Biomaterials (v.30, #6)

The gene-expression and phenotypic response of hFOB 1.19 osteoblasts to surface-modified titanium and zirconia by Bernhard Setzer; Maria Bächle; Marc C. Metzger; Ralf J. Kohal (979-990).
The osteoblastic cell-line hFOB 1.19 with the potential to proliferate and differentiate revealed that cellular differentiation is not affected by material and roughness on newly developed zirconia implant materials. Materials under investigation were surfaces machined titanium (Ti-m), modified titanium (TiUnite®), machined zirconia (TZP-A-m), modified zirconia (ZiUnite®), machined alumina-toughened zirconia (ATZ-m) and modified alumina-toughened zirconia (ATZ-mod). After surface description by scanning electron microscopy (SEM) and atomic force microscopy (AFM), cellular proliferation (EZ4U, Casy1) and differentiation were examined after days 1, 3, 7, 14, 21, and 28. Osteogenic differentiation was visualized by alkaline phosphatase staining, mineralization assay (alizarin red) and by expression analysis (RT-PCR) of bone- and extracellular matrix-related genes. Proliferation on rough surfaces was reduced on both titanium and zirconia. Cell-attachment and cytoskeleton organization documented by confocal laser scanning microscopy (CLSM) elucidated attenuated cell attachment within the first 4 h to be the reason for impaired proliferation. A specific up-regulation of m-RNAs in an early event (RUNX2, NELL-1, RUNX3, and BMP7) and a late event (Integrin B3) could be observed on TiUnite® and ZiUnite®. For titanium an up-regulation of IBSP and Integrin B1 could be described at day 21. In total, differentiation was neither affected by material nor by roughness.
Keywords: Zirconia; hFOB 1.19; Cell differentiation; RT-PCR; SEM; CLSM;

Different mechanisms of spinal fusion using equine bone protein extract, rhBMP-2 and autograft during the process of anterior lumbar interbody fusion by Xuenong Zou; Lijin Zou; Casper Foldager; Michael Bendtsen; Wenzhou Feng; Cody E. Bünger (991-1004).
To elucidate the molecular mechanisms of spinal fusion with different graft materials during an anterior lumbar interbody fusion, we examined the gene-expression profiles after implantation of equine bone protein extract, rhBMP-2 and autograft using microarray technology and data analysis, including hierarchical clustering, self-organizing maps (SOM), KEGG pathway and Biological process GO analyses in a porcine model. The results suggest that equine bone protein extract exhibited a more similar expression pattern with autograft than that of rhBMP-2. rhBMP-2 recruits progenitor cells, proliferation and differentiation possibly by inducing various factors including PGHS-2, IFGBP-2, VEGF and chemokines and then leads to preferable membranous ossification and bone remodeling. Conversely, equine bone protein extract results in endochondral ossification via upregulation of cartilage-related genes. Ossification by inducing direct osteoblastic differentiation and obviating the cartilaginous intermediate phases may increase spinal fusion rate.
Keywords: Bone graft; Equine bone protein extract; BMP (bone morphogenetic protein); Gene expression; GeneChip;

Effect of partial hydrolysis of octacalcium phosphate on its osteoconductive characteristics by Naohisa Miyatake; Koshi N. Kishimoto; Takahisa Anada; Hideki Imaizumi; Eiji Itoi; Osamu Suzuki (1005-1014).
The present study was designed to investigate whether the stoichiometry of octacalcium phosphate OCP affects its osteoconductive and immune response characteristics in rat bone marrow. Those characteristics of synthetic, well-grown OCP but with a non-stoichiometric composition were compared with those of a slightly hydrolyzed OCP (low crystalline OCP: LC-OCP), the fully hydrolyzed apatitic product of OCP or biodegradable β-tricalcium phosphate (β-TCP) ceramic, by their implantation in rat tibia for 56 days. The physicochemical aspect of implants and biological responses were analyzed by X-ray diffraction, histomorphometry, immunohistochemistry and expression of mRNA around the implants. The remarkable findings were that: (1) the highest bone formation rate was obtained for β-TCP whereas the lowest for LC-OCP at Day 14; (2) the rates were reversed and reached the highest for LC-OCP until Day 56; (3) the early expression of ostoeoclast markers TRAP and cathepsin-K was suppressed with LC-OCP; (4) the expression of inflammatory markers IL-β1 and TNF-α was suppressed with LC-OCP. The results confirmed that the partially hydrolyzed OCP with Ca/P molar ratio 1.37 (LC-OCP) enhances bone formation most, suppressing early osteoclast activity and reducing inflammation.
Keywords: Octacalcium phosphate; Partial hydrolysis; Osteconduction; Biodegradation; Bone marrow;

