Biomaterials (v.31, #13)

Structure–property relationships of silk-modified mesoporous bioglass scaffolds by Chengtie Wu; Yufeng Zhang; Yufang Zhu; Thor Friis; Yin Xiao (3429-3438).
Porous mesopore-bioglass (MBG) scaffolds have been proposed as a new class of bone regeneration materials due to their apatite-formation and drug-delivery properties; however, the material's inherent brittleness and high degradation and surface instability are major disadvantages, which compromise its mechanical strength and cytocompatibility as a biological scaffold. Silk, on the other hand, is a native biomaterial and is well characterized with respect to biocompatibility and tensile strength. In this study we set out to investigate what effects blending silk with MBG had on the physiochemical, drug-delivery and biological properties of MBG scaffolds with a view to bone tissue engineering applications. Transmission electron microscopy (TEM), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were the methods used to analyze the inner microstructure, pore size and morphology, and composition of MBG scaffolds, before and after addition of silk. The effect of silk modification on the mechanical property of MBG scaffolds was determined by testing the compressive strength of the scaffolds and also compressive strength after degradation over time. The drug-delivery potential was evaluated by the release of dexamethasone (DEX) from the scaffolds. Finally, the cytocompatibility of silk-modified scaffolds was investigated by the attachment, morphology, proliferation, differentiation and bone-relative gene expression of bone marrow stromal cells (BMSCs). The results showed that silk modification improved the uniformity and continuity of pore network of MBG scaffolds, and maintained high porosity (94%) and large-pore size (200–400 μm). There was a significant improvement in mechanical strength, mechanical stability, and control of burst release of DEX in silk-modified MBG scaffolds. Silk modification also appeared to provide a better environment for BMSC attachment, spreading, proliferation, and osteogenic differentiation on MBG scaffolds.
Keywords: Mesopore bioglass; Scaffold; Silk; Drug delivery; Cytocompatibility;

The biocompatibility of NiTi shape memory alloys (SMA) has made possible applications in self-expandable cardio-vascular stents, stone extraction baskets, catheter guide wires and other invasive and minimally invasive biomedical devices. The NiTi intermetallic alloy spontaneously forms a thin passive layer of TiO2, which provides its biocompatibility. The oxide layer is thought to form as the Ti in the alloy surface reacts with oxygen, resulting in a depletion of Ti in the subsurface region – experimental evidence indicates formation of a Ni-rich layer below the oxide film. In this paper, we study the initial stages of oxide growth on the (110) surface of the NiTi alloy to understand the formation of alloy/oxide interface. We initially adsorb atomic and molecular oxygen on the (110) surface and then successively add O2 molecules, up to 2 monolayer of O2. Oxygen adsorption always results in a large energy gain. With atomic oxygen, Ti is pulled out of the surface layer leaving behind a Ni-rich subsurface region. Molecular O2, on the other hand adsorbs dissociatively and pulls a Ti atom farther out of the surface layer. The addition of further O2 up to 1 monolayer is also dissociative and results in complete removal of Ti from the initial surface layer. When further O2 is added up to 2 monolayer, Ti is pulled even further out of the surface and a single thin layer of composition O–Ti–O is formed. The electronic structure shows that the metallic character of the alloy is unaffected by interaction with oxygen and formation of the oxide layer, consistent with the oxide layer being a passivant.
Keywords: Density functional theory; NiTi; TiO2; biocompatibility; oxidation;

In order to be used as a material for neural prostheses, polyimide has to withstand the body environment over years. To obtain more information about the long-term stability of this material, we tested three commercially available polyimides (PI2611 – HD-Microsystems (type: BPDA-PPD), U-Varnish-S – UBE (type: BPDA-PPD), Durimide 7510 – Fujifilm (type: information not provided)). Specimens were stored in phosphate buffered saline (PBS) at 37 °C to simulate body temperature and at elevated temperatures of 60 °C and 85 °C to accelerate aging. During the course of 20 months, stress-strain curves were measured monthly by tensile testing. From these curves the Young's modulus, the stress and strain at break, the stress at 10% strain as well as the fracture energy were extracted and used to characterize and to statistically evaluate the mechanical material properties. Mass loss was determined by weighing polyimide foils (Upilex25S - UBE) and optical measurements were conducted to examine changes in chemical structure and crystallinity. At 37 °C and 60 °C no change in material behavior could be observed, except for an increase of the Young's modulus of the BPDA-PPD type stored at 60 °C. This demonstrates the long-term stability of all tested polyimides with respect to PBS. All extracted quantities mentioned above, as well as the mass, decreased in specimens stored in PBS at 85 °C. As a comparison, BPDA-PPD type specimens stored at 85 °C in deionized water showed no change in any property but behaved similarly to the reference material.
Keywords: Neural prostheses; Polyimide degradation; Accelerated aging; Mechanical properties; Phosphate buffered saline;

A circular cross-section PDMS microfluidics system for replication of cardiovascular flow conditions by Lindsey K. Fiddes; Neta Raz; Suthan Srigunapalan; Ethan Tumarkan; Craig A. Simmons; Aaron R. Wheeler; Eugenia Kumacheva (3459-3464).
Since the inception of soft lithography, microfluidic devices for cardiovascular research have been fabricated easily and cost-effectively using the soft lithography method. The drawback of this method was the fabrication of microchannels with rectangular cross-sections, which did not replicate the circular cross-sections of blood vessels. This article presents a novel, straightforward approach for the fabrication of microchannels with circular cross-sections in poly(dimethylsiloxane) (PDMS), using soft lithography. The method exploits the polymerization of the liquid silicone oligomer around a gas stream when both of them are coaxially introduced in the microchannel with a rectangular cross-section. We demonstrate (i) the ability to control the diameter of circular cross-sections of microchannels from ca. 40–100 μm; (ii) the fabrication of microchannels with constrictions, and (iii) the capability to grow endothelial cells on the inner surface of the microchannels.
Keywords: Microchannels; Flow; Cardiovascular system; Circular cross-section; Patterning; Seeding of endothelial cells;

The electron beam deposition of titanium on polyetheretherketone (PEEK) and the resulting enhanced biological properties by Cheol-Min Han; Eun-Jung Lee; Hyoun-Ee Kim; Young-Hag Koh; Keung N. Kim; Yoon Ha; Sung-Uk Kuh (3465-3470).
The surface of polyetheretherketone (PEEK) was coated with a pure titanium (Ti) layer using an electron beam (e-beam) deposition method in order to enhance its biocompatibility and adhesion to bone tissue. The e-beam deposition method was a low-temperature coating process that formed a dense, uniform and well crystallized Ti layer without deteriorating the characteristics of the PEEK implant. The Ti coating layer strongly adhered to the substrate and remarkably enhanced its wettability. The Ti-coated samples were evaluated in terms of their in vitro cellular behaviors and in vivo osteointegration, and the results were compared to a pure PEEK substrate. The level of proliferation of the cells (MC3T3-E1) was measured using a methoxyphenyl tetrazolium salt (MTS) assay and more than doubled after the Ti coating. The differentiation level of cells was measured using the alkaline phosphatase (ALP) assay and also doubled. Furthermore, the in vivo animal tests showed that the Ti-coated PEEK implants had a much higher bone-in-contact (BIC) ratio than the pure PEEK implants. These in vitro and in vivo results suggested that the e-beam deposited Ti coating significantly improved the potential of PEEK for hard tissue applications.
Keywords: Polyetheretherketone (PEEK); Ti coating; Biocompatibility; e-beam;

