Biomaterials (v.32, #29)

Minimal surface scaffold designs for tissue engineering by Sebastian C. Kapfer; Stephen T. Hyde; Klaus Mecke; Christoph H. Arns; Gerd E. Schröder-Turk (6875-6882).
Triply-periodic minimal surfaces are shown to be a more versatile source of biomorphic scaffold designs than currently reported in the tissue engineering literature. A scaffold architecture with sheetlike morphology based on minimal surfaces is discussed, with significant structural and mechanical advantages over conventional designs. These sheet solids are porous solids obtained by inflation of cubic minimal surfaces to sheets of finite thickness, as opposed to the conventional network solids where the minimal surface forms the solid/void interface. Using a finite-element approach, the mechanical stiffness of sheet solids is shown to exceed that of conventional network solids for a wide range of volume fractions and material parameters. We further discuss structure–property relationships for mechanical properties useful for custom-designed fabrication by rapid prototyping. Transport properties of the scaffolds are analyzed using Lattice-Boltzmann computations of the fluid permeability. The large number of different minimal surfaces, each of which can be realized as sheet or network solids and at different volume fractions, provides design flexibility essential for the optimization of competing design targets.
Keywords: Bone tissue engineering; Finite element analysis; Mechanical properties; Scaffold; Rapid prototyping;

Osteoblast and monocyte responses to 444 ferritic stainless steel intended for a Magneto-Mechanically Actuated Fibrous Scaffold by Vera N. Malheiro; Rose L. Spear; Roger A. Brooks; Athina E. Markaki (6883-6892).
The rationale behind this work is to design an implant device, based on a ferromagnetic material, with the potential to deform in vivo promoting osseointegration through the growth of a healthy periprosthetic bone structure. One of the primary requirements for such a device is that the material should be non-inflammatory and non-cytotoxic. In the study described here, we assessed the short-term cellular response to 444 ferritic stainless steel; a steel, with a very low interstitial content and a small amount of strong carbide-forming elements to enhance intergranular corrosion resistance. Two different human cell types were used: (i) foetal osteoblasts and (ii) monocytes. Austenitic stainless steel 316L, currently utilised in many commercially available implant designs, and tissue culture plastic were used as the control surfaces. Cell viability, proliferation and alkaline phosphatase activity were measured. In addition, cells were stained with alizarin red and fluorescently-labelled phalloidin and examined using light, fluorescence and scanning electron microscopy. Results showed that the osteoblast cells exhibited a very similar degree of attachment, growth and osteogenic differentiation on all surfaces. Measurement of lactate dehydrogenase activity and tumour necrosis factor alpha protein released from human monocytes indicated that 444 stainless steel did not cause cytotoxic effects or any significant inflammatory response. Collectively, the results suggest that 444 ferritic stainless steel has the potential to be used in advanced bone implant designs.
Keywords: Stainless steel; Osteoblast; Monocyte; Cell proliferation; Cell viability; Cytotoxicity;

Uniform zwitterionic polymer hydrogels with a nonfouling and functionalizable crosslinker using photopolymerization by Louisa R. Carr; Yibo Zhou; Jordan E. Krause; Hong Xue; Shaoyi Jiang (6893-6899).
We reported previously the design and synthesis of a zwitterionic carboxybetaine dimethacrylate crosslinker, and we showed that its use with zwitterionic carboxybetaine methacrylate led to nonfouling hydrogels with high mechanical properties and high hydration. Now, we use photopolymerization to improve the uniformity of the polymer network, resulting in drastically improved mechanical properties (compressive modulus up to 90 MPa). Furthermore, we designed and synthesized a new functionalizable carboxybetaine dimethacrylate crosslinker, enabling functionalization of the higher strength hydrogels against a nonfouling background. Additionally, the biostability of the carboxybetaine hydrogel systems was tested, and it was found that these hydrogels are stable in oxidative, acidic, and basic environments.
Keywords: Hydrogel; Crosslinking; RGD peptide; Compression;

Effects of TiO2 nanotubes with different diameters on gene expression and osseointegration of implants in minipigs by Na Wang; Hongyi Li; Wulong Lü; Jinghui Li; Jinshu Wang; Zhenting Zhang; Yiran Liu (6900-6911).
Titanium dioxide (TiO2) nanotubes can accelerate the adhesion and differentiation of osteoblasts, yet little is known how this nano-modified implant surface affects osseointegration at molecular level in vivo. The aim of this study was to investigate the effects of TiO2 nanotubes with different diameters (30 nm, 70 nm and 100 nm) on biological attachment mechanism of implants to bone in vivo by studying the gene expression and bone formation around the implants. The histological features and fluorochrome labeling changes of bone around implants on the non-decalcified sections (at 3, 5 and 8 weeks after implantation) were investigated by using traditional light- and fluorescent microscopy, and the gene expression of alkaline phosphatase (ALP), osterix (Osx), collagen-I (Col-I) and tartrate-resistant acid phosphatase (TRAP) was examined by using real-time PCR at 1, 2, 3, 4 and 5 weeks after implantation. Comparing with machined titanium implants, a significant increase in bone–implant contact (BIC) and gene expression levels was found in the bone attached to implants with TiO2 nanotubes, especially with 70 nm diameter nanotubes. At the same time, the sequential fluorescent labeling images illustrated dynamic bone deposition. In conclusion, TiO2 nanotubes can modulate bone formation events at the bone–implant interface as to reach favorable molecular response and osseointegration; in addition, the diameters of nanotubes can be precisely controlled in order to obtain better bone formation.
Keywords: TiO2 nanotubes; Gene expression; Osseointegration; In vivo; Implant; Minipig;

Engineered polymer-media interfaces for the long-term self-renewal of human embryonic stem cells by Elizabeth F. Irwin; Rohini Gupta; Derek C. Dashti; Kevin E. Healy (6912-6919).
We have developed a synthetic polymer interface for the long-term self-renewal of human embryonic stem cells (hESCs) in defined media. We successfully cultured hESCs on hydrogel interfaces of aminopropylmethacrylamide (APMAAm) for over 20 passages in chemically-defined mTeSR™1 media and demonstrated pluripotency of multiple hESC lines with immunostaining and quantitative RT-PCR studies. Results for hESC proliferation and pluripotency markers were both qualitatively and quantitatively similar to cells cultured on Matrigel™-coated substrates. Mechanistically, it was resolved that bovine serum albumin (BSA) in the mTeSR™1 media was critical for cell adhesion on APMAAm hydrogel interfaces. This study uniquely identified a robust long-term culture surface for the self-renewal of hESCs without the use of biologic coatings (e.g., peptides, proteins, or Matrigel™) in completely chemically-defined media that employed practical culturing techniques amenable to clinical-scale cell expansion.
Keywords: Biointerface; Hydrogel; Human embryonic stem cell; Self-renewal; BSA; QCM-D;

Notch signaling has been recognized as a key pathway to regulate the proliferation and differentiation of hematopoietic stem cells (HSC). In this study, the orientation-regulated immobilization of a Notch ligand was designed to achieve the efficient Notch ligand–receptor recognition for the ex vivo proliferation of a bone marrow cell population containing HSC. Protein A was chemically conjugated onto aminated glass substrates, followed by immobilizing a recombinant chimeric protein of Jagged1 and Fc domain (Jagged1-Fc) through the biospecific binding between protein A and Fc domain. Protein A adsorption was suppressed for the Jagged1-Fc-immobilized substrates, in contrast to the Jagged1-Fc-coated ones, indicating the orientation-regulated immobilization of Jagged1-Fc for the substrates. Mouse lineage negative cells (Lin) were cultured on the Jagged1-Fc-immobilized substrates. Flow cytometric analyses demonstrated that c-Kit+, Sca-1+, Lin, and CD34 cells of an HSC population was significantly proliferated on the Jagged1-Fc-immobilized substrates 6 days after culture, whereas no proliferation was observed for the Jagged1-Fc-coated substrates in a random manner or Jagged1-Fc-immobilized ones with a Notch signaling inhibitor. It is concluded that the orientation-regulated immobilization of Jagged1-Fc increased the efficiency of Jagged1 to recognize the Notch receptors, resulting in the promoted ex vivo proliferation of the HSC population.
Keywords: Surface modification; Protein; Scaffold; Stem cell; Cell proliferation;

Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes by Guo-Shiang Huang; Lien-Guo Dai; Betty L. Yen; Shan-hui Hsu (6929-6945).
Stem cells can lose their primitive properties during in vitro culture. The culture substrate may affect the behavior of stem cells as a result of cell–substrate interaction. The maintenance of self-renewal for adult human mesenchymal stem cells (MSCs) by a biomaterial substrate, however, has not been reported in literature. In this study, MSCs isolated from human adipose (hADAS) and placenta (hPDMC) were cultured on chitosan membranes and those further modified by hyaluronan (chitosan-HA). It was observed that the MSCs of either origin formed three-dimensional spheroids that kept attached on the membranes. Spheroid formation was associated with the increased MMP-2 expression. Cells on chitosan-HA formed spheroids more quickly and the size of spheroids were larger than on chitosan alone. The expression of stemness marker genes (Oct4, Sox2, and Nanog) for MSCs on the materials was analyzed by the real-time RT-PCR. It was found that formation of spheroids on chitosan and chitosan-HA membranes helped to maintain the expression of stemness marker genes of MSCs compared to culturing cells on polystyrene dish. The maintenance of stemness marker gene expression was especially remarkable in hPDMC spheroids (vs. hADAS spheroids). Blocking CD44 by antibodies prevented the spheroid formation and decreased the stemness gene expression moderately; while treatment by Y-27632 compound inhibited the spheroid formation and significantly decreased the stemness gene expression. Upon chondrogenic induction, the MSC spheroids showed higher levels of Sox9, aggrecan, and collagen type II gene expression and were stained positive for glycosaminoglycan and collagen type II. hPDMC had better chondrogenic differentiation potential than hADAS upon induction. Our study suggested that the formation of adhered spheroids on chitosan and chitosan-HA membranes may sustain the expression of stemness marker genes of MSCs and increase their chondrogenic differentiation capacity. The Rho/Rho-associated kinase (ROCK) signaling pathway may be involved in spheroid formation.► MSCs form adherent spheroids on chitosan and chitosan-hyaluronan membranes. ► Spheroid formation of MSCs upregulates the stemness marker gene expression. ► The above effects are more pronounced for MSCs isolated from placenta. ► Blocking CD44 or Rho/ROCK inhibits spheroid formation. ► The chondrogenic differentiation potential is enhanced through spheroid formation.
Keywords: MSC; Hyaluronan; Spheroid; Stemness gene; Chondrogenesis;

Despite significant advances in stem cell differentiation and tissue engineering, directing progenitor cells into three-dimensionally (3D) organized, native-like complex structures with spatially-varying mechanical properties and extra-cellular matrix (ECM) composition has not yet been achieved. The key innovations needed to achieve this would involve methods for directing a single stem cell population into multiple, spatially distinct phenotypes or lineages within a 3D scaffold structure. We have previously shown that specific combinations of natural and synthetic biomaterials can direct marrow-derived stem cells (MSC) into varying phenotypes of chondrocytes that resemble cells from the superficial, transitional, and deep zones of articular cartilage. In this current study, we demonstrate that layer-by-layer organization of these specific biomaterial compositions creates 3D niches that allow a single MSC population to differentiate into zone-specific chondrocytes and organize into a complex tissue structure. Our results indicate that a three-layer polyethylene glycol (PEG)-based hydrogel with chondroitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) incorporated into the top layer (superficial zone, PEG:CS:MMP-pep), CS incorporated into the middle layer (transitional zone, PEG:CS) and hyaluronic acid incorporated in the bottom layer (deep zone, PEG:HA), creates native-like articular cartilage with spatially-varying mechanical and biochemical properties. Specifically, collagen II levels decreased gradually from the superficial to the deep zone, while collagen X and proteoglycan levels increased, leading to an increasing gradient of compressive modulus from the superficial to the deep zone. We conclude that spatially-varying biomaterial compositions within single 3D scaffolds can stimulate efficient regeneration of multi-layered complex tissues from a single stem cell population.
Keywords: Hydrogels; Layer-by-layer; Bone marrow stem cells; Articular cartilage; Composite tissue engineering;

In the degenerative disc, overproduction of reactive oxygen species (ROS) involves in apoptosis and senescence of nucleus pulposus (NP) cells that could accelerate the degenerative process. Ferulic acid (FA) has been reported to have an excellent antioxidant property. In the study, injectable thermosensitive chitosan/gelatin/glycerol phosphate (C/G/GP) hydrogel was applied as a controlled release system for FA delivery. The study was aimed to evaluate possible therapeutic effects of FA-incorporated C/G/GP hydrogel on hydrogen peroxide (H2O2)-induced oxidative stress NP cells. The results showed that the release of FA from C/G/GP hydrogel could decrease the H2O2-induced oxidative stress. Post-treatment of FA-incorporated C/G/GP hydrogel on H2O2-induced oxidative stress NP cells showed up-regulation of Aggrecan and type II collagen and down-regulation of MMP-3 in mRNA level. The results of sulfated-glycosaminoglycans (GAGs) to DNA ratio and alcian blue staining revealed that the GAGs production of H2O2-induced oxidative stress NP cells could reach to normal level. The results of caspase-3 activity and TUNEL staining indicated that FA-incorporated C/G/GP hydrogel decreased the apoptosis of H2O2-induced oxidative stress NP cells. The results suggested that the C/G/GP hydrogel was very suitable for sustained delivery of FA. The FA-incorporated C/G/GP hydrogel would be used to treat the degenerative disc in the early stage before it developed into the latter irreversible stages.
Keywords: Nucleus pulposus; Ferulic acid; Oxidative stress; Hydrogel; Antioxidant;

Construction of the recellularized corneal stroma using porous acellular corneal scaffold by Jianhui Xiao; Hucheng Duan; Zhao Liu; Zheng Wu; Yuqing Lan; Wei Zhang; Chaoyang Li; Fen Chen; Qiang Zhou; Xiaoran Wang; Junqi Huang; Zhichong Wang (6962-6971).
Acellular porcine cornea stroma (APCS) prepared using pancreatic phospholipase A2 was proven to be promising corneal scaffold. However, its dense ultrastructure provides insufficient space that prevents the seeded cells from organizing into a functional tissue. In this report, freezing dry APCS (FD-APCS) biomaterials containing pores with different sizes were fabricated at different pre-freezing temperatures of −10, −80 or −198 °C, and the percentage of large pores (equivalent circle diameter ≥ 10 μm) was 93.55%, 69.36%, 35.79%, while the small pores (<10 μm) were account for 6.45%, 30.64%, 64.21%, respectively. Both porosity and specific surface area increased in FD-APCS fabricated with decreased pre-freezing temperature, and they were dramatically higher than those in APCS. The three FD-APCS groups displayed higher permeability than APCS, and the −10 °C FD-APCS possessed the highest permeability. The keratocytes seeded in the FD-APCS construct survived well in vitro, and maximal cell proliferation was observed in the −10 °C FD-APCS. The light transmittance of the FD-APCS-transplanted cornea after interlamellar keratoplasty in rabbit eyes displayed no significant difference from the APCS-transplanted or native cornea. This study indicated that the porous FD-APCS prepared using pancreatic phospholipase A2 is capable of serving as potential scaffold for constructing tissue-engineered cornea with biological properties.
Keywords: Corneal tissue engineering; Decellularization; Scaffold; Freezing dry; Porosity;

When injured, tendons tend to heal but with poor structure and compromised function. Tissue engineering is a promising approach to enhancing the quality of healing tendons. Our group and others have identified tendon stem cells (TSCs), a type of tendon-specific stem cells which may be optimal for cellular interventions seeking to restore normal structure and function to injured tendons. However, in vitro expanding of TSCs on regular plastic cell culture dishes only yields a limited number of TSCs before they lose the stemness, i.e., the self-renewal capability and multipotency. In this study, we developed a substrate material for TSCs, engineered tendon matrix (ETM) from decellularized tendon tissues. We showed that ETM in vitro was able to stimulate TSC proliferation and better preserve the stemness of TSCs than plastic culture surfaces. In vivo, implantation of ETM-TSC composite promoted tendon-like tissue formation whereas implantation of TSCs alone led to little such tissue formation. Together, the findings of this study indicate that ETM may be used to effectively expand TSCs in vitro and with TSCs, to enhance repair of injured tendons in vivo.
Keywords: Tendon matrix; Stemness; Tendon stem cells; Differentiation; Regeneration;

