H.S. Koh; Thomas Yong; C.K. Chan; S. Ramakrishna (3574-3582).
Cell interactions with scaffolds are important for cell and tissue development in the process of repairing and regeneration of damaged tissue. Scaffolds that mimic extracellular matrix (ECM) surface topography, mechanical stiffness, and chemical composition will be advantageous to promote enhanced cell interactions. Electrospinning can easily produce nano-structured synthetic polymer mats with architecture that structurally resembles the ECM of tissue. Although electrospinning can produce sub-micron fibrous scaffolds, modification of electrospun scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation and differentiation. Incorporation of laminin, a neurite promoting ECM protein, onto the nanofibers is an alternative to further mimic the biochemical properties of the nervous tissue to create a biomimetic scaffold. In this study, we investigated the feasibility to functionalize scaffolds by coupling laminin onto poly(l-lactic acid) (PLLA) nanofibers. Laminin was successfully added to nanofibers using covalent binding, physical adsorption or blended electrospinning procedures. PC12 cell viability and neurite outgrowth assays confirmed that the functionalized nanofibers were able to enhance axonal extensions. Significantly, compared to covalent immobilization and physical adsorption, blended electrospinning of laminin and synthetic polymer is a facile and efficient method to modify nanofibers for the fabrication of a biomimetic scaffold. Using these functionalization techniques, nanofibers can be effectively modified with laminin for potential use in peripheral nerve regeneration applications.
Keywords: Nerve regeneration; Tissue engineering; Laminin; Neurite; Electrospinning; Nanofibers;

A promising new direction for contrast-enhanced magnetic resonance (MR) imaging involves tracking the migration and biodistribution of superparamagnetic iron oxide (SPIO)-labeled cells in vivo. Despite the large number of cell labeling studies that have been performed with SPIO particles of differing size and surface charge, it remains unclear which SPIO configuration provides optimal contrast in non-phagocytic cells. This is largely because contradictory findings have stemmed from the variability and imprecise control over surface charge, the general need and complexity of transfection and/or targeting agents, and the limited number of particle configurations examined in any given study. In the present study, we systematically evaluated the cellular uptake of SPIO in non-phagocytic T cells over a continuum of particle sizes ranging from 33 nm to nearly 1.5 μm, with precisely controlled surface properties, and without the need for transfection agents. SPIO labeling of T cells was analyzed by flow cytometry and contrast enhancement was determined by relaxometry. SPIO uptake was dose-dependent and exhibited sigmoidal charge dependence, which was shown to saturate at different levels of functionalization. Efficient labeling of cells was observed for particles up to 300 nm, however, micron-sized particle uptake was limited. Our results show that an unconventional highly cationic particle configuration at 107 nm maximized MR contrast of T cells, outperforming the widely utilized USPIO (<50 nm).
Keywords: Molecular imaging; MRI; Ultrasmall superparamagnetic iron oxide; Standard superparamagnetic iron oxide; Micron-sized paramagnetic iron oxide; Nanoparticles;

Regeneration of bone is driven by the action of numerous biomolecules. However, most osteobiologic devices mainly depend on delivery of a single molecule. The present studies were directed at investigating a polymeric system that enables localized, alternating delivery of two or more biomolecules. The osteotropic biomolecules studied were simvastatin hydroxyacid (Sim) and parathyroid hormone (1–34) (PTH(1–34)), and the antimicrobial peptide cecropin B (CB) was also incorporated. Loaded microspheres were made using the complexation polymer system of cellulose acetate phthalate and Pluronic F-127 (blend ratio, 7:3). By alternating layers of the different types of microspheres, 10-layer devices were made to release CB and Sim, CB and PTH, or Sim and PTH. In vitro experiments showed five discrete peaks for each molecule over a release period of approximately two weeks. MC3T3-E1 osteoblastic cells alternately exposed to the osteotropic biomolecules showed enhanced proliferation and early osteoblastic activity. Alternating delivery of 10 nm Sim and either 500 pg/ml or 5 ng/ml PTH showed additive effects compared to the CB/Sim or CB/PTH devices. These implantable formulations may be useful for alternating delivery of different biomolecules to stimulate concurrent biological effects in focal tissue regeneration applications.
Keywords: Controlled release; Alternating release; Simvastatin; Parathyroid hormone (1–34); Cecropin B;