The effect of ultraviolet functionalization of titanium on integration with bone by Hideki Aita; Norio Hori; Masato Takeuchi; Takeo Suzuki; Masahiro Yamada; Masakazu Anpo; Takahiro Ogawa (1015-1025).
Titanium implants are used as a reconstructive anchor in orthopedic and dental diseases and problems. Recently, ultraviolet (UV) light-induced photocatalytic activity of titanium has earned considerable and broad interest in environmental and clean-energy sciences. This study determines whether UV treatment of titanium enhances its osteoconductive capacity. Machined and acid-etched titanium samples were treated with UV for various time periods up to 48 h. For both surfaces, UV treatment increased the rates of attachment, spread, proliferation and differentiation of rat bone marrow-derived osteoblasts, as well as the capacity of protein adsorption, by up to threefold. In vivo histomorphometry in the rat model revealed that new bone formation occurred extensively on UV-treated implants with virtually no intervention by soft tissue, maximizing bone–implant contact up to nearly 100% at week 4 of healing. An implant biomechanical test revealed that UV treatment accelerated the establishment of implant fixation 4 times. The rates of protein adsorption and cell attachment strongly correlated with the UV dose-responsive atomic percentage of carbon on TiO2, but not with the hydrophilic status. The data indicated that UV light pretreatment of titanium substantially enhances its osteoconductive capacity, in association with UV-catalytic progressive removal of hydrocarbons from the TiO2 surface, suggesting a photofunctionalization of titanium enabling more rapid and complete establishment of bone–titanium integration.
Keywords: Osseointegration; Photocatalysis; Dental implant; Arthroplasty; Femoral stem; Hydrocarbon;

Sulfated glyco-block copolymers with specific receptor and growth factor binding to support cell adhesion and proliferation by Zeynep Oezyuerek; Katja Franke; Mirko Nitschke; Roland Schulze; Frank Simon; Klaus-Jochen Eichhorn; Tilo Pompe; Carsten Werner; Brigitte Voit (1026-1035).
We report on the successful preparation of thin glyco-block copolymer films with a combined thermoresponsive and heparin-like functionality. The copolymers were synthesized from poly(N-isopropylacrylamide) and glucose units and were covalently fixed onto glass supports by means of low pressure plasma cross-linking. The thin films retain the thermoresponsive characteristics of poly(N-isopropylacrylamide) with a transition temperature around 33 °C. Additionally, it could be shown that sulfation of the glucose moieties introduces a heparin-like functionality to the films. An increase in binding of basic fibroblast growth factor (bFGF) as well as specific adhesion of endothelial cells and hematopoietic progenitor cells could be demonstrated. The functional coupling of bFGF to the glyco-block copolymer surfaces was further proven by the dose-dependent response of endothelial cell proliferation. The results show that the newly synthesized glyco-block copolymers allow for the preparation of biomimetic surfaces with dual functionalities of thermoresponsive and heparin-like characteristics for the application in cell culture experiments with specific binding and release of heparin-binding growth factors and cell adhesion receptors.
Keywords: Cell adhesion; Copolymer; Fibroblast growth factor; Heparin; Polysaccharide;

In vitro behavior of neural stem cells in response to different chemical functional groups by Yong-Juan Ren; Han Zhang; Hua Huang; Xiu-Mei Wang; Zi-You Zhou; Fu-Zhai Cui; Yi-Hua An (1036-1044).
Neural stem cells (NSCs) cultured on glass surfaces modified by different chemical groups, including hydroxyl (–OH), sulfonic (–SO3H), amino (–NH2), carboxyl (–COOH), mercapto (–SH) and methyl (–CH3) groups, are shown here to commit to phonotypes with extreme sensitivity to surface chemical groups. The adhering NSCs at the level of single cells exhibited morphological changes in response to different chemical groups. NSCs on –SO3H surfaces had the largest contact area and the most flattened morphology, while those on –CH3 surfaces exhibited the smallest contact area and the most rounded morphology. After 5 days of culture, the migration of NSCs from their original aggregates onto these test surfaces followed the trend: –NH2>–COOH = –SH≫–SO3H>–CH3>–OH. The expression of specific markers, including nestin, β-Tubulin-III, glial fibrillary acidic protein and O4, were used to examine NSCs lineage specification. The –SO3H surfaces favored NSCs differentiation into oligodendrocytes, while NSCs in contact with –COOH, –NH2, –SH and –CH3 had the ability to differentiate into neurons, astrocytes and oligodendrocytes. Compared to –COOH surfaces, –NH2 seemed to promote neuronal differentiation. These chemically modified surfaces exhibited regulation of NSCs on adhesion, migration and differentiation potential, providing chemical means for the design of biomaterials to direct NSCs lineage specification for neural tissue engineering.
Keywords: Neural stem cells; Surface chemistry; Adhesion; Migration; Differentiation;

A microarray approach to the identification of polyurethanes for the isolation of human skeletal progenitor cells and augmentation of skeletal cell growth by Rahul S. Tare; Ferdous Khan; Guilhem Tourniaire; Suzanne M. Morgan; Mark Bradley; Richard O.C. Oreffo (1045-1055).
The present study has examined the efficacy of a polymer microarray platform to screen a library of polyurethanes for applications such as human skeletal progenitor cell isolation and surface modification of tissue engineering scaffolds to enhance skeletal cell growth and differentiation. Analysis of polyurethane microarrays incubated with adult human bone marrow-derived STRO-1+ skeletal progenitor cells identified 31 polyurethanes (from the entire library of 120 polyurethanes) capable of binding to the STRO-1+ cells. Four polyurethanes (out of the 31 identified in the previous screen) were able to selectively immobilise cells of the STRO-1+ fraction from the heterogeneous human bone marrow mononuclear cell population. These four polyurethanes were highly selective for the STRO-1+ fraction of human bone marrow as they failed to bind STRO-1+ immature osteoblast-like MG63 cells, the STRO-1+ fraction of human fetal skeletal cells and differentiated osteoblast-like SaOs cells. Culture of human bone marrow-derived STRO-1+ cells on fibres of Polyglycolic acid (PGA) fleece surface modified by polyurethane adsorption, in osteogenic conditions, enhanced the expression of early osteogenic genes. Similarly, surface modification of PGA fleece fibres by polyurethane adsorption increased the responsiveness of MG63 cells, cultured on this scaffold, to 1,25 dihydroxy Vitamin D3, as demonstrated by enhanced Osteocalcin expression.
Keywords: Microarray; Polyurethanes; Skeletal progenitors; STRO-1; MG63; Fetal skeletal cells;