Galactosylated cellulosic sponge for multi-well drug safety testing by Bramasta Nugraha; Xin Hong; Xuejun Mo; Looling Tan; Wenxia Zhang; Po-Mak Chan; Chiang Huen Kang; Yan Wang; Lu Thong Beng; Wanxin Sun; Deepak Choudhury; Jeffrey M. Robens; Michael McMillian; Jose Silva; Shannon Dallas; Choon-Hong Tan; Zhilian Yue; Hanry Yu (6982-6994).
Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier structures when in extended culture. When the spheroid diameter exceeds 200 μm, cells in the inner core experience hypoxia and limited access to nutrients and drugs. Here we report the development of a thin galactosylated cellulosic sponge to culture hepatocytes in multi-well plates as 3D spheroids, and constrain them within a macroporous scaffold network to maintain spheroid size and prevent detachment. The hydrogel-based soft sponge conjugated with galactose provided suitable mechanical and chemical cues to support rapid formation of hepatocyte spheroids with a mature hepatocyte phenotype. The spheroids tethered in the sponge showed excellent maintenance of 3D cell morphology, cell–cell interaction, polarity, metabolic and transporter function and/or expression. For example, cytochrome P450 (CYP1A2, CYP2B2 and CYP3A2) activities were significantly elevated in spheroids exposed to β-naphthoflavone, phenobarbital, or pregnenolone-16α-carbonitrile, respectively. The sponge also exhibits minimal drug absorption compared to other commercially available scaffolds. As the cell seeding and culture protocols are similar to various high-throughput 2D cell-based assays, this platform is readily scalable and provides an alternative to current hepatocyte platforms used in drug safety testing applications.
Keywords: Cellulose; Galactose; Hydrogel; Sponge; Cytochrome P450; Drug induction;

A comparison between adipose tissue and dental pulp as sources of MSCs for tooth regeneration by Chia-Nung Hung; Kwei Mar; Hao-Chen Chang; Yi-Lun Chiang; Huai-Yun Hu; Chia-Chi Lai; Rei-Min Chu; Chang M. Ma (6995-7005).
In this study, several in vivo and in vitro comparisons were performed to test the possibility of using adipose-derived stem cells (ADSCs), a more convenient cell source than dental pulp stem cells (DPSCs), in tooth regeneration. Using an efficient, non-engineering implantation method, we first demonstrated that both implants of ADSCs and DPSCs were able to grow self-assembled new teeth in adult rabbit extraction sockets with high success rate. The stem cells were necessary because the implants grew no tooth without them. A stepwise comparison showed that the regenerated teeth from these two types of adult stem cells were living with nerves and vascular system and remarkably similar to a normal tooth in many details. Further strictly controlled, side-by-side comparisons between the two types of stem cells also showed that the expression patterns of gene markers and the broad differentiation potentials induced by specific methods in vitro were very similar. Although a few differences were found, they did not affect the tested tooth regeneration in vivo or differentiation in vitro. Furthermore, rabbit ADSCs had a higher growth rate and a better senescence resistance in culture. All these findings suggest that ADSCs, one of the richest adult stem cells in mammals, are very similar and useful as DPSCs for regenerative dentistry.
Keywords: Adipose-derived stem cells (ADSC); Dental pulp stem cells (DPSC); Mesenchymal stem cell; Transplantation; Tooth regeneration;

Prediction of spatio-temporal bone formation in scaffold by diffusion equation by Alireza Roshan-Ghias; Arne Vogel; Lalaonirina Rakotomanana; Dominique P. Pioletti (7006-7012).
Developing a successful bone tissue engineering strategy entails translation of experimental findings to clinical needs. A major leap forward toward this goal is developing a quantitative tool to predict spatial and temporal bone formation in scaffold. We hypothesized that bone formation in scaffold follows diffusion phenomenon. Subsequently, we developed an analytical formulation for bone formation, which had only three unknown parameters: C, the final bone volume fraction, α , the so-called scaffold osteoconduction coefficient, and h, the so-called peri-scaffold osteoinduction coefficient. The three parameters were estimated by identifying the model within vivo data of polymeric scaffolds implanted in the femoral condyle of rats. In vivo data were obtained by longitudinal micro-CT scanning of the animals. Having identified the three parameters, we used the model to predict the course of bone formation in two previously published in vivo studies. We found the predicted values to be consistent with the experimental ones. Bone formation into a scaffold can then adequately be described through diffusion phenomenon. This model allowed us to spatially and temporally predict the outcome of tissue engineering scaffolds with only 3 physically relevant parameters.
Keywords: Mathematical modeling; Bone formation; Animal model; Micro-CT imaging;

Wnt5a-mediating neurogenesis of human adipose tissue-derived stem cells in a 3D microfluidic cell culture system by Jeein Choi; Sohyeun Kim; Jinsun Jung; Youngbin Lim; Kyungsun Kang; Seungsu Park; Sookyung Kang (7013-7022).
In stem cell biology, cell plasticity refers to the ability of stem cells to differentiate into a variety of cell lineages. Recently, cell plasticity has been used to refer to the ability of a given cell type to reversibly de-differentiate, re-differentiate, or transdifferentiate in response to specific stimuli. These processes are regulated by multiple intracellular and extracellular growth and differentiation factors, including low oxygen. Our recent study showed that 3D microfluidic cell culture induces activation of the Wnt5A/β-catenin signaling pathway in hATSCs (human Adipose Tissue-derived Stem Cells). This resulted in self renewal and transdifferentiation of hATSCs into neurons. To improve neurogenic potency of hATSCs in response to low oxygen and other unknown physical factors, we developed a gel-free 3D microfluidic cell culture system (3D-μFCCS). The functional structure was developed for the immobilization of 3D multi-cellular aggregates in a microfluidic channel without the use of a matrix on the chip. Growth of hATSCs neurosphere grown on a chip was higher than the growth of control cells grown in a culture dish. Induction of differentiation in the Chip system resulted in a significant increase in the induction of neuronal-like cell structures and the presentation of TuJ or NF160 positive long neuritis compared to control cells after active migration from the center of the microfluidic channel layer to the outside of the microfluidic channel layer. We also observed that the chip neurogenesis system induced a significantly higher level of GABA secreting neurons and, in addition, almost 60% of cells were GABA + cells. Finally, we observed that 1 month of after the transplantation of each cell type in a mouse SCI lesion, chip cultured and neuronal differentiated hATSCs exhibited the ability to effectively transdifferentiate into NF160 + motor neurons at a high ratio. Interestingly, our CHIP/PCR analysis revealed that HIF1α-induced hATSCs neurogenesis on the chip. This induction was a result of the direct binding of HIF1α to the regulatory regions of the Oct4 and β-catenin genes in nucleus. In the Chip culture of hATSCs that we developed, a low oxygen microenvironment was induced. The low oxygen level induced HIF1α expression, which resulted in increased expression of Wnt5A/β-catenin and Oct4 via the direct binding of HIF1α to the regulatory regions of β-catenin and Oct4.
Keywords: 3D microfluidic cell culture; human Adipose Tissue-derived Stem Cells; Wnt5A/ β-cateninsignaling; HIF1α;

The stimulation of osteogenic differentiation of human adipose-derived stem cells by ionic products from akermanite dissolution via activation of the ERK pathway by Huijie Gu; Fangfang Guo; Xiao Zhou; Lunli Gong; Yun Zhang; Wanyin Zhai; Lei Chen; Lian Cen; Shuo Yin; Jiang Chang; Lei Cui (7023-7033).
Our previous study indicates that akermanite, a type of Ca-, Mg-, Si-containing bioceramic, can promote the osteogenic differentiation of hASCs. To elucidate the underlying mechanism, we investigated the effect of the extract from akermanite, on proliferation and osteogenic differentiation of hASCs. The original extract was obtained at 200 mg akermanite/ml LG-DMEM and further diluted with LG-DMEM. The final extracts were denoted as 1/2, 1/4, 1/8, 1/16, and 1/32 extracts based on the concentrations of the original extract. The LDH assay and live/dead stain were used to reveal the cytotoxicity of the different extracts on hASCs, while the DNA assay was carried out to quantitatively evaluate the proliferation of cells after being cultured with the extracts for 1, 3 and 7 days. Flow cytometry for cell cycle analysis was carried out on cells cultured in two media (GM and 1/2 extract) in order to further analyze the effect of the extract on cell proliferation behaviors. Osteogenic differentiation of hASCs cultured in the extracts was detected by ALP expression and calcium deposition, and further confirmed by real-time PCR analysis. It was shown that Ca, Mg and Si ions in the extract could suppress the LDH release and proliferation of hASCs, whereas promote their osteogenic differentiation. Such effects were concentration-dependent with the 1/4 extract (Ca 2.36 mM, Mg 1.11 mM, Si 1.03 mM) being the optimum in promoting the osteogenic differentiation of hASCs. An immediate increase in ERK was observed in cells cultured in the 1/4 extract and such osteogenic differentiation of hASCs promoted by released ions could be blocked by MEK1-specific inhibitor, PD98059. Briefly, Ca, Mg and Si ions extracted from akermanite in the concentrations of 2.36, 1.11, 1.03 mM, respectively, could facilitate the osteogenic differentiation of hASCs via an ERK pathway, and suppress the proliferation of hASCs without significant cytotoxicity.
Keywords: Akermanite; Adipose-derived stem cells; Ions; Osteogenesis; Bone tissue engineering; MAPK;