Biocompatible, hyaluronic acid modified silicone elastomers by Johan G. Alauzun; Stuart Young; Renita D'Souza; Lina Liu; Michael A. Brook; Heather D. Sheardown (3471-3478).
Although silicones possess many useful properties as biomaterials, their hydrophobicity can be problematic. To a degree, this issue can be addressed by surface modification with hydrophilic polymers such as poly(ethylene glycol), but the resulting structures are usually not conducive to cell growth. In the present work, we describe the synthesis and characterization of covalently linked hyaluronic acid (HA) (35 kDa) to poly(dimethylsiloxane) (PDMS) elastomer surfaces. HA is of interest because of its known biological properties; its presence on a surface was expected to improve the biocompatibility of silicone materials for a wide range of bioapplications. HA was introduced with a coupling agent in two steps from high-density, tosyl-modified, poly(ethylene glycol) tethered silicone surfaces. All materials synthesized were characterized by water contact angle, ATR-FTIR, XPS and 13C solid state NMR spectroscopy. Biological interactions with these modified silicone surfaces were assessed by examining interactions with fibrinogen as a model protein as well as determining the in vitro response of fibroblast (3T3) and human corneal epithelial cells relative to unmodified poly(dimethylsiloxane) controls. The results suggest that HA modification significantly enhances cell interactions while decreasing protein adsorption and may therefore be effective for improving biocompatibility of PDMS and other materials.
Keywords: Hyaluronic acid; Poly(dimethylsiloxane) elastomer; Protein adsorption; Cell interactions; Tosyl-modified poly-(ethylene glycol);

Characterization of topographical effects on macrophage behavior in a foreign body response model by Sulin Chen; Jacqueline A. Jones; Yongan Xu; Hong-Yee Low; James M. Anderson; Kam W. Leong (3479-3491).
Current strategies to limit macrophage adhesion, fusion and fibrous capsule formation in the foreign body response have focused on modulating material surface properties. We hypothesize that topography close to biological scale, in the micron and nanometric range, provides a passive approach without bioactive agents to modulate macrophage behavior. In our study, topography-induced changes in macrophage behavior was examined using parallel gratings (250 nm–2 μm line width) imprinted on poly(ε-caprolactone) (PCL), poly(lactic acid) (PLA) and poly(dimethyl siloxane) (PDMS). RAW 264.7 cell adhesion and elongation occurred maximally on 500 nm gratings compared to planar controls over 48 h. TNF-α and VEGF secretion levels by RAW 264.7 cells showed greatest sensitivity to topographical effects, with reduced levels observed on larger grating sizes at 48 h. In vivo studies at 21 days showed reduced macrophage adhesion density and degree of high cell fusion on 2 μm gratings compared to planar controls. It was concluded that topography affects macrophage behavior in the foreign body response on all polymer surfaces examined. Topography-induced changes, independent of surface chemistry, did not reveal distinctive patterns but do affect cell morphology and cytokine secretion in vitro, and cell adhesion in vivo particularly on larger size topography compared to planar controls.
Keywords: Microtopography; Nanotopography; Foreign body response; Nanostructured biomaterials; Inflammation and wound healing; Cytokines;

Template synthesized poly(ɛ-caprolactone) nanowire surfaces for neural tissue engineering by Samuel L. Bechara; Anna Judson; Ketul C. Popat (3492-3501).
Tissue engineering therapies targeted at nerve regeneration in spinal cord injuries (SCI) have broad social and economic benefits to the American population. Due to the complicated pathophysiology of SCI, there are very few options available for functional regeneration of the spinal column. Nanotechnology offers interesting avenues to explore tissue engineering in SCI. In this study, we have developed a novel solvent free nanotemplating technique for fabricating poly(ɛ-caprolactone) (PCL) surfaces with controlled arrays of high aspect ratio substrate-bound nanowires for the growth and maintenance of differentiated states of neuronal cells. PC12 cells were used to evaluate the ability of nanowire surfaces to promote neuronal phenotypic behavior. Cell adhesion, proliferation and viability were investigated for up to 4 days of culture using fluorescence microscopy, scanning electron microscopy (SEM) and MTT activity. Our results indicate significantly higher cell adhesion and subsequent proliferation and viability of PC12 cells cultured on nanowire surfaces as compared to control surfaces without any nanoarchitecture. Further, the adhered cells were maintained in a differentiated state for 7 days and neuronal network formation and expression of neuronal markers were investigated using fluorescence microscopy, SEM and immunofluorescence. Cells on nanowire surfaces expressed key neuronal markers and demonstrated neuronal phenotypic behavior as compared to the cells on control surfaces.
Keywords: poly(ɛ-caprolactone) nanowires; Neural tissue engineering; Neuronal network; Nanotemplating; PC12 cells;

Biodegradable magnetic-fluorescent magnetite/poly(dl-lactic acid-co-α,β-malic acid) composite nanoparticles for stem cell labeling by Liang Wang; Koon-Gee Neoh; En-Tang Kang; Borys Shuter; Shih-Chang Wang (3502-3511).
Bifunctional superparamagnetic magnetite/poly(dl-lactic acid-co-α,β-malic acid) composite nanoparticles (PLMA–MNPs) detectable by both magnetic resonance imaging (MRI) and fluorescence microscopy were synthesized by coating Fe3O4 nanoparticles with biodegradable poly(dl-lactic acid-co-α,β-malic acid) copolymer (PLMA) with covalently bound fluorescein isothiocyanate (FITC). The FITC modified PLMA–MNPs (FITC–PLMA–MNPs) have a hydrodynamic diameter of 100 nm and an anionic surface. MTT assays of mouse macrophages, 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) incubated with these nanoparticles indicated that these nanoparticles did not possess significant cytotoxicity. Furthermore, the osteogenic and adipogenic differentiations of the hMSCs were not influenced by the labeling process. As a result of the high R 2 (164.8 mm −1  s−1) and R 2/R 1 ratio (32) of the FITC–PLMA–MNPs, the labeled hMSCs can be detected by a clinical 3T MRI scanner at an in vitro detection threshold of about 1200 cells. The green fluorescence associated with the FITC can be readily observed. Such nanoparticles can potentially be used as a T 2-weighted contrast agent and fluorescent agent for stem cell labeling.
Keywords: Nanoparticles; Biodegradable; MRI; Stem cells; Poly(dl-lactic acid-co-α,β-malic acid);