The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells by Alex J. Shortt; Genevieve A. Secker; Richard J. Lomas; Stacy P. Wilshaw; John N. Kearney; Stephen J. Tuft; Julie T. Daniels (1056-1065).
Human amniotic membrane (HAM) is employed as a substrate for the ex-vivo expansion of limbal epithelial cells (LECs) used to treat corneal epithelial stem cell deficiency in humans. The optimal method of HAM preparation for this purpose is unknown. This study evaluated the ability of different preparations of stored HAM to serve as substrates for LEC expansion ex-vivo. The effect of removing the amniotic epithelial cells (decellularisation) from HAM prior to seeding of LECs, the effect of glycerol cryopreservation and the effect of peracetic acid (PAA) sterilization and antibiotic disinfection were evaluated using different HAM test groups. Human LECs were cultured on each preparation and the following outcomes were assessed: confluence of growth, cell density, cell morphology and expression of the putative LESC markers deltaN-p63alpha and ABCG2. Removing amniotic epithelial cells prior to seeding of LECs resulted in a higher percentage of confluence but a lower cell density than intact HAM suggesting that decellularisation does not increase proliferation, but rather that it facilitates migration of LECs resulting in larger cells. Decellularisation did not affect the percentage of cells expressing the putative LESC markers deltaN-p63alpha (≤4% in both intact and acellular groups) and ABCG2 (≤3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (≤6%) and ABCG2 (≤8%). HAM frozen at −80 °C in Hank's Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (≤68%) and ABCG2 (≤62%) expression (p  < 0.001). The use of PAA or antibiotics to decontaminate HAM does not appear to affect this function. The variables affecting the ability of HAM to serve as a substrate for LEC expansion ex-vivo are poorly understood. The use of glycerol as a cryoprotectant impairs this ability whereas simple frozen HAM appears to work extremely well for this purpose.
Keywords: Stem cell; Niche; Amniotic membrane; Cornea; Limbus; Somatic stem cell biology;

The geometric control of E14 and R1 mouse embryonic stem cell pluripotency by plasma polymer surface chemical gradients by Nicola Wells; Melissa A. Baxter; Jeremy E. Turnbull; Patricia Murray; David Edgar; Kristina L. Parry; David A. Steele; Robert D. Short (1066-1070).
Plasma polymer surfaces were fabricated such that the cell response to a range of carboxylic acid concentrations on a single sample could be investigated. Surface chemical gradients from hydrophobic plasma polymerised octadiene (OD) to a more hydrophilic plasma polymerised acrylic acid (AA) were formed on glass coverslips. Surface characterisation of the chemical gradients was performed using X-ray photoelectron spectroscopy to determine elemental composition. Following culture of E14 and R1 mouse embryonic stem cells (mES) in differing culture media, cell pluripotency was determined by alkaline phosphatase staining. The results demonstrate that for these cell lines the capacity for self-renewal is maintained if the cells are restricted in their spreading to <120 μm2.
Keywords: Cell culture; Cell spreading; Cell viability; Plasma polymerisation;

In vitro analog of human bone marrow from 3D scaffolds with biomimetic inverted colloidal crystal geometry by Joan E. Nichols; Joaquin Cortiella; Jungwoo Lee; Jean A. Niles; Meghan Cuddihy; Shaopeng Wang; Joseph Bielitzki; Andrea Cantu; Ron Mlcak; Esther Valdivia; Ryan Yancy; Matthew L. McClure; Nicholas A. Kotov (1071-1079).
In vitro replicas of bone marrow can potentially provide a continuous source of blood cells for transplantation and serve as a laboratory model to examine human immune system dysfunctions and drug toxicology. Here we report the development of an in vitro artificial bone marrow based on a 3D scaffold with inverted colloidal crystal (ICC) geometry mimicking the structural topology of actual bone marrow matrix. To facilitate adhesion of cells, scaffolds were coated with a layer of transparent nanocomposite. After seeding with hematopoietic stem cells (HSCs), ICC scaffolds were capable of supporting expansion of CD34+ HSCs with B-lymphocyte differentiation. Three-dimensional organization was shown to be critical for production of B cells and antigen-specific antibodies. Functionality of bone marrow constructs was confirmed by implantation of matrices containing human CD34+ cells onto the backs of severe combined immunodeficiency (SCID) mice with subsequent generation of human immune cells.
Keywords: Scaffolds; Tissue engineering; Bone marrow; Colloidal crystals; Stem cell;