Polymers for the rapid and effective activation and aggregation of platelets by Anne Hansen; Loraine McMillan; Alex Morrison; Juraj Petrik; Mark Bradley (7034-7041).
Platelets are responsible for plugging sites of vascular injury, where upon activation they spread out and become cross-linked, preventing further blood loss. It is desirable to control the activation process on demand for applications such as the rapid staunching of blood flow following trauma. Polymers are the material of choice in many biological areas, with physical properties that allow control of morphology as well as ease of functionalisation and production. Herein, polymer microarrays were used to screen a complex human fluid (platelet rich plasma) to identify polyacrylates that could be used to modulate platelet activation. Several polymers were identified which rapidly activated platelets as determined by CD61P binding and subsequent confirmation by scanning electron microcopy analysis. This approach enabled a direct comparison between the natural agonist collagen and synthetic polymers with respect to the activation status of the platelets as well as the number of bound platelets. Further investigations under physiological flow demonstrated that the static microarray experiments gave viable candidates for potential medical applications while specific protein binding to the polymers was identified as a possible mode of action. The approach demonstrates the ability of polymer microarrays to identify new polymers for specific biological activation events and in this case allowed the identification of materials that allowed higher levels of platelets to bind in advanced activation states than the natural standard collagen in static and flow studies.
Keywords: Collagen; Copolymer; Haemostasis; Platelet activation; Platelet adhesion; Wound dressing;

Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix by Ren Lang; Matthew M. Stern; Leona Smith; Yan Liu; Shantaram Bharadwaj; Guihua Liu; Pedro M. Baptista; Christopher R. Bergman; Shay Soker; James J. Yoo; Anthony Atala; Yuanyuan Zhang (7042-7052).
There is currently no optimal system to expand and maintain the function of human adult hepatocytes in culture. Recent studies have demonstrated that specific tissue-derived extracellular matrix (ECM) can serve as a culture substrate and that cells tend to proliferate and differentiate best on ECM derived from their tissue of origin. The goal of this study was to investigate whether three-dimensional (3D) ECM derived from porcine liver can facilitate the growth and maintenance of physiological functions of liver cells. Optimized decellularization/oxidation procedures removed up to 93% of the cellular components from porcine liver tissue and preserved key molecular components in the ECM, including collagen-I, -III, and -IV, proteoglycans, glycosaminoglycans, fibronectin, elastin, and laminin. When HepG2 cells or human hepatocytes were seeded onto ECM discs, uniform multi-layer constructs of both cell types were formed. Dynamic culture conditions yielded better cellular infiltration into the ECM discs. Human hepatocytes cultured on ECM discs expressed significantly higher levels of albumin over a 21-day culture period compared to cells cultured in traditional polystyrene cultureware or in a collagen gel “sandwich”. The culture of hepatocytes on 3D liver-specific ECM resulted in considerably improved cell growth and maintained cell function; therefore, this system could potentially be used in liver tissue regeneration, drug discovery or toxicology studies.
Keywords: ECM (extracellular matrix); Liver; 3D; Cell culture; Hepatocyte; Albumin;

The effect of composition of calcium phosphate composite scaffolds on the formation of tooth tissue from human dental pulp stem cells by Liqin Zheng; Fei Yang; Hong Shen; Xuefeng Hu; Chihiro Mochizuki; Mitsunobu Sato; Shenguo Wang; Yanding Zhang (7053-7059).
Different approaches towards making 3-dimensional (3-D) bioengineered tooth for future replacement therapy have been developed including scaffold-based tooth regeneration. However, selection of optimal scaffold for future clinical application remains a challenge. In the present study, we tested biocompatibility of four different types of 3-D scaffolds for tooth-tissue regeneration, including a pure poly(lactide-co-glycolide) (PLGA) (70/30, mol/mol) scaffold and three types of calcium phosphate contained composites scaffolds that were 50 wt% of PLGA combined with 50 wt% of hydroxyapatite (HA), tricalcium phosphate (TCP) or calcium carbonate hydroxyapatite (CDHA) respectively. These scaffolds were fabricated by the particle leaching in combination with phase separation technology. Surface modification of these scaffolds was further performed by an ammonia plasma treatment and anchorage of collagen technology. Effect of composition of the composite scaffolds on proliferation of human dental pulp stem cells (DPSCs) was accessed using in vitro MTT assay and in vivo BrdU labeling. Differentiation capability of the DPSCs in the scaffolds was analyzed by measurement of the levels of calcified tissue formation and ALP activity. Our results showed that while the calcium phosphate contained compound is able to support regeneration of tooth tissue effectively, the PLGA/TCP scaffold is more appropriate for the proliferation and differentiation of DPSCs. Furthermore, seeding of dissociated 4-dpn rat tooth bud cells on the PLGA/TCP scaffold generated dentin- and pulp-like tissues. Our results demonstrate that the PLGA/TCP scaffold is superior to the other three scaffolds for tooth-tissue regeneration, especially for dentin formation.
Keywords: Scaffold; Calcium phosphate contained composites scaffolds; PLGA/TCP composite scaffolds; Bioengineered tooth;

The effect of decellularized bone/bone marrow produced by high-hydrostatic pressurization on the osteogenic differentiation of mesenchymal stem cells by Yoshihide Hashimoto; Seiichi Funamoto; Tsuyoshi Kimura; Kwangwoo Nam; Toshiya Fujisato; Akio Kishida (7060-7067).
Decellularized bone/bone marrow was prepared to provide a microenvironment mimicking that of the bone marrow for three-dimensional culture in vitro. Bone/bone marrows were hydrostatically pressed at 980 MPa at 30 °C for 10 min to dismantle the cells. Then, they were washed with EGM-2 and further treated in an 80% EtOH to remove the cell debris and lipid, respectively. After being rinsed and shaken with PBS again, treated bone/bone marrows were stained with hematoxylin and eosin (H-E) to assess the efficacy of decellularization. Cells were determined to have been completely removed through H-E staining of their sections and DNA quantification. Rat mesenchymal stem cells (rMSCs) were seeded on the decellularized bone/bone marrows and cultured for 21 days. The adhesion of rMSCs on or into decellularized bone/bone marrows was confirmed and proliferated over time in culture. The osteogenic differentiation effect of decellularized bone/bone marrows on rMSCs in the presence or absence of dexamethasone was investigated. Decellularized bone/bone marrows without dexamethasone significantly increased alkaline phosphatase (ALP) activity, indicating promoted osteogenic differentiation of rMSCs. In an animal study, when decellularized bone/bone marrows were implanted into the rat subcutaneous, no immune reaction occurred and clusters of the hematopoietic cells could be observed, suggesting the decellularized bone/bone marrows can provide a microenvironment in vivo.
Keywords: Decellularization; Extracellular matrix; Bone marrow; Scaffold; Hematopoiesis;

Proliferation, differentiation and gene expression of osteoblasts in boron-containing associated with dexamethasone deliver from mesoporous bioactive glass scaffolds by Chengtie Wu; Richard Miron; Anton Sculean; Stefan Kaskel; Thomas Doert; Renate Schulze; Yufeng Zhang (7068-7078).
Boron is one of the trace elements in the human body which plays an important role in bone growth. Porous mesopore bioactive glass (MBG) scaffolds are proposed as potential bone regeneration materials due to their excellent bioactivity and drug-delivery ability. The aims of the present study were to develop boron-containing MBG (B-MBG) scaffolds by sol–gel method and to evaluate the effect of boron on the physiochemistry of B-MBG scaffolds and the response of osteoblasts to these scaffolds. Furthermore, the effect of dexamethasone (DEX) delivery in B-MBG scaffold system was investigated on the proliferation, differentiation and bone-related gene expression of osteoblasts. The composition, microstructure and mesopore properties (specific surface area, nano-pore volume and nano-pore distribution) of B-MBG scaffolds have been characterized. The effect of boron contents and large-pore porosity on the loading and release of DEX in B-MBG scaffolds were also investigated. The results have shown that the incorporation of boron into MBG scaffolds slightly decreases the specific surface area and pore volume, but maintains well-ordered mesopore structure and high surface area and nano-pore volume compared to non-mesopore bioactive glass. Boron contents in MBG scaffolds did not influence the nano-pore size distribution or the loading and release of DEX. B-MBG scaffolds have the ability to maintain a sustained release of DEX in a long-term span. Incorporating boron into MBG glass scaffolds led to a controllable release of boron ions and significantly improved the proliferation and bone-related gene expression (Col I and Runx2) of osteoblasts. Furthermore, the sustained release of DEX from B-MBG scaffolds significantly enhanced alkaline phosphatase (ALP) activity and gene expressions (Col I, Runx2, ALP and BSP) of osteoblasts. These results suggest that boron plays an important role in enhancing osteoblast proliferation in B-MBG scaffold system and DEX-loaded B-MBG scaffolds show great potential as a release system to enhance osteogenic property for bone tissue engineering application.
Keywords: Mesopore bioactive glass; Scaffold; Boron; Drug delivery; Proliferation; Gene expression;