The induction of bone formation in rat calvarial defects and subcutaneous tissues by recombinant human BMP-2, produced in Escherichia coli by Ji-Hyun Lee; Chang-Sung Kim; Kyung-Hee Choi; Ui-Won Jung; Jeong-Ho Yun; Seong-Ho Choi; Kyoo-Sung Cho (3512-3519).
We investigated the ability of recombinant human bone morphogenetic protein-2, produced from Escherichia coli (ErhBMP-2), to form orthotopic and ectopic bone in rat models. BMP-2 was expressed in E. coli and extracted from the inclusion bodies. Critical-sized calvarial defects and subcutaneous pouches were created in rats, and an absorbable collagen sponge (ACS) was loaded with different doses of ErhBMP-2 for implantation. ACS alone and sham surgery controls were also included. Implant sites were evaluated by histological and/or histometric analyses following a 2- or 8-week healing interval.In the calvarial defect model, enhanced bone formation was observed with all doses of ErhBMP-2, while only limited amounts of new bone were found in controls. In the ectopic subcutaneous implant model, bone formation was clearly observed in all animals treated with ErhBMP-2 at 2 weeks. However, at 8 weeks, less new bone formation was detected than at 2 weeks. Nevertheless, the remaining new bone showed an advanced degree of bone remodeling and more maturity than that observed at 2 weeks. These results showed that ErhBMP-2 was osteoinductive under controlled in vivo conditions. Thus, ErhBMP-2 has definite potential as an alternative to rhBMP-2 produced in a eukaryotic system for clinical use.
Keywords: BMP (bone morphogenetic protein); Bone regeneration; Bone tissue engineering; Histomorphometry; In vivo test;

Hydrated xenogeneic decellularized tracheal matrix as a scaffold for tracheal reconstruction by Nathaniel T. Remlinger; Caitlin A. Czajka; Mark E. Juhas; David A. Vorp; Donna B. Stolz; Stephen F. Badylak; Sebastien Gilbert; Thomas W. Gilbert (3520-3526).
Tracheal injury is a rare but complex problem. Primary tracheal reconstructions are commonly performed, but complications such as tension and inadequate vascular supply limit the length of surgical resection. The objective of the present study was to determine whether a hydrated, decellularized porcine tracheal extracellular matrix showed the potential to serve as a functional tracheal replacement graft. Porcine tracheas were decellularized and evaluated to characterize their biochemical composition and biomechanical behavior. Hydrated decellularized tracheal matrix (HDTM) grafts (>5 cm) were implanted heterotopically beneath the strap muscle and wrapped in the omentum in a canine model for 2 and 8 weeks followed by histologic and mechanical analysis. HDTM patches (2 × 3 cm) were also used in a patch tracheoplasty model. The repair site was evaluated bronchoscopically and radiographically, and the grafts were analyzed by histologic methods to evaluate epithelialization and persistence of the cartilage rings. The present study showed that HDTM maintains mechanical characteristics necessary for function under physiologic loading conditions even after 8 weeks of heterotopic implantation. After orthotopic implantation, the grafts were shown to support development of a columnar, pseudostratified, ciliated epithelium, but the cartilage structures showed histologic evidence of degradation and limited new cartilage formation. The results of the study showed tracheal ECM scaffolds support the formation of site-specific epithelium and provide sufficient mechanical integrity withstand physiologic pressures in the short-term. However, for long-term success, it appears that pre-implantation to allow vascularization or preseeding of the graft with chondrocytes will be necessary.
Keywords: Trachea; Cartilage; ECM (extracellular matrix); Epithelium; In vivo test; Mechanical properties;

Dose-dependent effect of adipose-derived adult stem cells on vertical bone regeneration in rabbit calvarium by Francesco Pieri; Enrico Lucarelli; Giuseppe Corinaldesi; Nicolò Nicoli Aldini; Milena Fini; Annapaola Parrilli; Barbara Dozza; Davide Donati; Claudio Marchetti (3527-3535).
Previous in vivo studies have shown a limited potential for vertical bone regeneration using osteoconductive scaffolds alone. In the present study, we investigated whether the association of adipose-derived adult stem cells (ASCs) with anorganic bovine bone (ABB) scaffold improved bone formation and implant osseointegration in a vertical guided bone regeneration model. Two pre-formed titanium domes were placed on the calvaria of 12 rabbits. Four treatment modalities were evenly distributed among the 24 domes: ABB alone, and ABB containing 3 × 105, 3 × 106, or 3 × 107 cells/graft. After 1 month, the domes were removed and one titanium implant was placed into each augmented site. One month after the second operation, the animals were killed and biopsy specimens were examined by histomorphometric and micro-CT analyses. Results indicated that at all concentrations, the ASC-loaded groups showed significantly more new bone formation and higher mean values of bone–implant contact and bone density inside threads than the ABB group. Furthermore, ASCs demonstrated a dose–response relationship, with the highest dose chosen inducing more robust bone regeneration. This study suggests that the delivery of ASCs on ABB might effectively increase vertical bone regeneration and implant osseointegration, versus ABB alone.
Keywords: Bone tissue engineering; Adipose-derived stem cells; Bone regeneration; Hydroxyapatite; Dental implants; Histomorphometry;

The effect of fiber alignment and heparin coating on cell infiltration into nanofibrous PLLA scaffolds by Kyle T. Kurpinski; Jacob T. Stephenson; Randall Raphael R. Janairo; Hanmin Lee; Song Li (3536-3542).
Biodegradable nanofibers simulate the fibril structure of natural extracellular matrix, and provide a cell-friendly microenvironment for tissue regeneration. However, the effects of nanofiber organization and immobilized biochemical factors on cell infiltration into three-dimensional scaffolds are not well understood. For example, cell infiltration into an electrospun nanofibrous matrix is often limited due to relatively small pore size between the fibers. Here we showed that biophysical and biochemical modification of nanofibrous scaffolds facilitated endothelial cell infiltration in three-dimensional scaffolds in vitro and in vivo. Aligned nanofibers significantly enhanced cell infiltration into the nanofibrous matrices in vitro. In a full-thickness dermal wound model, the nanofiber scaffolds enhanced epidermal skin cell migration across the wound when compared to a control group without scaffold. Aligned nanofibers promoted the infiltration of endothelial cells into the scaffolds. Furthermore, heparin-coated nanofibers also increased cell infiltration significantly. These results shed light on the importance of biophysical and biochemical properties of nanofibers in the regulation of cell infiltration into three-dimensional scaffolds and tissue remodeling.
Keywords: Electrospinning; Nanofibers; Tissue engineering; Wound healing; Heparin; Endothelial cells;