Preparation and characterization of bioactive mesoporous wollastonite – Polycaprolactone composite scaffold by Jie Wei; Fangping Chen; Jung-Woog Shin; Hua Hong; Chenglong Dai; Jiancan Su; Changsheng Liu (1080-1088).
A well-defined mesoporous structure of wollastonite with high specific surface area was synthesized using surfactant P123 (triblock copolymer) as template, and its composite scaffolds with poly(ɛ-caprolactone) (PCL) were fabricated by a simple method of solvent casting-particulate leaching. The measurements of the water contact angles suggest that the incorporation of either mesoporous wollastonite (m-WS) or conventional wollastonite (c-WS) into PCL could improve the hydrophilicity of the composites, and the former was more effective than the later. The bioactivity of the composite scaffold was evaluated by soaking the scaffolds in a simulated body fluid (SBF) and the results show that the m-WS/PCL composite (m-WPC) scaffolds can induce a dense and continuous layer of apatite after soaking for 1 week, as compared with the scattered and discrete apatite particles on the c-WS/PCL composite (c-WPC) scaffolds. The m-WPC had a significantly enhanced apatite-forming bioactivity compared with the c-WPC owing to the high specific surface area and pore volume of m-WS. In addition, attachment and proliferation of MG63 cells on m-WPC scaffolds were significantly higher than that of c-WPC, revealing that m-WPC scaffolds had excellent biocompatibility. Such improved properties of m-WPC should be helpful for developing new biomaterials and may have potential use in hard tissue repair.
Keywords: Mesoporous wollastonite; Composite scaffold; Hydrophilicity; Bioactivity; Cell attachment and proliferation;

Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability by Mikaël M. Martino; Mayumi Mochizuki; Dominique A. Rothenfluh; Sandra A. Rempel; Jeffrey A. Hubbell; Thomas H. Barker (1089-1097).
The extracellular matrix (ECM) exerts powerful control over many cellular phenomena, including stem cell differentiation. As such, design and modulation of ECM analogs to ligate specific integrin is a promising approach to control cellular processes in vitro and in vivo for regenerative medicine strategies. Although fibronectin (FN), a crucial ECM protein in tissue development and repair, and its RGD peptide are widely used for cell adhesion, the promiscuity with which they engage integrins leads to difficulty in control of receptor-specific interactions. Recent simulations of force-mediated unfolding of FN domains and sequences analysis of human versus mouse FN suggest that the structural stability of the FN's central cell-binding domains (FN III9–10) affects its integrin specificity. Through production of FN III9–10 variants with variable stabilities, we obtained ligands that present different specificities for the integrin α5β1 and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of α5β1 integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that α5β1 has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for α5β1 versus αvβ3 results in significantly enhanced osteogenic differentiation of MSCs in 2D and in a clinically relevant 3D fibrin matrix system, although attachment/spreading and proliferation were comparable with that on full-length FN. This work shows how integrin-dependant cellular interactions with the ECM can be engineered to control stem cell fate, within a system appropriate for both 3D cell culture and tissue engineering.
Keywords: Fibronectin; Integrins; Recombinant protein; Hydrogel; Mesenchymal stem cell; Osteogenesis;

A three-dimensional model of vasculogenesis by Mani T. Valarmathi; Jeffrey M. Davis; Michael J. Yost; Richard L. Goodwin; Jay D. Potts (1098-1112).
Postnatal bone marrow contains various subpopulations of resident and circulating stem cells (HSCs, BMSCs/MSCs) and progenitor cells (MAPCs, EPCs) that are capable of differentiating into one or more of the cellular components of the vascular bed in vitro as well as contribute to postnatal neo-vascularization in vivo. When rat BMSCs were seeded onto a three-dimensional (3-D) tubular scaffold engineered from topographically aligned type I collagen fibers and cultured either in vasculogenic or non-vasculogenic media for 7, 14, 21 or 28 days, the maturation and co-differentiation into endothelial and/or smooth muscle cell lineages were observed. Phenotypic induction of these substrate-grown cells was assayed at transcript level by real-time PCR and at protein level by confocal microscopy. In the present study, the observed upregulation of transcripts coding for vascular phenotypic markers is reminiscent of an in vivo expression pattern. Immunolocalization of vasculogenic lineage-associated markers revealed typical expression patterns of vascular endothelial and smooth muscle cells. These endothelial cells exhibited high metabolism of acetylated low-density lipoprotein. In addition to the induced monolayers of endothelial cells, the presence of numerous microvascular capillary-like structures was observed throughout the construct. At the level of scanning electron microscopy, smooth-walled cylindrical tube-like structures with smooth muscle cells and/or pericytes attached to its surface were elucidated. Our 3-D culture system not only induces the maturation and differentiation of BMSCs into vascular cell lineages but also supports microvessel morphogenesis. Thus, this unique in vitro model provides an excellent platform to study the temporal and spatial regulation of postnatal de novo vasculogenesis, as well as attack the lingering limit in developing engineered tissues, that is perfusion.
Keywords: Bone marrow stromal cells; Mesenchymal stem cells; Vasculogenesis; Angiogenesis; Vascular tissue engineering;