A computational analysis of the insertion of carbon nanotubes into cellular membranes by Siegfried Höfinger; Manuel Melle-Franco; Tommaso Gallo; Andrea Cantelli; Matteo Calvaresi; José A.N.F. Gomes; Francesco Zerbetto (7079-7085).
Carbon nanotubes have been proposed to serve as nano-vehicles to deliver genetic or therapeutic material into the interior of cells because of their capacity to cross the cell membrane. A detailed picture of the molecular mode of action of such a delivery is, however, difficult to obtain because of the concealing effects of the cell membrane. Here we report a systematic computational study of membrane insertion of individual carbon nanotubes and carbon nanotube bundles using two entirely different and unrelated techniques. First a static scan of the environmental free energy is carried out based on a membrane mimicry approach and different insertion geometries are assessed. Then the dynamics is investigated with a coarse-grained approach that was previously used in the study of the integration dynamics of nanoparticles into the bilayer. The results of both models point, for unfunctionalized carbon nanotubes, at a preference for the horizontal orientation inside the internal hydrophobic layer of the cell membrane. Finally, the energetics of the formation of bundles of carbon nanotubes is studied. The cellular membrane promotes aggregation of carbon nanotubes in its hydrophobic core and modifies the structural stability of the bundles.
Keywords: Carbon nanotubes; Membrane insertion; Membrane mimicry; Dissipative particle dynamics;

Several porcine-derived acellular biologic grafts are increasingly used in abdominal wall reconstruction due to the limitations of synthetic meshes in many clinical situations. However, relatively little is known so far about their comparative mechanical characteristics and performance after defect repair. We therefore investigated three most commonly used porcine-derived acellular biomaterials, small intestine submucosa (P-SIS), pericardium (P-PC) and acellular dermal matrix (P-ADM) immediately after prepared, and their effectiveness, biomechanical and histological characteristics in repairing full-thickness abdominal defect in a rat model. P-PC had the best native performance in the burst strength, tensile strength and ball burst among the three porcine-derived scaffolds. P-SIS showed a significantly higher water vapor transmission in comparison with P-PC or P-ADM. Abdominal wall defects in rats were all satisfied repaired with P-SIS, P-PC or P-ADM. No laxity or fistula was observed in the repaired abdominal wall in the P-SIS group up to 8 weeks after surgery. However, there was a tendency for high postoperative abdominal eventration in the P-ADM and P-PC groups as compared with the P-SIS group. With regard to overall aspects of the postoperative laxity, intra-abdominal adhesion formation, tensile stress, stretchability, and degree of tissue ingrowth in terms of collagen deposition and neovascularization, P-SIS exhibits clear advantages over P-PC as well as P-ADM after large abdominal wall defect reconstruction.
Keywords: Porcine-derived biologic grafts; Small intestine submucosa; Pericardium; Dermal matrix; Abdominal defect repair;

The role of fibrin E on the modulation of endothelial progenitors adhesion, differentiation and angiogenic growth factor production and the promotion of wound healing by Francisco Caiado; Tânia Carvalho; Fernanda Silva; Catarina Castro; Nuno Clode; Julian F. Dye; Sérgio Dias (7096-7105).
Severe skin loss constitutes a major unsolved clinical problem worldwide. For this reason, in the last decades there has been a major push towards the development of novel therapeutic approaches to enhance skin wound healing. Neo-vessel formation through angiogenesis is a critical step during the wound healing process. Besides the contribution of pre-existing endothelial cells (EC), endothelial progenitor cells (EPCs) have also been implicated in wound healing acting either by differentiating into EC that incorporate the neo-vessels, or via the production of paracrine factors that improve angiogenesis. Here we tested the importance of different extracellular matrices (ECM) in regulating the angiogenic and wound healing potential of cord blood-derived EPC (CB-EPC). We compared the properties of several ECM and particularly of fibrin fragment E (FbnE) in regulating EPC adhesion, proliferation, differentiation and healing-promotion in vitro and in vivo. Our results show that CB-EPCs have increased adhesion and endothelial differentiation when plated on FbnE compared to collagens, fibronectin or fibrin. Using integrin neutralizing antibodies, we show that CB-EPC adhesion to FbnE is mediated by integrin α5β1. Gene expression analysis of CB-EPCs plated on different substrates revealed that CB-EPC grown on FbnE shows increased expression of paracrine factors such as VEGF-A, TGF-β1, SDF-1, IL-8 and MIP-1α. Accordingly, conditioned media from CB-EPC grown on FbnE induced EC tube formation and monocyte migration in vitro. To test the wound healing effects of FbnE in vivo we used an FbnE enriched scaffold in a cutaneous wound healing mouse model. In accordance with our in vitro data, co-administration of the FbnE enriched scaffold with CB-EPC significantly accelerated wound closure and wound vascularization, compared FbnE enriched scaffold alone or to using collagen-based scaffolds. Our results show that FbnE modulates several CB-EPC properties in vivo and in vitro, and as such promotes wound healing. We suggest the use of FbnE-based scaffolds represents a promising approach to resolve wound healing complications arising from different pathologies.► We tested the effect of fibrin fragment E (FbnE) on the vasculogenic and angiogenic properties of endothelial progenitor cells (EPCs). ► FbnE enhances EPC adhesion, via integrin α5β1 and endothelial differentiation compared to other substrates. ► FbnE promotes angiogenic and chemotactic factor production by EPCs. ► Co-administration of EPCs with a FbnE enriched scaffold into mouse cutaneous wounds increases vascularization and promotes wound closure.
Keywords: Endothelial progenitors; Wound healing; Fibrin E; Angiogenesis;

Discontinuities in the human bone–PDL–cementum complex by Jonathan M. Hurng; Michael P. Kurylo; Grayson W. Marshall; Samuel M. Webb; Mark I. Ryder; Sunita P. Ho (7106-7117).
A naturally graded interface due to functional demands can deviate toward a discontinuous interface, eventually decreasing the functional efficiency of a dynamic joint. It is this characteristic feature in a human bone–tooth fibrous joint bone–PDL–tooth complex that will be discussed through histochemistry, and site-specific high resolution microscopy, micro tomography(Micro XCT™), X-ray fluorescence imaging and wet nanoindentation techniques. Results demonstrated two causes for the occurrence of 5–50 μm narrowed PDL-space: 1) microscopic scalloped regions at the PDL-insertion sites and macro-scale stratified layers of bone with rich basophilic lines, and 2) macroscopic bony protrusions. Narrowed PDL-complexes illustrated patchy appearance of asporin, and when imaged under wet conditions using an atomic force microscope (AFM), demonstrated structural reorganization of the PDL, collagen periodicity, organic-dominant areas at the PDL–cementum and PDL–bone entheses and within cementum and bone. Scanning electron microscopy (SEM) results confirmed AFM results. Despite the narrowed PDL, continuity between PDL and vasculature in endosteal spaces of bone was demonstrated using a Micro XCT™. The higher levels of Ca and P X-ray fluorescence using a microprobe were correlated with higher elastic modulus values of 0.1–1.4 and 0.1–1.2 GPa for PDL–bone and PDL–cementum using wet nanoindentation. The ranges in elastic modulus values for PDL–bone and PDL–cementum entheses in 150–380 μm wide PDL-complex were 0.1–1.0 and 0.1–0.6 GPa. Based on these results we propose that strain amplification at the entheses could be minimized with a gradual change in modulus profile, a characteristic of 150–380 μm wide functional PDL-space. However, a discontinuity in modulus profile, a characteristic of 5–50 μm wide narrowed PDL-space would cause compromised mechanotransduction. The constrictions or narrowed sites within the bone–tooth fibrous joint will become the new “load bearing sites” that eventually could cause direct local fusion of bone with cementum.
Keywords: Interfaces; Bone–PDL and cementum–PDL entheses; Attachment sites; Discontinuities; Gradients;