The osteoblastic differentiation of dental pulp stem cells and bone formation on different titanium surface textures by Carlo Mangano; Alfredo De Rosa; Vincenzo Desiderio; Riccardo d'Aquino; Adriano Piattelli; Francesco De Francesco; Virginia Tirino; Francesco Mangano; Gianpaolo Papaccio (3543-3551).
Bone Tissue Engineering (BTE) and Dental Implantology (DI) require the integration of implanted structures, with well characterized surfaces, in bone. In this work we have challenged acid-etched titanium (AET) and Laser Sintered Titanium (LST) surfaces with either human osteoblasts or stem cells from human dental pulps (DPSCs), to understand their osteointegration and clinical use capability of derived implants. DPSCs and human osteoblasts were challenged with the two titanium surfaces, either in plane cultures or in a roller apparatus within a culture chamber, for hours up to a month. During the cultures cells on the titanium surfaces were examined for histology, protein secretion and gene expression. Results show that a complete osteointegration using human DPSCs has been obtained: these cells were capable to quickly differentiate into osteoblasts and endotheliocytes and, then, able to produce bone tissue along the implant surfaces. Osteoblast differentiation of DPSCs and bone morphogenetic protein production was obtained in a better and quicker way, when challenging stem cells with the LST surfaces. This successful BTE in a comparatively short time gives interesting data suggesting that LST is a promising alternative for clinical use in DI.
Keywords: Titanium; Surface texturing; Osteodifferentiation; DPSCs; Osteoblasts;

Multiscale osteointegration as a new paradigm for the design of calcium phosphate scaffolds for bone regeneration by Sheeny K. Lan Levengood; Samantha J. Polak; Matthew B. Wheeler; Aaron J. Maki; Sherrie G. Clark; Russell D. Jamison; Amy J. Wagoner Johnson (3552-3563).
The role of macropore size (>100 μm) and geometry in synthetic scaffolds for bone regeneration has been studied extensively, but successful translation to the clinic has been slow. Significantly less attention has been given to porosity at the microscale (0.5–10 μm). While some have shown that microporosity in calcium phosphate (CaP)-based scaffolds can improve rate and extent of bone formation in macropores, none has explored microporosity as an additional and important space for bone ingrowth. Here we show osteointegration of biphasic calcium phosphate (BCP) scaffolds at both the macro and micro length scales. Bone, osteoid, and osteogenic cells fill micropores in scaffold rods and osteocytes are embedded in mineralized matrix in micropores, without the addition of growth factors. This work further highlights the importance of considering design parameters at the microscale and demonstrates the possibility for a bone–scaffold composite with no “dead space.” Embedded osteocytes distributed throughout microporous rods may form a mechanosensory network, which would not be possible in scaffolds without microporosity. Multiscale osteointegration has the potential to greatly improve overall performance of these scaffolds through an improvement of mechanical properties, load transfer, and stability in the long and short term, and represents a new paradigm for scaffold design.
Keywords: Bone ingrowth; Calcium phosphate; Porosity; Microstructure; Osteointegration;

Potent in vitro chondrogenesis of CD105 enriched human adipose-derived stem cells by Ting Jiang; Wei Liu; Xiaojie Lv; Hengyun Sun; Lu Zhang; Yu Liu; Wen Jie Zhang; Yilin Cao; Guangdong Zhou (3564-3571).
Adipose-derived stem cells (ASCs) are considered as a promising cell source for cartilage regeneration. However, the heterogeneity of this cell source may affect their ability in cartilage formation. It is therefore necessary to establish an efficient method for isolating the cells that have chondrogenic potential. To date, no specific markers have been reported to be able to isolate such a cell population from human adipose tissue. In recent studies, endoglin (CD105) has been known as a relatively specific marker for identifying mesenchymal stem cells, but no studies show it is related to chondrogenic potential of human ASCs. In this study, human cells from adipose tissue were isolated, cultured, and sorted according to CD105 expression. The sorted cells were then subjected to adipogenic, osteogenic, and chondrogenic induction to confirm their multi-potentiality. In adipogenic conditions, CD105− cells showed stronger Oil Red staining and higher expression of adipose-specific genes compared to CD105+ cells. By contrast, CD105+ cells exhibited better osteogenic potential with stronger Alizarin Red staining and higher expression of osteogenic specific genes than CD105− cells. Noticeably, CD105+ cells also exhibited a much stronger chondrogenic potential than CD105− cells, with stronger collagen II staining and higher gene expression of collagen II and aggrecan. Most importantly, CD105+ cells could form a homogeneous cartilage-like tissue when seeded into a biodegradable scaffold and cultured in chondrogenic media for 8 weeks. These results indicate that sorting of ASC subpopulation with CD105 as a marker may allow better in vitro chondrogenesis and thus provide an important implications for cartilage regeneration and reconstruction using autologous cells from adipose tissue.
Keywords: Adipose-derived stem cells (ASCs); Endoglin (CD105); Cell sorting; Chondrogenic potential; Chondrogenesis;

Comparison of mesenchymal stem cells from bone marrow and adipose tissue for bone regeneration in a critical size defect of the sheep tibia and the influence of platelet-rich plasma by Philipp Niemeyer; Katharina Fechner; Stefan Milz; Wiltrud Richter; Norbert P. Suedkamp; Alexander T. Mehlhorn; Simon Pearce; Philip Kasten (3572-3579).
Aim of the present study was to compare the osteogenic potential of bone marrow derived mesenchymal stem cells (BMSC) and adipose-tissue derived stem cells (ASC) and to evaluate the influence of platelet-rich plasma (PRP) on the osteogenic capacity of ASC in a large animal model.Ovine BMSC (BMSC-group) and ASC (ASC-group) were seeded on mineralized collagen sponges and implanted into a critical size defect of the sheep tibia (n = 5 each). In an additional group, platelet-rich plasma (PRP) was used in combination with ASC (PRP-group). Unloaded mineralized collagen (EMPTY-group) served as control (n = 5 each). Radiographic evaluation was performed every 2 weeks, after 26 weeks histological analysis was performed.Radiographic evaluation revealed a significantly higher amount of newly formed bone in the BMSC-group compared to the ASC-group at week 10 and compared to EMPTY-group from week 12 (all p < 0.05). A superiority on radiographic level concerning bone formation of the PRP-group versus the empty control group was found (p < 0.05), but not for the ASC-group. Histological analysis confirmed radiographic evaluation finding analogous significances.In conclusion, ASC seem to be inferior to BMSC in terms of their osteogenic potential but that can partially be compensated by the addition of PRP.
Keywords: Bone, tissue engineering; Bone regeneration; Platelet; Growth factors; Stem cell; Adipose tissue;

The periosteum plays an indispensable role in both bone formation and bone defect healing. In this study we constructed an artificial in vitro periosteum by incorporating osteogenic differentiated bone marrow stromal cells (BMSCs) and cobalt chloride (CoCl2)-treated BMSCs. The engineered periostea were implanted both subcutaneously and into skull bone defects in SCID mice to investigate ectopic and orthotopic osteogenesis and vascularization. After two weeks in subcutaneous and four weeks in bone defect areas, the implanted constructs were assessed for ectopic and orthotopic osteogenesis and vascularization by micro-CT, histomorphometrical and immunohistochemical methods. The results showed that CoCl2 pre-treated BMSCs induced higher degree of vascularization and enhanced osteogenesis within the implants in both ectopic and orthotopic areas. This study provided a novel approach using BMSCs sourced from the same patient for both osteogenic and pro-angiogenic purposes in constructing tissue engineered periosteum to enhance vascularized osteogenesis.
Keywords: Periosteum; Tissue engineering; Osteoblasts; Vascularization; Cobalt chloride;