Modulation of hepatocyte phenotype in vitro via chemomechanical tuning of polyelectrolyte multilayers by Alice A. Chen; Salman R. Khetani; Sunyoung Lee; Sangeeta N. Bhatia; Krystyn J. Van Vliet (1113-1120).
It is increasingly appreciated that since cell and tissue functions are regulated by chemomechanical stimuli, precise control over such stimuli will improve the functionality of tissue models. However, due to the inherent difficulty in decoupling these cues as presented by extracellular materials, few studies have explored the independent modulation of biochemical and mechanical stimuli towards the manipulation of sustained cellular processes. Here, we demonstrate that both mechanical compliance and ligand presentation of synthetic, weak polyelectrolyte multilayers (PEMs) can be tuned independently to influence the adhesion and liver-specific functions of primary rat hepatocytes over extended in vitro culture (two weeks). These synthetic PEMs exhibited elastic moduli E ranging over 200 kPa <  E  < 142 MPa, as much as one thousand-fold more compliant than tissue-culture polystyrene (E  ∼ 2.5 GPa). The most compliant of these PEM substrata promoted hepatocyte adhesion and spheroidal morphology. Subsequent modification of PEMs with type I collagen and the proteoglycan decorin did not alter substrata compliance, but enhanced the retention of spheroids on surfaces and stabilized hepatic functions (albumin and urea secretion, CYP450 detoxification activity). Decorin exhibited unique compliance-mediated effects on hepatic functions, down-regulating the hepatocyte phenotype when presented on highly compliant substrata while up-regulating hepatocyte functions when presented on increasingly stiffer substrata. These results show that phenotypic functions of liver models can be modulated by leveraging synthetic polymers to study and optimize the interplay of biochemical and mechanical cues at the cell–material interface. More broadly, these results suggest an enabling approach for the systematic design of functional tissue models applied to drug screening, cell-based therapies and fundamental studies in development, physiology and disease.
Keywords: Hepatocyte; Polyelectrolyte multilayers; Compliance; Surface modification; Chemomechanics;

Repair of thoracic spinal cord injury by chitosan tube implantation in adult rats by Xiaoguang Li; Zhaoyang Yang; Aifeng Zhang; Tailing Wang; Weichang Chen (1121-1132).
Spinal cord injury (SCI) is a common outcome of traffic accidents and trauma with severe consequences. There has been no cure for such a condition. We performed experiments to evaluate the feasibility of implanting a chitosan tube filled with semifluid type I collagen into the site of surgically induced SCI to facilitate functional recovery. After a segment of the spinal cord, 4 mm in length and 2/3 of the spinal cord across its width, at the ninth thoracic level of an adult rat was dissected and removed, the biodegradable chitosan tube was implanted into the lesioned site. One year later, we found that axons from the proximal spinal cord regenerated, traversed the dissected area inside the tube and reentered the distal spinal cord, leading to functional restoration of the essentially paralyzed hind limbs. The nerve regeneration and functional recovery were confirmed by immunohistochemistry, electron microscopy, nerve tracing and Basso–Beattie–Bresnahan behavioral evaluation. Such beneficial outcomes were not observed in the control groups, in which either no tube was implanted or the implanted tube had no collagen filling. We conclude that the newly designed tube implant promotes both axon regeneration and functional recovery following SCI. A similar approach may have clinical implications in humans.
Keywords: Spinal cord injury; Axon regeneration; Function recovery; Collagen; Chitosan;

Collagen-based fibrous scaffold for spatial organization of encapsulated and seeded human mesenchymal stem cells by S.Z. Yow; C.H. Quek; Evelyn K.F. Yim; C.T. Lim; K.W. Leong (1133-1142).
Living tissues consist of groups of cells organized in a controlled manner to perform a specific function. Spatial distribution of cells within a three-dimensional matrix is critical for the success of any tissue-engineering construct. Fibers endowed with cell-encapsulation capability would facilitate the achievement of this objective. Here we report the synthesis of a cell-encapsulated fibrous scaffold by interfacial polyelectrolyte complexation (IPC) of methylated collagen and a synthetic terpolymer. The collagen component was well distributed in the fiber, which had a mean ultimate tensile strength of 244.6 ± 43.0 MPa. Cultured in proliferating medium, human mesenchymal stem cells (hMSCs) encapsulated in the fibers showed higher proliferation rate than those seeded on the scaffold. Gene expression analysis revealed the maintenance of multipotency for both encapsulated and seeded samples up to 7 days as evidenced by Sox 9, CBFA-1, AFP, PPARγ2, nestin, GFAP, collagen I, osteopontin and osteonectin genes. Beyond that, seeded hMSCs started to express neuronal-specific genes such as aggrecan and MAP2. The study demonstrates the appeal of IPC for scaffold design in general and the promise of collagen-based hybrid fibers for tissue engineering in particular. It lays the foundation for building fibrous scaffold that permits 3D spatial cellular organization and multi-cellular tissue development.
Keywords: Mesenchymal stem cells; Cell encapsulation; Fibrous scaffold; Collagen; Stem cell tissue engineering; 3D cell patterning;

Acellularization of embryoid bodies via physical disruption methods by Alyssa V. Ngangan; Todd C. McDevitt (1143-1149).
Embryonic stem cells (ESCs) are capable of differentiating into all somatic cell types and have therefore attracted significant interest for use in tissue repair and regeneration therapies. Transplanted ESCs can not only integrate into compromised tissues, but can also stimulate endogenous regeneration via secreted factors. In this study, several acellularization protocols were applied to spheroids of differentiating ESCs, termed embryoid bodies (EBs), to develop a potential route to deliver ESC-derived molecules, independent of cells, to damaged tissues. The objective of this study was to physically disrupt EBs via lyophilization or freeze–thaw cycling, and in combination with DNase treatment, determine the efficacy of acellularization based upon cell viability, DNA removal, and protein retention. Mechanical disruption and DNase treatment of EBs efficiently inhibited viability and removed DNA while retaining protein content to produce an acellular EB matrix. The EB-derived acellular matrices permitted attachment and repopulation of the constructs by 3T3 fibroblasts in vitro. Overall, these studies demonstrate that effective mechanical means to acellularize EBs may be used in order to further elucidate the composition and function of embryonic extracellular matrices and serve as novel naturally-derived scaffolds for tissue repair and regeneration.
Keywords: Acellularization; Decellularization; Embryoid bodies; Extracellular matrix; Embryonic stem cells; Differentiation;