A highly organized three-dimensional alginate scaffold for cartilage tissue engineering prepared by microfluidic technology by Chen-Chie Wang; Kai-Chiang Yang; Keng-Hui Lin; Hwa-Chang Liu; Feng-Huei Lin (7118-7126).
Osteoarthritis is a degenerative disease and frequently involves the knee, hip and phalangeal joints. Current treatments used in small cartilage defects including multiple drilling, abrasion arthroplasty, mosaicplasty, and autogenous chondrocyte transplantation, however, there are problems needed to be solved. The standard treatment for severe osteoarthritis is total joint arthroplasty. The disadvantages of this surgery are the possibility of implant loosening. Therefore, tissue engineering for cartilage regeneration has become a promising topic. We have developed a new method to produce a highly organized single polymer (alginate) scaffold using microfluidic device. Scanning electron microscope and confocal fluoroscope examinations showed that the scaffold has a regular interconnected porous structure in the scale of 250 μm and high porosity. The scaffold is effective in chondrocyte culture; the cell viability test (WST-1 assay), cell toxicity (lactate dehydrogenase assay), cell survival rate, extracellular matrix production (glycosaminoglycans contents), cell proliferation (DNA quantification), and gene expression (real-time PCR) all revealed good results for chondrocyte culture. The chondrocytes can maintain normal phenotypes, highly express aggrecan and type II collagen, and secrete a great deal of extracellular matrix when seeded in the alginate scaffold. This study demonstrated that a highly organized alginate scaffold can be prepared with an economical microfluidic device, and this scaffold is effective in cartilage tissue engineering.
Keywords: Alginate; Scaffold; Chondrocyte; Microfluidic device;

A review of NIR dyes in cancer targeting and imaging by Shenglin Luo; Erlong Zhang; Yongping Su; Tianmin Cheng; Chunmeng Shi (7127-7138).
The development of multifunctional agents for simultaneous tumor targeting and near infrared (NIR) fluorescence imaging is expected to have significant impact on future personalized oncology owing to the very low tissue autofluorescence and high tissue penetration depth in the NIR spectrum window. Cancer NIR molecular imaging relies greatly on the development of stable, highly specific and sensitive molecular probes. Organic dyes have shown promising clinical implications as non-targeting agents for optical imaging in which indocyanine green has long been implemented in clinical use. Recently, significant progress has been made on the development of unique NIR dyes with tumor targeting properties. Current ongoing design strategies have overcome some of the limitations of conventional NIR organic dyes, such as poor hydrophilicity and photostability, low quantum yield, insufficient stability in biological system, low detection sensitivity, etc. This potential is further realized with the use of these NIR dyes or NIR dye-encapsulated nanoparticles by conjugation with tumor specific ligands (such as small molecules, peptides, proteins and antibodies) for tumor targeted imaging. Very recently, natively multifunctional NIR dyes that can preferentially accumulate in tumor cells without the need of chemical conjugation to tumor targeting ligands have been developed and these dyes have shown unique optical and pharmaceutical properties for biomedical imaging with superior signal-to-background contrast index. The main focus of this article is to provide a concise overview of newly developed NIR dyes and their potential applications in cancer targeting and imaging. The development of future multifunctional agents by combining targeting, imaging and even therapeutic routes will also be discussed. We believe these newly developed multifunctional NIR dyes will broaden current concept of tumor targeted imaging and hold promise to make an important contribution to the diagnosis and therapeutics for the treatment of cancer.
Keywords: Near infrared dyes; Cancer targeting; Imaging; Multifunctional agents;

Theragnostics polymer nanoparticles (NPs) loaded simultaneously with anticancer drug docetaxel (Dtxl) and superparamagnetic iron oxide (SPIO) nanocrystals were developed for both cancer therapy and ultrasensitive MRI. These multifunctional polymer vesicles were formed by carboxy-terminated poly(lactic-co-glycolic) acid using a single emulsion evaporation method. The active tumor-targeting single chain prostate stem cell antigen antibodies (scAbPSCA) were conjugated on the surface of polymer vesicles by using functional poly(ethylene glycol). The diameter of NPs was about 147 nm and the SPIO and drug encapsulation efficacy was 23% and 6.02%, respectively. Vibration simple magnetometer and X-ray diffraction proved that the superparamagnetic behavior of SPIO was not changed during NPs formation and modification. The NPs exhibited a triphasic drug release pattern in vitro over 30 days. Enhanced cellular uptake ability and antiproliferative effect of the targeted NPs in prostate cancer PC3 cell line by using the confocal laser scanning microscopy and cytotoxicity assay were observed. Moreover, the Prussian blue staining and the MRI assay in vitro demonstrated that the NPs have a high SPIO clustering effect. Therefore, these stable and tumor-targeting polymer NPs could be promising multifunctional vesicles for simultaneous targeting imaging, drug delivery and real time monitoring of therapeutic effect.
Keywords: Theragnostics; Docetaxel; Superparamagnetic iron oxide; Antibody; Magnetic resonance imaging; Prostate cancer;

The use of the fusion protein RGD-HSA-TIMP2 as a tumor targeting imaging probe for SPECT and PET by Naeun Choi; Sung-Min Kim; Kwan Soo Hong; Gyunggoo Cho; Jee-Hyun Cho; Chulhyun Lee; Eun Kyoung Ryu (7151-7158).
The human serum albumin tissue inhibitor of metalloproteinase 2 (HSA-TIMP2) is known to possess antitumor activity, which has been attributed to its ability to inhibit endothelial cell proliferation by binding to integrin receptors. In this study, a fusion protein, cyclic arginine-glycine-aspartate (RGD)-HSA-TIMP2, formed by conjugating HSA-TIMP2 with a RGD peptide, and its 123I- and 68Ga-labeled compounds, were synthesized and evaluated with in vivo tumor imaging using single photon emission computed tomography (SPECT) and positron emission tomography (PET). RGD-HSA-TIMP2 was synthesized by covalent bonding of the RGD peptide to the side chain amino groups of HSA-TIMP2 from a two-step reaction involving from activation with N-succinimidyl iodoacetate. This conjugation improved the anticancer effect of HSA-TIMP2 in cancer cells. The 123I- and 68Ga-labeled fusion proteins were prepared and subsequently injected into the tail veins of mice bearing human glioblastoma cancer U87MG xenografts for SPECT and PET imaging and biodistribution studies. Tumor uptake of radioligand was high in both the PET images and in the biodistribution studies at 3 h after injection. These studies demonstrated that the new fusion protein has potential not only as an anticancer agent but also as a radioligand for the diagnosis of tumors.
Keywords: HSA-TIMP2; RGD; RGD-HSA-TIMP2; Diagnosis; Tumor; Imaging probe;

The in vivo cellular microenvironment is regulated by a complex interplay of soluble factors and signaling molecules secreted by cells and it plays a critical role in the growth and development of normal and diseased tissues. In vitro systems that can recapitulate the microenvironment at the cellular level are needed to investigate the influence of autocrine signaling and extracellular matrix effects on tissue homeostasis, regeneration, disease development and progression. In this study, we report the use of microbubble technology as a means to culture cells in a controlled microenvironment in which cells can influence their function through autocrine signaling. Microbubbles (MB) are small spherical cavities about 100–300 μm in diameter formed in hydrophobic polydimethylsiloxane (PDMS) with ∼60–100 μm circular openings and aspect ratio ∼3.0. We demonstrate that the unique architecture of the microbubble compartment is advantaged for cell culture using HaCaT cells, an immortalized keratinocyte cell line. We observe that HaCaT cells, seeded in microbubbles (15–20 cells/MB) and cultured under standard conditions, adopt a compact 3D spheroidal morphology. Within 2–3 days, the cells transition to a sheeting morphology. Through experimentation and simulation we show that this transition in morphology is due to the unique architecture of the microbubble compartment which enables cells to condition their local microenvironment. The small media volume per cell and the development of shallow concentration gradients allow factors secreted by the cells to rise to bioactive levels. The kinetics of the morphology transition depends on the number of cells seeded per microbubble; higher cell seeding induces a more rapid transition. HaCaT cells seeded onto PDMS cured in 96-well plates also form compact spheroids but they do not undergo a transition to a sheeting morphology even after several weeks of culture. The importance of soluble factor accumulation in driving this morphology transition in microbubbles is supported by the observation that spheroids do not form when cells – seeded into microbubbles or onto PDMS cured in 96-well plates – are cultured in media conditioned by HaCaT cells grown in standard tissue culture plate. We observed that the addition of TGF-β1 to the growth media induced cells to proliferate in a sheeting morphology from the onset both on PDMS cured in 96-well plates and in microbubbles. TGF-β1 is a morphogen known to regulate epithelial-to-mesenchymal transition (EMT). Studies of the role of Ca2+ concentration and changes in E-cadherin expression additionally support an EMT-like HaCaT morphology transition. These findings taken together validate the microbubble compartment as a unique cell culture platform that can potentially transform investigative studies in cell biology and in particular the tumor microenvironment. Targeting the tumor microenvironment is an emerging area of anti-cancer therapy.
Keywords: Cell spreading; Polydimethylsiloxane; Cell culture; Transforming growth factor (TGF);