The use of high-hydrostatic pressure treatment to decellularize blood vessels by Seiichi Funamoto; Kwangwoo Nam; Tsuyoshi Kimura; Ayako Murakoshi; Yoshihide Hashimoto; Kazuo Niwaya; Soichiro Kitamura; Toshiya Fujisato; Akio Kishida (3590-3595).
A decellularization method using high-hydrostatic pressure (HHP) technology (>600 MPa) is described. The HHP disrupts the cells inside the tissue. The cell debris can be eliminated with a simple washing process, producing clean, decellularized tissue. In this study, porcine aortic blood vessel was decellularized by HHP. The mechanical properties and in vivo performance of the decellularized tissue were evaluated. Mechanical properties of the decellularized tissue were not altered by the HHP treatment. Reduced inflammation of the decellularized tissue was confirmed by xenogenic transplant experimentation. An allogenic transplantation study showed that decellularized blood vessel endured the arterial blood pressure, and there was no clot formation on the luminal surface. In addition, cellular infiltration into the vessel wall was observed 4 weeks after implantation, suggesting that HHP treatments could be applied widely as a high-quality decellularization method.
Keywords: Arterial tissue engineering; Vascular graft; Mechanical property; Transplantation;

Heparin-based hydrogel as a matrix for encapsulation and cultivation of primary hepatocytes by Mihye Kim; Ji Youn Lee; Caroline N. Jones; Alexander Revzin; Giyoong Tae (3596-3603).
Primary hepatocytes are commonly used as liver surrogates in toxicology and tissue engineering fields, therefore, maintenance of functional hepatocytes in vitro is an important topic of investigation. This paper sought to characterize heparin-based hydrogel as a three-dimensional scaffold for hepatocyte culture. The primary rat hepatocytes were mixed with a prepolymer solution comprised of thiolated heparin and acrylated poly(ethylene glycol) (PEG). Raising the temperature from 25° to 37 °C initiated Michael addition reaction between the thiol and acrylated moieties and resulted in formation of hydrogel with entrapped cells. Analysis of liver-specific products, albumin and urea, revealed that the heparin hydrogel was non-cytotoxic to cells and, in fact, promoted hepatic function. Hepatocytes entrapped in the heparin-based hydrogel maintained high levels of albumin and urea synthesis after three weeks in culture. Because heparin is known to bind growth factors, we incorporated hepatocyte growth factor (HGF)–an important liver signaling molecule - into the hydrogel. HGF release from heparin hydrogel matrix was analyzed using enzyme linked immunoassay (ELISA) and was shown to occur in a controlled manner with only 40% of GF molecules released after 30 days in culture. Importantly, hepatocytes cultured within HGF-containing hydrogels exhibited significantly higher levels of albumin and urea synthesis compared to cells cultured in the hydrogel alone. Overall, heparin-based hydrogel showed to be a promising matrix for encapsulation and maintenance of difficult-to-culture primary hepatocytes. In the future, we envision employing heparin-based hyrogels as matrices for in vitro differentiation of hepatocytes or stem cells and as vehicles for transplantation of these cells.
Keywords: Liver; Hepatocyte cultivation; Growth factors; Hydrogels; Heparin;

Rat hepatocyte aggregate formation on discrete aligned nanofibers of type-I collagen-coated poly(l-lactic acid) by Zhang-Qi Feng; Xue-Hui Chu; Ning-Ping Huang; Michelle K. Leach; Gan Wang; Yi-Chun Wang; Yi-Tao Ding; Zhong-Ze Gu (3604-3612).
Primary hepatocytes cultured in three dimensional tissue constructs composed of multicellular aggregates maintain normal differentiated cellular function in vitro while cultured monolayers do not. Here, we report a technique to induce hepatocyte aggregate formation using type-I collagen-coated poly(l-lactic acid) (PLLA) discrete aligned nanofibers (disAFs) by providing limited cell-substrate adhesion strength and restricting cell migration to uniaxial movement. Kinetics of aggregate formation, morphology and biochemical activities of rat hepatocyte aggregates were tested over a 15 day culture period. Evidence was provided that physical cues from disAFs quickly induced the formation of aggregates. After 3 days in culture, 88.3% of free hepatocytes on disAFs were incorporated into aggregates with an average diameter of 61 ± 18 μm. Hepatocyte aggregates formed on disAFs displayed excellent cell retention, cell activity and stable functional expression in terms of albumin secretion, urea synthesis and phase I and II (CYP1A and UGT) metabolic enzyme activity compared to monolayer culture of hepatocytes on tissue culture plastic (TCP) with type-I collagen as well as on meshes of type-I collagen-coated PLLA random nanofibers (meshRFs). These results suggest that disAFs may be a suitable method to maintain large-scale hepatic cultures with high activity for tissue engineering research and potential therapeutic applications, such as bioartificial liver devices.
Keywords: Hepatocyte; Cell culture; Scaffold; Nanotopography; Collagen; Bioartificial liver;

Biohybrid thin films for measuring contractility in engineered cardiovascular muscle by Patrick W. Alford; Adam W. Feinberg; Sean P. Sheehy; Kevin K. Parker (3613-3621).
In vitro cardiovascular disease models need to recapitulate tissue-scale function in order to provide in vivo relevance. We have developed a new method for measuring the contractility of engineered cardiovascular smooth and striated muscle in vitro during electrical and pharmacological stimulation. We present a growth theory-based finite elasticity analysis for calculating the contractile stresses of a 2D anisotropic muscle tissue cultured on a flexible synthetic polymer thin film. Cardiac muscle engineered with neonatal rat ventricular myocytes and paced at 0.5 Hz generated stresses of 9.2 ± 3.5 kPa at peak systole, similar to measurements of the contractility of papillary muscle from adult rats. Vascular tissue engineered with human umbilical arterial smooth muscle cells maintained a basal contractile tone of 13.1 ± 2.1 kPa and generated another 5.1 ± 0.8 kPa when stimulated with endothelin-1. These data suggest that this method may be useful in assessing the efficacy and safety of pharmacological agents on cardiovascular tissue.
Keywords: Soft tissue biomechanics; tissue biomechanics; Cardiac tissue engineering; Cardiomyocyte; Smooth muscle cell; Mechanical properties;

The role of collagen reorganization on mammary epithelial morphogenesis in a 3D culture model by Eugen Dhimolea; Maricel V. Maffini; Ana M. Soto; Carlos Sonnenschein (3622-3630).
Collagen-based three-dimensional (3D) in vitro models that recapitulate the structural and functional context of normal and malignant tissues provide a relevant surrogate to animal models in the study of developmental and carcinogenic processes. Human breast epithelial MCF10A cells embedded in a collagen gel formed both acinar and tubular structures only when the gel was detached (floating) from the cell culture plate's well, and allowed to be contracted by the cells. Epithelial phenotype depended upon the time and the location within the gel, as ducts formed exclusively on the upper layer of the gel while ductal branching occurred earlier in the central area of the gel, and gradually progressed toward the periphery. The addition of fibroblasts accelerated tubulogenesis. MCF10A cells facilitated the organization of thick collagen fibers packed into large bundles at the tip of the ducts and parallel to the direction of ductal elongation. In gels that were not detached from the well's wall, MCF10A cells organized in monolayer and collagen fibers were aligned along the axis of outstretched sprouts stemming from those cellular aggregates. Partial gel release induced uniaxial tubulogenesis associated with orderly aligned collagen fibers. These results suggest that proper collagen organization is necessary for epithelial morphogenesis to occur, and that biomechanical interactions between fibers and cells mediated duct formation, elongation and branching.
Keywords: MCF10A; Tubulogenesis; Collagen fibers; Picrosirius red; Floating gels; Biophysical forces;