Self-assembled star-shaped chlorin-core poly(ɛ-caprolactone)–poly(ethylene glycol) diblock copolymer micelles for dual chemo-photodynamic therapies by Cheng-Liang Peng; Ming-Jium Shieh; Ming-Hsien Tsai; Cheng-Chung Chang; Ping-Shan Lai (3599-3608).
Amphiphilic 4-armed star-shaped chlorin-core diblock copolymers based on methoxy poly(ethylene glycol) (mPEG) and poly(ɛ-caprolactone) (PCL) were synthesized and characterized in this study. The synthesized photosensitizer-centered amphiphilic star block copolymer that forms assembled micelle-like structures can be used in a photodynamic therapy (PDT)-functionalized drug delivery system. Moreover, the hydrophobic chemotherapeutic agent, paclitaxel, can be trapped in the hydrophobic inner core of micelles. In our results, the star-polymer-formed micelle exhibited efficient singlet oxygen generation, whereas the hydrophobic photosensitizer failed due to aggregation in aqueous solution. The chlorin-core micelle without paclitaxel loading exhibited obvious phototoxicity in MCF-7 breast cancer cells with 7 J/cm2 or 14 J/cm2 light irradiation at a chlorin concentration of 125 μg/ml. After paclitaxel loading, the size of micelle increased from 71.4 nm to 103.2 nm. Surprisingly, these micelles were found to improve the cytotoxicity of paclitaxel significantly in MCF-7 cells after irradiation through a synergistic effect evaluated by median effect analysis. This functionalized micellar delivery system is a potential dual carrier for the synergistic combination of photodynamic therapy and chemotherapy for the treatment of cancer.
Keywords: Star-shaped copolymer; Micelle; Photodynamic therapy; Paclitaxel; Cancer therapy;

Silk polymer-based adenosine release: Therapeutic potential for epilepsy by Andrew Wilz; Eleanor M. Pritchard; Tianfu Li; Jing-Quan Lan; David L. Kaplan; Detlev Boison (3609-3616).
Adenosine augmentation therapies (AAT) make rational use of the brain's own adenosine-based seizure control system and hold promise for the therapy of refractory epilepsy. In an effort to develop an AAT compatible with future clinical application, we developed a novel silk protein-based release system for adenosine. Adenosine releasing brain implants with target release doses of 0, 40, 200, and 1000 ng adenosine per day were prepared by embedding adenosine containing microspheres into nanofilm-coated silk fibroin scaffolds. In vitro, the respective polymers released 0, 33.4, 170.5, and 819.0 ng adenosine per day over 14 days. The therapeutic potential of the implants was validated in a dose-response study in the rat model of kindling epileptogenesis. Four days prior to the onset of kindling, adenosine releasing polymers were implanted into the infrahippocampal cleft and progressive acquisition of kindled seizures was monitored over a total of 48 stimulations. We document a dose-dependent retardation of seizure acquisition. In recipients of polymers releasing 819 ng adenosine per day, kindling epileptogenesis was delayed by one week corresponding to 18 kindling stimulations. Histological analysis of brain samples confirmed the correct location of implants and electrodes. We conclude that silk-based delivery of around 1000 ng adenosine per day is a safe and efficient strategy to suppress seizures.
Keywords: Silk; Controlled drug release; Drug delivery; Adenosine; Epilepsy; Brain;

Cationic lipid bilayer coated gold nanoparticles-mediated transfection of mammalian cells by Peicai Li; Dan Li; Lixue Zhang; Gaiping Li; Erkang Wang (3617-3624).
Here, we demonstrated dimethyldioctadecylammonium bromide (DODAB), a cationic lipid, bilayer coated Au nanoparticles (AuNPs) could efficiently deliver two types of plasmid DNA into human embryonic kidney cells (HEK 293) in the presence of serum. The transfection efficiency of AuNPs was about five times higher than that of DODAB. The interaction of AuNPs with DNA was characterized with dye intercalation assay and agarose gel electrophoresis. The morphology of the complex of AuNPs with DNA was observed with scanning electron microscope (SEM). The intracellular trafficking of the complex was monitored with transmission electron microscope (TEM). Based on experimental results, the possible mechanism was proposed and the barriers in the process of transfection were discussed. This work demonstrates a simple way to increase the transfection efficiency of cationic lipid through changing the stability of the complex of cationic lipid with DNA. It may provide some insights into understanding and controlling the interaction of cationic lipid with DNA. It also provides a novel way to construct gold nanoparticles-based gene vectors and some insights into learning the process of nanomaterials-mediated transfection.
Keywords: Gold nanoparticles; Dimethyldioctadecylammonium bromide; Transfection; Trafficking; Cytotoxicity;