The effect of stromal components on the modulation of the phenotype of human bronchial epithelial cells in 3D culture by Steven C. Pageau; Olga V. Sazonova; Joyce Y. Wong; Ana M. Soto; Carlos Sonnenschein (7169-7180).
The stroma plays an important role in the development and progression of human diseases. Pulmonary diseases such as asthma, fibrosis and cancer are thought to be the result of altered communications between the epithelial and stromal tissue compartments. In order to study these epithelial–mesenchymal interactions, we developed a three dimensional (3D) in vitro model of the human airway that mimics bronchial morphology and function. This model consists of a type-I collagen matrix, normal human fetal lung fibroblasts (IMR-90) or primary human adult lung cancer-associated fibroblasts (LuCAFs), and a surface epithelium of normal human bronchial epithelial cells (HBECs). When cultured at an air–liquid interface (ALI), the epithelial component generated a well-differentiated pseudo-stratified bronchial epithelium that contained basal, ciliated, and non-ciliated (secretory) epithelial cells. IMR-90 and LuCAFs differentially altered the phenotype of HBECs in distinct ways. While IMR-90 permitted HBECs to form a typical respiratory surface epithelium, LuCAFs promoted HBECs to invade the collagen gel forming both epithelial nodules and cysts, suggesting that LuCAFs may alter the HBEC phenotype by modifying biomechanical signals conveyed through the extracellular matrix (ECM). Furthermore, LuCAFs secreted soluble factors that induced HBECs to express genes associated with immune responses, apoptosis, mitosis, cell survival, differentiation and cancer.
Keywords: Co-culture; Collagen; ECM (extracellular matrix); Fibroblast; Lung;

Self-assembled nanoparticles based on hyaluronic acid-ceramide (HA-CE) and Pluronic® for tumor-targeted delivery of docetaxel by Hyun-Jong Cho; Hong Yeol Yoon; Heebeom Koo; Seung-Hak Ko; Jae-Seong Shim; Ju-Hee Lee; Kwangmeyung Kim; Ick Chan Kwon; Dae-Duk Kim (7181-7190).
Hyaluronic acid-ceramide (HA-CE)-based self-assembled nanoparticles were developed for intravenous docetaxel (DCT) delivery. In this study, physicochemical properties, cellular uptake efficiency, and in vivo targeting capability of the nanoparticles developed were investigated. DCT-loaded nanoparticles composed of HA-CE and Pluronic 85 (P85) with a mean diameter of 110–140 nm were prepared and their morphological shapes were assessed using transmission electron microscopy (TEM). DCT release from nanoparticle was enhanced with increasing P85 concentrations in our in vitro model. Blank nanoparticles exhibited low cytotoxicity in U87-MG, MCF-7 and MCF-7/ADR cell lines. From cellular uptake studies, the nanoparticles developed enhanced the intracellular DCT uptake in the CD44-overexpressing cell line (MCF-7). The nanoparticles were shown to be taken up by the HA–CD44 interaction according to DCT and coumarin 6 (C6) cellular uptake studies. The multidrug resistance (MDR)-overcoming effects of DCT-loaded HA-CE/P85-based nanoparticles were also observed in cytotoxicity tests in MCF-7/ADR cells. Following the intravenous injection of DCT-loaded cyanine 5.5 (Cy5.5)-conjugated nanoparticles in MCF-7/ADR tumor-bearing mice, its in vivo targeting for CD44-overexpressing tumors was identified by non-invasive near-infrared (NIR) fluorescence imaging. These results indicate that the HA-CE-based nanoparticles prepared may be a promising anti-cancer drug delivery system through passive and active tumor targeting.► Nanoparticles based on hyaluronic acid-ceramide and Pluronic® were developed. ► Docetaxel was delivered to tumor via hyaluronic acid and CD44 receptor interaction. ► In vitro multidrug resistance overcoming effect was also achieved.
Keywords: Hyaluronic acid-ceramide; Poloxamer; Nanoparticle; Docetaxel; CD44;

Gold nanoparticle-incorporated human red blood cells (RBCs) for X-ray dynamic imaging by Sungsook Ahn; Sung Yong Jung; Eunseok Seo; Sang Joon Lee (7191-7199).
Time-resolved dynamic imaging of bio-fluids can provide valuable information for clinical diagnosis and treatment of circulatory disorders. Quantitative information on non-transparent blood flows can be directly obtained by particle-tracing dynamic X-ray imaging, which needs better spatial resolution and enhanced image contrast compared to static imaging. For that use, tracer particles tagging along the flow streams are critically required. In this study, taking the advantage of high X-ray absorption, gold nanoparticles (AuNPs) are incorporated into human red blood cells (RBC) to produce contrast-enhanced tracers designed for dynamic X-ray imaging of blood flows. RBCs are advantageous tracers for blood flow measurements since they are natural and primary components of blood. The loading efficiency of AuNPs into RBCs is investigated in terms of the surface properties of the AuNPs. The AuNP-incorporated RBC provides a potential in the dynamic X-ray imaging of blood flows which can be used for clinical applications.
Keywords: Gold nanoparticles; Red blood cells; Flow tracer; Dynamic X-ray imaging;

New core–shell structured NaYF4:Yb3+,Tm3+@FexOy nanocrystals, with 20 nm Yb3+,Tm3+-co-doped NaYF4 nanocrystals as a core and 5 nm FexOy nanocrystals as a shell, have been synthesized and characterized by TEM and XRD analysis. These core–shell nanocrystals exhibit excellent near-infrared upconversion luminescence (UCL) emission at 800 nm under excitation by a continuous-wave 980 nm laser and superparamagnetic properties with a saturation magnetization (Ms) of ∼12 emu/g. Water-soluble nanocrystals were obtained by surface ligand exchange of oleic acid-coated precursor nanocrystals, and their internalization within living cells has been investigated by laser scanning UCL microscopy. Furthermore, the obtained core–shell nanocrystals have been applied in dual-modality T2-enhanced magnetic resonance (MR) and UCL imaging in vivo of the lymphatic system. Moreover, the toxicity of NaYF4:Yb3+,Tm3+@FexOy nanocrystals has also been evaluated by MTT assay, IC50 values, and histological analysis of lymphatic node sections.
Keywords: Core-shell nanocrystals; Upconversion luminescence; Magnetic resonance imaging; Multimodality imaging;

Receptor-targeted iron oxide nanoparticles for molecular MR imaging of inflamed atherosclerotic plaques by Chuqiao Tu; Thomas S.C. Ng; Hargun K. Sohi; Heather A. Palko; Adrian House; Russell E. Jacobs; Angelique Y. Louie (7209-7216).
In a number of literature reports iron oxide nanoparticles have been investigated for use in imaging atherosclerotic plaques and found to accumulate in plaques via uptake by macrophages, which are critical in the process of atheroma initiation, propagation, and rupture. However, the uptake of these agents is non-specific; thus the labeling efficiency for plaques in vivo is not ideal. We have developed targeted agents to improve the efficiency for labeling macrophage-laden plaques. These probes are based on iron oxide nanoparticles coated with dextran sulfate, a ligand of macrophage scavenger receptor type A (SR-A). We have sulfated dextran-coated iron oxide nanoparticles (DIO) with sulfur trioxide, thereby targeting our nanoparticle imaging agents to SR-A. The sulfated DIO (SDIO) remained mono-dispersed and had an average hydrodynamic diameter of 62 nm, an r 1 relaxivity of 18.1 mM−1 s−1, and an r 2 relaxivity of 95.8 mM−1 s−1 (37 °C, 1.4 T). Cell studies confirmed that these nanoparticles were nontoxic and specifically targeted to macrophages. In vivo MRI after intravenous injection of the contrast agent into an atherosclerotic mouse injury model showed substantial signal loss on the injured carotid at 4 and 24 h post-injection of SDIO. No discernable signal decrease was seen at the control carotid and only mild signal loss was observed for the injured carotid post-injection of non-sulfated DIO, indicating preferential uptake of the SDIO particles at the site of atherosclerotic plaque. These results indicate that SDIO can facilitate MRI detection and diagnosis of vulnerable plaques in atherosclerosis.
Keywords: Nanoparticles; Imaging agents; MRI (magnetic resonance imaging); Molecular imaging; Macrophages; Inflammation;