Controlled delivery of therapeutic agents from medical devices can improve their safety and effectiveness in vivo, by ameliorating the surrounding tissue responses and thus maintaining the functional integrity of the devices. Previously, we presented a new method for providing simultaneous controlled delivery from medical devices, by surface assembly of biodegradable polymer nanoparticles (NPs) encapsulating fluorescent dyes. Here, we continue our investigation with NPs loaded with therapeutic agents, dexamethasone (DEX) or plasmid DNA, and evaluated the bioactivity of the released molecules with macrophage cells associated with inflammation. Over a period of one week, NPs encapsulating DEX released 24.9 ± 0.8 ng from the probe surface and was successful at suppressing macrophage cell growth by 40 ± 10%. This percentage of suppression corresponded to ∼100% drug delivery efficiency, in comparison with the unencapsulated drug. DNA NP coatings, in contrast, released ∼1 ng of plasmid DNA and were effective at transfecting macrophage cells to express the luciferase gene at 300 ± 200 relative luminescence/mg total protein. This amount of luciferase activity corresponded to 100% gene delivery efficiency. Thus, NP coatings were capable of providing continuous release of bioactive agents in sufficient quantities to induce relevant biological effects in cell culture studies. These coatings also remained intact, even after 14 days of incubation with phosphate buffered saline. Although the maximum loading for NP coatings is inherently lower than the more established matrix coating, our study suggests that the NP coatings are a more versatile and efficient approach toward drug delivery or gene delivery from a medical device surface and are perhaps best suited for continuous release of highly potent therapeutic agents.
Keywords: Nanoparticle; Self-assembly; Biocompatibility; Dexamethasone; Plasmid DNA;

Use of vitamin E to protect cross-linked UHMWPE from oxidation by Reto Lerf; Daniel Zurbrügg; Daniel Delfosse (3643-3648).
Wear and oxidative degradation may limit the life span of UHMWPE implants. Cross-linking and stabilisation by vitamin E are proposed to overcome wear and degradation. The present investigation takes a close look to the oxidative behaviour of cross-linked and stabilised UHMWPE. First, the consolidated vitamin E stabilised UHMWPE was qualified in terms of microstructure and homogeneity of the distribution of the additive to be suitable for oxidation profiles over the entire section. Then cross-linked samples with five different concentrations of vitamin E (nil to 1.0%) underwent two different ageing protocols. The first was under pressurized oxygen at 70 °C, as defined in the ASTM F 2003 standard with a prolonged period of 60 days, the second was in 5% aqueous hydrogen peroxide solution with iron (III) chloride as catalyst at 50 °C. The first accelerated ageing protocol showed that a vitamin E concentration as low as 0.05% is effective to protect irradiated highly cross-linked UHMWPE against oxidation when exposed direct to oxygen. Vitamin E stabilised, highly cross-linked UHMWPE exhibits therefore no oxidation potential origination from the irradiation treatment. Analysis of samples treated by the second chemical ageing yielded, that vitamin E is effective to prolong initial stability against a supplementary attack of hydrogen peroxide and reactive radicals. The time period of stability against the aggressive hydrogen peroxide solution increases with increasing vitamin E content. However, even 0.05% have a marked stabilisation effect. Therefore, such small additions of vitamin E are effective to protect the UHMWPE material against a supplementary exposure to in vivo oxidation after the irradiation treatment. In conclusion, vitamin E shields cross-linked UHMWPE for orthopaedic application against oxidation in the heat of consolidation, during irradiation treatment and finally while implanted in the human body.
Keywords: Vitamin E; Highly cross-linked ultra high molecular weight polyethylene; Oxidation; Joint replacement; Artificial accelerated ageing;

Many surgical methods and hemostatic agents can be used to achieve and maintain hemostasis in surgical fields. Numerous clinical situations exist where current treatment modalities are neither effective nor practical. Assessment of new hemostats primarily targets efficacy. However, the biocompatibility and healing properties associated with hemostats are crucial for regulatory approval and product acceptance. Standard biocompatibility and healing studies may not be appropriate for hemostats containing active biologics. Liver defects in NTac:NIH-Whn (athymic) and Sprague Dawley® ™ Outbred (immunocompetent) rats were treated with Fibrin Pad (absorbable matrix containing human-derived biologics) or the matrix only. Defects were evaluated at 14 and 28 days post-implantation. As expected, Fibrin Pad in immunocompetent rats induced a cellular immune response. Unexpectedly, biologically significant decreases in healing, material absorption, and local fibrin degradation were also present. Evaluation of Fibrin Pad in immunocompetent animal models must consider potentially significant alterations in healing, material absorption, and local fibrin degradation, in addition to the expected immune response; none of which may be relevant when Fibrin Pad is used in the clinical setting. These considerations are essential when standard efficacy and biocompatibility studies assessing Fibrin Pad are submitted for regulatory consideration or utilized as pre-clinical translational studies.
Keywords: Hemostasis; Thrombin; Fibrinogen; Immune response; Animal model; Biocompatibility;

Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles by Chunbai He; Yiping Hu; Lichen Yin; Cui Tang; Chunhua Yin (3657-3666).
To elucidate the effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles (NPs), rhodamine B (RhB) labeled carboxymethyl chitosan grafted NPs (RhB-CMCNP) and chitosan hydrochloride grafted NPs (RhB-CHNP) were developed as the model negatively and positively charged polymeric NPs, respectively. These NPs owned well defined particle sizes (150–500 nm) and Zeta potentials (−40 mV – +35 mV). FITC labeled protamine sulfate (FITC-PS) loaded RhB-CMCNP and camptothecin (CPT) loaded RhB-CHNP with high encapsulation efficiency were prepared. The fluorescence stability in plasma and towards I was investigated, and the result indicated it was sufficient for qualitative and quantitative analysis. NPs with high surface charge and large particle size were phagocytized more efficiently by murine macrophage. Slight particle size and surface charge differences and different cell lines had significant implications in the cellular uptake of NPs, and various mechanisms were involved in the uptake process. In vivo biodistribution suggested that NPs with slight negative charges and particle size of 150 nm were tended to accumulate in tumor more efficiently. These results could serve as a guideline in the rational design of drug nanocarriers with maximized therapeutic efficacy and predictable in vivo properties, in which the control of particle size and surface charge was of significance.
Keywords: Polymeric nanoparticles; Fluorescence labeling; Particle size; Surface charge; Cellular uptake; Biodistribution;