Microfeature guided skeletal muscle tissue engineering for highly organized 3-dimensional free-standing constructs by Mai T. Lam; Yen-Chih Huang; Ravi K. Birla; Shuichi Takayama (1150-1155).
Engineering tissue similar in structure to their natural equivalents is a major challenge and crucial to function. Despite attempts to engineer skeletal muscle, it is still difficult to effectively mimic tissue architecture. Rigid scaffolds can guide cell alignment but have the critical drawback of hindering mechanical function of the resultant tissue. We present a method for creating highly ordered tissue-only constructs by using rigid microtopographically patterned surfaces to first guide myoblast alignment, followed by transfer of aligned myotubes into a degradable hydrogel and self-organization of the ordered cells into a functional, 3-dimensional, free-standing construct independent of the initial template substrate. Histology revealed an intracellular organization resembling that of native muscle. Aligned cell constructs exhibited a 2-fold increase in peak force production compared to controls. Effective specific force, or force normalized over cross-sectional area, was increased by 23%. This template, transfer, and self-organization strategy is envisioned to be broadly useful in improving construct function and clinical applicability for highly ordered tissues like muscle.
Keywords: Poly(dimethylsiloxane); Micropatterning; Silicone elastomer; Muscle; Laminin; Hydrogel;

The effect of self-assembling peptide nanofiber scaffolds on mouse embryonic fibroblast implantation and proliferation by Irene R. Dégano; Lluís Quintana; Marta Vilalta; David Horna; Nuria Rubio; Salvador Borrós; Carlos Semino; Jerónimo Blanco (1156-1165).
Development of new materials for tissue engineering can be facilitated by the capacity to efficiently monitor in vivo the survival, proliferation and differentiation behaviour of cells implanted in different target tissues. We present here the application of a previously developed platform that allows to monitor in real time the survival and proliferative behaviour of implanted cells in two anatomical sites: subcutaneous and intramuscular. Basically, the system is based on the use of a non-invasive bioluminescence imaging (BLI) technique to detect luciferase expressing C57BL/6 cells, mouse embryonic fibroblasts, seeded in two sets of scaffolds: 1, a RAD16-I self-assembling peptide nanofiber matrix and 2, a composite consisted of the same RAD16-I nanofibers contained into a microporous biorubber scaffold. Interestingly, our results indicated considerable differences in the behaviour of implanted cells in each scaffold type. We observed that the self-assembling peptide scaffold alone foster cell survival and promotes cell proliferation where the composite scaffold not. Since self-assembling peptide scaffolds presents value stiffness proximal to the implanted tissues it is suggestive to think that harder materials will provide a physical constriction for cells to proliferate as well as mechanical discontinuity. We therefore propose that it is important to close match the implantation environment with the cell/material constructs in order to obtain the best response of the cells, illustrating the convenience of this strategy for the development of new tissue engineering platforms.
Keywords: Cell proliferation; In vivo imaging; Luciferase; Nanofiber scaffold;

Directional BMP-2 for functionalization of titanium surfaces by Kenji Kashiwagi; Toru Tsuji; Kiyotaka Shiba (1166-1175).
Efficient immobilization of biomacromolecules on material surfaces is a key to development in areas of regenerative medicine and tissue engineering. However, strong and irreversible immobilization of cytokines on surfaces often diminishes their biological functionality. A destructive hydrophobic interaction between the material surface and the biomolecule may underlie this inactivation. Alternatively, dissociation of the cytokine from the material may be necessary for signal transduction. Here we propose a new method for immobilizing cytokines on material surfaces: a material-binding artificial peptide is used to mediate reversible interaction between the cytokine and the material surface. We created artificial proteins that contained three copies of a Ti-binding motif, and fused them to the N-terminal of BMP-2. The engineered BMP-2 showed reversible binding to Ti surfaces and induced BMP signaling activity. When a hydrophobic protein devoid of the Ti-binding motif was fused to BMP-2, the protein tightly bound to Ti surfaces but showed little BMP activity, confirming the importance of the mode of immobilization.
Keywords: Artificial protein; BMP (bone morphogenetic protein); Interface; Motif-programming; Osseointegration; Titanium;

We developed a new strategy to prepare folate-decorated nanoparticles of biodegradable polymers for Quantum dots (QDs) formulation for targeted and sustained imaging for cancer diagnosis at its early stage. Poly(lactide)-vitamin E TPGS (PLA-TPGS) copolymer and vitamin E TPGS-carboxyl (TPGS-COOH) copolymer were synthesized. Their blend at various weight ratio was used to prepare folate-decorated nanoparticles (NPs) for QDs formulation to improve their imaging effects and reduce their side effects. The TPGS-COOH on the NP surface was designed to conjugate folate-NH2 with advantage to make the targeting effect adjustable. The size of such NPs was found in the range of 280–300 nm. In vitro cellular uptakes of such NPs were investigated with confocal laser scanning microscopy (CLSM), which demonstrated much higher internalization of the folate-decorated QDs-loaded PLA-TPGS/TPGS-COOH NPs by MCF-7 breast cancer cells which are of over-expression of folate receptors than the cellular uptake by NIH 3T3 fibroblast cells which are of low expression of folate receptors. Compared with the free QDs, the QDs formulated in the PLA-TPGS/TPGS-COOH NPs showed lower in vitro cytotoxicity for both of MCF-7 cells and NIH 3T3 cells. Additionally, our findings indicated that under same conditions, cytotoxicity of QDs formulated in the PLA-TPGS/TPGS-COOH NPs is lower for normal cells such as NIH 3T3 cells than that for breast cancer such as MCF-7 breast cancer cells due to folate targeting effect. Targeted imaging by QDs formulated in folate-decorated PLA-TPGS/TPGS-COOH nanoparticles with better effects and less side effects is feasible.
Keywords: Cancer diagnosis; Cancer nanotechnology; Confocal laser scanning microscopy; Molecular imaging; Nanomedicine; Vitamin E TPGS;

Protective effects of fullerenol C60(OH)24 against doxorubicin-induced cardiotoxicity and hepatotoxicity in rats with colorectal cancer by Rade Injac; Martina Perse; Manica Cerne; Nejka Potocnik; Natasa Radic; Biljana Govedarica; Aleksandar Djordjevic; Anton Cerar; Borut Strukelj (1184-1196).
The effects of fullerenol C60(OH)24 (Frl) at doses of 25, 50, and 100 mg/kg/week (for a time-span of 3 weeks) on heart and liver tissue after doxorubicin (Dox)-induced toxicity in rats with colorectal cancer were investigated. In the present study, we used an in vivo Wistar male rat model to explore whether Frl could protect against Dox-induced (1.5 mg/kg/week for 3 weeks) chronic cardio- and hepato- toxicity and compared the effect with a well-known antioxidant, vitamin C (100 mg/kg/week for 3 weeks). According to macroscopic, microscopic, hematological, biochemical, physiological, pharmacological, and pharmacokinetic results, we confirmed that, at all examined doses, Frl exhibits a protective influence on the heart and liver tissue against chronic toxicity induced by Dox.
Keywords: Doxorubicin; Cardiotoxicity; Hepatotoxicity; Fullerenol; Oxidative stress; Colorectal cancer;

Simultaneous intracellular delivery of targeting antibodies and functional nanoparticles with engineered protein G system by Yong Taik Lim; Mi Young Cho; Jung Min Lee; Sang Jeon Chung; Bong Hyun Chung (1197-1204).
Cellular internalization of functional nanoparticles that have optical and magnetic properties is very important in the cellular imaging and manipulation of specifically targeted biomolecules. In this study, a robust method to deliver functional nanoparticles and targeting antibodies into cells was suggested. The engineered protein G system, which contains an affinity tag and a cell penetration peptide in the N- and C-terminals, respectively, can capture surface-modified nanoparticles and antibodies without chemical reaction, and then non-invasively deliver them into the cells. Finally, gold-coated iron oxide nanoparticle/engineered protein G hybrid systems were successfully employed as multifunctional cargo systems for the targeting, imaging, and manipulation of mitochondria.
Keywords: Nanoparticle; Gold; Engineered protein; Intracellular delivery; Surface modification; Cell imaging;

The mechanical properties of individual, electrospun fibrinogen fibers by Christine R. Carlisle; Corentin Coulais; Manoj Namboothiry; David L. Carroll; Roy R. Hantgan; Martin Guthold (1205-1213).
We used a combined atomic force microscopic (AFM)/fluorescence microscopic technique to study the mechanical properties of individual, electrospun fibrinogen fibers in aqueous buffer. Fibers (average diameter 208 nm) were suspended over 12 μm-wide grooves in a striated, transparent substrate. The AFM, situated above the sample, was used to laterally stretch the fibers and to measure the applied force. The fluorescence microscope, situated below the sample, was used to visualize the stretching process. The fibers could be stretched to 2.3 times their original length before breaking; the breaking stress was 22 × 106  Pa. We collected incremental stress–strain curves to determine the viscoelastic behavior of these fibers. The total stretch modulus was 17.5 × 106  Pa and the relaxed elastic modulus was 7.2 × 106  Pa. When held at constant strain, electrospun fibrinogen fibers showed a fast and slow stress relaxation time of 3 and 55 s.Our fibers were spun from the typically used 90% 1,1,1,3,3,3-hexafluoro-2-propanol (90-HFP) electrospinning solution and re-suspended in aqueous buffer. Circular dichroism spectra indicate that α-helical content of fibrinogen is ∼70% higher in 90-HFP than in aqueous solution.These data are needed to understand the mechanical behavior of electrospun fibrinogen structures. Our technique is also applicable to study other nanoscopic fibers.
Keywords: AFM; Fluorescence; Biomimetic material; Fibrinogen; Mechanical properties; Viscoelasticity;

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor. Development of drug delivery technologies facilitating controlled release of GDNF is critical to applying GDNF in treating neurodegenerative diseases. We previously developed 3D collagen microspheres and demonstrated enhanced GDNF secretion after encapsulation of HEK293 cells, which were transduced to overexpress GDNF in these microspheres. However, the entrapped HEK293 cells were able to migrate out of the collagen microspheres, making it undesirable for clinical applications. In this report, we investigate two new carrier designs, namely collagen–alginate composite gel and collagen microspheres embedded in alginate gel in preventing cell leakage, maintaining cell growth and controlling GDNF secretion in the HEK293 cells. We demonstrated that inclusion of alginate gel in both designs is efficient in preventing cell leakage to the surrounding yet permitting the GDNF secretion, although the cellular growth rate is reduced in an alginate concentration dependent manner. Differential patterns of GDNF secretion in the two designs were demonstrated. The collagen–alginate composite gel maintains a more or less constant GDNF secretion over time while the collagen microspheres embedded in alginate gel continue to increase the secretion level of GDNF over time. This study contributes towards the development of cell-based GDNF delivery devices for the future therapeutics of neurodegenerative diseases.
Keywords: GDNF; HEK293 cells; Microsphere; Collagen; Microencapsulation; Alginate;