Water-soluble superparamagnetic manganese ferrite nanoparticles for magnetic resonance imaging by Hong Yang; Cuixia Zhang; Xiangyang Shi; He Hu; Xiaoxia Du; Yong Fang; Yanbin Ma; Huixia Wu; Shiping Yang (3667-3673).
We report here a thermal decomposition approach to the synthesis of water-soluble superparamagnetic manganese ferrite (MnFe2O4) nanoparticles (NPs) for magnetic resonance (MR) imaging applications. In this approach, tetraethylene glycol was utilized as a coordination and stabilization agent, rendering the NPs water-soluble and stable. The formed NPs had a diameter of 7 nm with a narrow size distribution, and were superparamagnetic with a saturated magnetization (Ms) of 39 emu/g. In vitro cytotoxicity test revealed that the MnFe2O4 NPs were biocompatible at a particle concentration below 200 μg/mL. The transverse relaxivity of MnFe2O4 NPs in water and cells after incubation were determined to be 189.3 mm −1  s−1 and 36.8 mm −1  s−1 based on iron concentration, respectively. In vivo MR imaging studies in conjunction with inductively coupled plasma-atomic emission spectroscopy showed that the MnFe2O4 NPs were preferentially accumulated in liver after intravenous injection for 4 h. This suggests that the developed MnFe2O4 NPs can serve as a sensitive MR imaging contrast agent for liver imaging. By appropriately modifying or functionalizing the surface of the NPs, these particles may be used for MR detection of other diseases.
Keywords: Manganese ferrite; Nanoparticles; MR imaging;

The influence of nano-scale surface roughness on bacterial adhesion to ultrafine-grained titanium by Vi K. Truong; Rimma Lapovok; Yuri S. Estrin; Stuart Rundell; James Y. Wang; Christopher J. Fluke; Russell J. Crawford; Elena P. Ivanova (3674-3683).
We discuss the effect of extreme grain refinement in the bulk of commercial purity titanium (CP, Grade-2) on bacterial attachment to the mechano-chemically polished surfaces of the material. The ultrafine crystallinity of the bulk was achieved by severe plastic deformation by means of equal channel angular pressing (ECAP). The chemical composition, wettability, surface topography and roughness of titanium surfaces were characterized using X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA) measurements, as well as atomic force microscopy (AFM) with 3D interactive visualization of the titanium surface morphology. It was found that physico-chemical surface characteristics of the as-received and the ECAP-modified CP titanium did not differ in any significant way, while the surface roughness at the nano-scale did. Optical profilometry performed on large scanning areas of approximately 225 μm × 300 μm showed that there was no significant difference between the roughness parameters R a and R q for surfaces in the two conditions, the overall level of roughness being lower for the ECAP-processed one. By contrast, topographic profile analysis at the nano-scale by AFM did reveal a difference in these parameters. This difference was sensitive to the size of the scanned surface area. A further two surface roughness parameters, skewness (R skw) and kurtosis (R kur), were also used to describe the morphology of titanium surfaces. It was found that the bacterial strains used in this study as adsorbates, viz. Staphylococcus aureus CIP 65.8 and Pseudomonas aeruginosa ATCC 9025, showed preference for surfaces of ECAP-processed titanium. S. aureus cells were found to have a greater propensity for attachment to surfaces of ECAP-modified titanium, while the attachment of P. aeruginosa, while also showing some preference for the ECAP-processed material, was less sensitive to the ECAP processing.
Keywords: Titanium surfaces; Equal channel angular pressing (ECAP); Bacterial adhesion; Staphylococcus aureus; Pseudomonas aeruginosa; Bioimplant materials;

Over the past decade, nanotechnology (or the use of materials with dimensions less than 100 nm in at least one direction) has been proposed to improve the lifespan of many biomedical devices, including orthopedic implants. Specifically, to improve the cytocompatibility properties of currently used orthopedic implants, nanotechnology has been used to create nanometer surface features (through anodization) on titanium. In addition to this approach, another therapeutic method widely investigated to heal bone fractures is through electrical stimulation. Here, the coupling of such nanotechnology approaches and electrical stimulation were studied to maximize bone cell functions on titanium. Results showed that compared to unstimulated conventional titanium, bone forming cell (osteoblast) proliferation and long-term functions (alkaline phosphatase synthesis, collagen type I synthesis and calcium deposition) were improved upon both the creation of an anodized nanotubular titanium surface and biphasic electrical stimulation. Most importantly, when electrical stimulation was combined with anodized nanotubular titanium features, osteoblast long-term functions were improved the most. Therefore, coupling the positive effects of anodized nanotubular titanium topographies with currently used therapeutic electrical stimulation should be further studied to improve orthopedic implants.
Keywords: Titanium; Surface modification; Nanotopography; Osseointegration; Osteoblasts;

Dual drug loaded superparamagnetic iron oxide nanoparticles for targeted cancer therapy by Fahima Dilnawaz; Abhalaxmi Singh; Chandana Mohanty; Sanjeeb K. Sahoo (3694-3706).
The primary inadequacy of chemotherapeutic drugs is their relative non-specificity and potential side effects to the healthy tissues. To overcome this, drug loaded multifunctional magnetic nanoparticles are conceptualized. We report here an aqueous based formulation of glycerol monooleate coated magnetic nanoparticles (GMO-MNPs) devoid of any surfactant capable of carrying high payload hydrophobic anticancer drugs. The biocompatibility was confirmed by tumor necrosis factor α assay, confocal microscopy. High entrapment efficiency ∼95% and sustained release of encapsulated drugs for more than two weeks under in vitro conditions was achieved for different anticancer drugs (paclitaxel, rapamycin, alone or combination). Drug loaded GMO-MNPs did not affect the magnetization properties of the iron oxide core as confirmed by magnetization study. Additionally the MNPs were functionalized with carboxylic groups by coating with DMSA (Dimercaptosuccinic acid) for the supplementary conjugation of amines. For targeted therapy, HER2 antibody was conjugated to GMO-MNPs and showed enhanced uptake in human breast carcinoma cell line (MCF-7). The IC50 doses revealed potential antiproliferative effect in MCF-7. Therefore, antibody conjugated GMO-MNPs could be used as potential drug carrier for the active therapeutic aspects in cancer therapy.
Keywords: Magnetic nanoparticles; Biocompatibility; Cytotoxicity; Targeted drug delivery; In vitro release; Cellular uptake;

Biodegradable polyamino acid-based polycations as safe and effective gene carrier minimizing cumulative toxicity by Keiji Itaka; Takehiko Ishii; Yoko Hasegawa; Kazunori Kataoka (3707-3714).
Gene delivery using cationic polymers has attracted much attention due to their potential advantages, such as large DNA loading capacity, ease of large-scale production, and reduced immunogenicity. We recently reported that polyplexes from poly[N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide] (P[Asp(DET)]), having an efficient endosomal escape due to pH-selective membrane destabilization, showed high transfection efficiency with minimal toxicity. Pharmacogenomic analysis demonstrated that P[Asp(DET)] also provided long-term security after transfection. We hypothesized that the biodegradability of P[Asp(DET)] played a significant role in achieving effective transfection. Gel permeation chromatography (GPC) and electrospray ionization mass spectrometry (ESI-MS) measurements of P[Asp(DET)] revealed their ability to undergo rapid degradation. In contrast, a derivative polycation, N-substituted polyglutamide (P[Glu(DET)]), showed no degradability, indicating that the degradation of P[Asp(DET)] was induced by a specific self-catalytic reaction between the PAsp backbone and the side-chain amide nitrogen. Degradation products of P[Asp(DET)] caused no cytotoxicity, even at high concentrations in the culture medium. Repeated transfection by administering the polyplexes for every 24 h showed that biodegradable P[Asp(DET)] provided a continuous increase in transgene expression, while non-degradable P[Glu(DET)] showed a decrease in transgene expression after 48 h, coupled with fluctuations in expression profiles of endogenous genes. In vivo intraperitoneal injection of P[Asp(DET)] induced minimal inflammatory cytokine induction to a level comparable to that of normal saline. These results indicate that the biodegradability of P[Asp(DET)] played a key role in achieving safe and sustained transgene expression, by minimizing cumulative toxicity caused by polycations remaining in cells or in the body.
Keywords: Non-viral gene carrier; Cationic polymer; Biodegradable polycation; Polyplex; Cumulative toxicity;

The adhesive properties of coacervated recombinant hybrid mussel adhesive proteins by Seonghye Lim; Yoo Seong Choi; Dong Gyun Kang; Young Hoon Song; Hyung Joon Cha (3715-3722).
Marine mussels attach to substrates using adhesive proteins. It has been suggested that complex coacervation (liquid–liquid phase separation via concentration) might be involved in the highly condensed and non-water dispersed adhesion process of mussel adhesive proteins (MAPs). However, as purified natural MAPs are difficult to obtain, it has not been possible to experimentally validate the coacervation model. In the present work, we demonstrate complex coacervation in a system including recombinant MAPs and hyaluronic acid (HA). Our recombinant hybrid MAPs, fp-151 and fp-131, can be produced in large quantities, and are readily purified. We observed successful complex coacervation using cationic fp-151 or fp-131, and an anionic HA partner. Importantly, we found that highly condensed complex coacervates significantly increased the bulk adhesive strength of MAPs in both dry and wet environments. In addition, oil droplets were successfully engulfed using a MAP-based interfacial coacervation process, to form microencapsulated particles. Collectively, our results indicate that a complex coacervation system based on MAPs shows superior adhesive properties, combined with additional valuable features including liquid/liquid phase separation and appropriate viscoelasticity. Our microencapsulation system could be useful in the development of new adhesive biomaterials, including self-adhesive microencapsulated drug carriers, for use in biotechnological and biomedical applications.
Keywords: Mussel adhesive protein; Hyaluronic acid; Coacervation; Bulk adhesion; Microencapsulation;

Synthesis, biophysical properties and pharmacokinetics of ultrahigh molecular weight tense and relaxed state polymerized bovine hemoglobins by Paul W. Buehler; Yipin Zhou; Pedro Cabrales; Yiping Jia; Guoyong Sun; David R. Harris; Amy G. Tsai; Marcos Intaglietta; Andre F. Palmer (3723-3735).
Hemoglobin-based oxygen carriers (HBOC) are currently being developed as red blood cell (RBC) substitutes for use in transfusion medicine. Despite significant commercial development, late stage clinical results of polymerized hemoglobin (PolyHb) solutions hamper development. We synthesized two types of PolyHbs with ultrahigh molecular weights: tense (T) state PolyHb (M W  = 16.59 MDa and P 50  = 41 mm Hg) and relaxed (R) state PolyHb (M W  = 26.33 MDa and P50  = 0.66 mm Hg). By maintaining Hb in either the T- or R-state during the polymerization reaction, we were able to synthesize ultrahigh molecular weight PolyHbs in distinct quaternary states with no tetrameric Hb, high viscosity, low colloid osmotic pressure and the ability to maintain O2 dissociation, CO association and NO dioxygenation reactions. The PolyHbs elicited some in vitro RBC aggregation that was less than 6% dextran (500 kDa) but more than 5% human serum albumin. In vitro, T-state PolybHb autoxidized faster than R-state PolybHb as expected from previously reported studies, conversely, when administered to guinea pigs as a 20% exchange transfusion, R-state PolybHb oxidized faster and to a greater extent than T-state PolybHb, suggesting a more complex oxidative processes in vivo. Our findings also demonstrate that T-state PolybHb exhibited a longer circulating half-life, slower clearance and longer systemic exposure time compared to R-state PolybHb.
Keywords: Hemoglobin; Red blood cell; Blood substitute; Oxygen carrier; Polymerized hemoglobin; Transfusion;

Bioactive hydrogels made from step-growth derived PEG–peptide macromers by Jordan S. Miller; Colette J. Shen; Wesley R. Legant; Jan D. Baranski; Brandon L. Blakely; Christopher S. Chen (3736-3743).
Synthetic hydrogels based on poly(ethylene glycol) (PEG) have been used as biomaterials for cell biology and tissue engineering investigations. Bioactive PEG-based gels have largely relied on heterobifunctional or multi-arm PEG precursors that can be difficult to synthesize and characterize or expensive to obtain. Here, we report an alternative strategy, which instead uses inexpensive and readily available PEG precursors to simplify reactant sourcing. This new approach provides a robust system in which to probe cellular interactions with the microenvironment. We used the step-growth polymerization of PEG diacrylate (PEGDA, 3400 Da) with bis-cysteine matrix metalloproteinase (MMP)-sensitive peptides via Michael-type addition to form biodegradable photoactive macromers of the form acrylate–PEG–(peptide–PEG)m-acrylate. The molecular weight (MW) of these macromers is controlled by the stoichiometry of the reaction, with a high proportion of resultant macromer species greater than 500 kDa. In addition, the polydispersity of these materials was nearly identical for three different MMP-sensitive peptide sequences subjected to the same reaction conditions. When photopolymerized into hydrogels, these high MW materials exhibit increased swelling and sensitivity to collagenase-mediated degradation as compared to previously published PEG hydrogel systems. Cell-adhesive acrylate–PEG–CGRGDS was synthesized similarly and its immobilization and stability in solid hydrogels was characterized with a modified Lowry assay. To illustrate the functional utility of this approach in a biological setting, we applied this system to develop materials that promote angiogenesis in an ex vivo aortic arch explant assay. We demonstrate the formation and invasion of new sprouts mediated by endothelial cells into the hydrogels from embedded embryonic chick aortic arches. Furthermore, we show that this capillary sprouting and three-dimensional migration of endothelial cells can be tuned by engineering the MMP-susceptibility of the hydrogels and the presence of functional immobilized adhesive ligands (CGRGDS vs. CGRGES peptide). The facile chemistry described and significant cellular responses observed suggest the usefulness of these materials in a variety of in vitro and ex vivo biologic investigations, and may aid in the design or refinement of material systems for a range of tissue engineering approaches.
Keywords: Polyethylene glycol; Hydrogel; Peptide; Cell encapsulation; Cell adhesion; Copolymer;