Non-viral endostatin plasmid transfection of mesenchymal stem cells via collagen scaffolds by Xiao-Dan Sun; Lily Jeng; Catherine Bolliet; Bjorn R. Olsen; Myron Spector (1222-1231).
Angiogenesis is critical in the early stage of reparative processes and tissue regeneration, but the persistence of a vascular network may interfere with later transformation/maturation in naturally avascular tissues such as articular cartilage. Our supposition is that the timed delivery of an anti-angiogenic factor in cartilage tissue engineering may facilitate the formation of hyaline cartilage by inducing the regression of vascularization. To this end our overall goal is to prepare an off-the-shelf scaffold containing the gene for a potent anti-angiogenic factor. The objective of this study was to investigate the use of a type I/III collagen scaffold for the non-viral transfection of marrow stromal cells (MSCs, also referred to as mesenchymal stem cells) with the plasmid encoding endostatin. Caprine MSCs were transfected by the naked plasmid alone and plasmid incorporated into a cationic lipid complex in three experiments: 1) cells were transfected in monolayer; 2) monolayer-transfected cells were grown in a collagen sponge-like scaffold; and 3) non-transfected cells were grown in a collagen scaffold containing the naked plasmid and endostatin lipoplex. Independent variables were the passage number of the cells and the plasmid loading. The amount of endostatin released by the cells into the medium was measured using an ELISA. The results demonstrated the overexpression of endostatin by MSCs growing in the endostatin lipoplex-supplemented collagen scaffolds. Endostatin released by the cell-seeded scaffolds reached a peak of 13 ng/ml for scaffolds incorporating as little as 20 μg of plasmid, at the 3-day collection period ending 5 days post-seeding. The accumulated endostatin synthesis over a 2-week period began to achieve what may be a therapeutic level. MSCs transfected with the endostatin gene in monolayer continued to express the gene when grown in the collagen scaffolds. The results demonstrate the promise of the non-viral delivery of the gene for this potent anti-angiogenic protein to MSCs via a collagen scaffold.
Keywords: Endostatin; Collagen; Scaffold; Stem cells;

The effect of the nonionic block copolymer pluronic P85 on gene expression in mouse muscle and antigen-presenting cells by Zagit Z. Gaymalov; Zhihui Yang; Vladimir M. Pisarev; Valery Yu Alakhov; Alexander V. Kabanov (1232-1245).
DNA vaccines can be greatly improved by polymer agents that simultaneously increase transgene expression and activate immunity. We describe here Pluronic P85 (P85), a triblock copolymer of ethylene oxide (EO) and propylene oxide (PO) EO26–PO40–EO26. Using a mouse model we demonstrate that co-administration of a bacterial plasmid DNA with P85 in a skeletal muscle greatly increases gene expression in the injection site and distant organs, especially the draining lymph nodes and spleen. The reporter expression colocalizes with the specific markers of myocytes and keratinocytes in the muscle, as well as dendritic cells (DCs) and macrophages in the muscle, lymph nodes and spleen. Furthermore, DNA/P85 and P85 alone increase the systemic expansion of CD11c+ (DC), and local expansion of CD11c+, CD14+ (macrophages) and CD49b+ (natural killer) cell populations. DNA/P85 (but not P85) also increases maturation of local DC (CD11c + CD86+, CD11c + CD80+, and CD11c + CD40+). We suggest that DNA/P85 promotes the activation and recruitment of the antigen-presenting cells, which further incorporate, express and carry the transgene to the immune system organs.
Keywords: Copolymer; Gene expression; Leukocyte; Pluronics;

Protamine sulfate/poly(l-aspartic acid) polyionic complexes self-assembled via electrostatic attractions for combined delivery of drug and gene by Han Cheng; Yong-Yong Li; Xuan Zeng; Yun-Xia Sun; Xian-Zheng Zhang; Ren-Xi Zhuo (1246-1253).
In this study, a series of self-assembled polyionic complexes (PICs) were prepared via electrostatic attraction between protamine sulfate (PS) and poly(l-aspartic acid) (PASP) or doxorubicin (DOX)-conjugated PASP (DOX-PASP). The size of the PICs measured by Nano-ZS ZEN3600 was around 200–300 nm at different weight ratios of PS/PASP. Transmission electron microscopy (TEM) showed that PS/PASP PICs displayed a regular spherical shape and no aggregation was observed. The cytotoxicity study indicated that the PICs did not exhibit apparent cytotoxicity in comparison with that of 25 kDa polyethylenimine (PEI). Gel retardation assay indicated that the PICs were able to bind DNA completely when weight ratio of PS/PASP was higher than 2:1. Luciferase assay and green fluorescent protein (GFP) detection were used to confirm that the PICs could be used as efficient non-viral gene vectors and they exhibited comparable transfection efficiency with the one of 25 kDa PEI. Furthermore, confocal laser scanning microscopy as well as suppression activity of DOX-conjugated PICs (DOX-PICs) showed that they could quickly release the loaded DOX into HeLa cells, indicating that PICs can be also used as carriers for combined delivery of drug and gene.
Keywords: Polyionic complex; Non-viral gene vector; Drug release; Combined delivery;