Cellular uptake, cytotoxicity, and ROS generation with silica/conducting polymer core/shell nanospheres by Yoon Seon Jeong; Wan-Kyu Oh; Sojin Kim; Jyongsik Jang (7217-7225).
The cellular response to conducting polymer (CP) nanospheres with similar physical properties was evaluated by in vitro cellular uptake and cytotoxicity in mouse macrophage RAW 264.7 and rat pheochromocytoma PC-12 cells. Four different CPs (polythiophene, poly(3,4-ethylenedioxythiophene), polyaniline, and polypyrrole) were deposited onto silica nanoparticles with a diameter of ca. 22 nm. Cellular uptake of silica/CP core/shell nanospheres in both cell lines was observed by transmission electron microscopy and they were internalized via phagocytosis and endocytosis. Cytotoxic effects were systemically assessed using live-cell microscopy, viability, oxidative stress, and lactate dehydrogenase assays. Silica/polythiophene core/shell nanospheres were the most toxic in both cell lines examined, because of the cellular effects of sulfur atoms. On the other hand, silica/polypyrrole core/shell nanospheres caused the lowest levels of toxicity in both cell lines. Furthermore, both rat and mouse cell viability was concentration-dependent with the nanospheres. These findings enhance nanotoxicological information regarding CP nanospheres when used with macrophage and neuronal cells, which may be useful in their application in bioelectronic and biomedical fields.► The cellular response to silica/conducting polymer core/shell nanospheres was investigated using macrophage and rat pheochromocytoma cells. ► Four different conducting polymers including polythiophene, poly(3,4-ethylenedioxythiophene), polyaniline, and polypyrrole were used as shell materials. ► Among the four types of nanospheres, silica/polythiophene core/shell nanospheres represented the highest toxicity in both cells mainly due to cellular effect of sulfur atoms.
Keywords: Conducting polymer; Core shell nanosphere; Cytotoxicity; Cell viability; Reactive oxygen species;

Single-phased luminescent mesoporous nanoparticles for simultaneous cell imaging and anticancer drug delivery by Weihua Di; Xinguang Ren; Haifeng Zhao; Naoto Shirahata; Yoshio Sakka; Weiping Qin (7226-7233).
Multifunctional materials for biological use have mostly been designed with composite or hybrid nanostructures in which two or more components are incorporated. The present work reports on a multifunctional biomaterial based on single-phased luminescent mesoporous lanthanide oxide nanoparticles that combine simultaneous drug delivery and cell imaging. A simple strategy based on solid-state-chemistry thermal decomposition process was employed to fabricate the spherical mesoporous Gd2O3:Eu nanoparticles with homogeneous size distribution. The porous nanoparticles developed by this strategy possess well-defined mesopores, large pore size and volume, and high specific surface area. The mesoporous features of nanoparticles impart the material with capabilities of loading and releasing the drug with a relatively high loading efficiency and a sustained release behavior of drugs. The DOX-loaded porous Gd2O3 nanoparticles are able to kill the cancer cells efficiently upon incubation with the human cervical carcinoma (HeLa) cells, indicating the potential for treatment of cancer cells. Meanwhile, the intrinsic luminescence of Gd2O3:Eu nanoparticles gives the function of optical imaging. Therefore, the drug release activity and effect of drugs on the cells can be effectively monitored via luminescence of nanoparticles themselves, realizing multifunctionality of simultaneous cell imaging and anticancer drug delivery in a single-phased nanoparticle.
Keywords: Lanthanide oxides; Mesopore; Luminescence; Cell imaging; Drug delivery; Cytotoxicity;

Cellular trafficking of low molecular weight heparin incorporated in layered double hydroxide nanoparticles in rat vascular smooth muscle cells by Zi Gu; Barbara E. Rolfe; Anita C. Thomas; Julie H. Campbell; G.Q. (Max) Lu; Zhi P. Xu (7234-7240).
This paper reports a clear elucidation of the pathway for the cellular delivery of layered double hydroxide (LDH) nanoparticles intercalated with anti-restenotic low molecular weight heparin (LMWH). Cellular uptake of LMWH-LDH conjugates into cultured rat vascular smooth muscle cells (SMCs) measured via flow cytometry was more than ten times greater than that of LMWH alone. Confocal and transmission electron microscopy showed LMWH-LDH conjugates taken up by endosomes, then released into the cytoplasm. We propose that LMWH-LDH is taken up via a unique ‘modified endocytic’ pathway, whereby the conjugate is internalized by SMCs in early endosomes, sorted in late endosomes, and quickly released from late endosomes/lysosomes, avoiding degradation. Treatment of cells with LMWH-LDH conjugates suppressed the activation of ERK1/2 in response to foetal calf serum (FCS) for up to 24 h, unlike unconjugated LMWH which had no significant effect at 24 h. Improved understanding of the intracellular pathway of LMWH-LDH nanohybrids in SMC will allow for refinement of design for LDH nanomedicine applications.
Keywords: Layered double hydroxide (LDH); Low molecular weight heparin (LMWH); Rat vascular smooth muscle cells; Nanoparticle; Endocytosis; Mitogen-activated protein kinase (MAPK) Signal transduction;

Effect of gold nanoparticle morphology on adsorbed protein structure and function by Jennifer E. Gagner; Marimar D. Lopez; Jonathan S. Dordick; Richard W. Siegel (7241-7252).
Many biomedical applications of gold nanoparticles (NPs) rely on proteins that are covalently attached or adsorbed on the NP surface. The biological functionality of the protein-NP conjugate depends on the protein’s ability to interact with target molecules, which is affected by NP characteristics such as size, curvature, aspect ratio, morphology, crystal structure, and surface chemistry. In the present study, the effect of gold nanoparticle morphology on the structure and function of adsorbed enzymes, lysozyme (Lyz) and α-chymotrypsin (ChT), has been investigated. Gold nanospheres (AuNS) were synthesized with diameters 10.6 ± 1 nm, and gold nanorods (AuNR) were synthesized with dimensions of (10.3 ± 2) × (36.4 ± 9) nm. Under saturating conditions, proteins adsorb with a higher surface density on AuNR when compared to AuNS. In the case of Lyz, adsorption on AuNS and AuNR resulted in a 10% and 15% loss of secondary structure, respectively, leading to conjugate aggregation and greatly reduced enzymatic activity. ChT retained most of its secondary structure and activity on AuNS and AuNR at low surface coverages; however, as protein loading approached monolayer conditions on AuNR, a 40% loss in secondary structure and 86% loss of activity was observed. Subsequent adsorption of ChT in multilayers on the AuNR surface allowed the conjugates to recover activity and remain stable. It is clear that AuNP morphology does affect adsorbed protein structure; a better understanding of these differences will be essential to engineer fully functional nanobioconjugates.► Gold nanospheres and nanorods of similar size and surface chemistry were synthesized. ► Lysozyme and α-chymotrypsin adsorption onto these gold nanoparticles was studied. ► The adsorbed protein surface coverage depended on nanoparticle morphology. ► Changes in protein structure and conjugate stability were protein specific.
Keywords: Protein adsorption; Protein-nanoparticle conjugates; Nanoparticle morphology; Surface energy;

Gene delivery to tumor cells by cationic polymeric nanovectors coupled to folic acid and the cell-penetrating peptide octaarginine by Qi-Ying Jiang; Li-Hua Lai; Jie Shen; Qing-Qing Wang; Fu-Jian Xu; Gu-Ping Tang (7253-7262).
Target ligand folic acid (FA) and cell-penetrating peptide octaarginine (R8) were coupled with the gene vectors (PEI600-CyD, PC) composed of β-cyclodextrin (β-CyD) and low-molecular-weight polyethylenimine (PEI, Mw 600) to form nanovectors for highly efficient gene delivery to tumor cells. The resultant ternary nanocomplexes of FA-PC/R8-PC/pDNA produced excellent gene transfaction abilities in the folate receptor (FR)-positive tumor cells in vitro and in vivo. The FR-mediated endocytosis and the R8-mediated transmembrane functionality together contributed to the high transfection levels. This study provides a promising means to produce gene nanovectors for in vivo applications.
Keywords: Gene delivery; Nanovector; PEI; Target; Cell-penetrating peptide;

Mannosyl-coated nanocomplexes from amphiphilic cyclodextrins and pDNA for site-specific gene delivery by Alejandro Díaz-Moscoso; Nicolas Guilloteau; Céline Bienvenu; Alejandro Méndez-Ardoy; José L. Jiménez Blanco; Juan M. Benito; Loïc Le Gourriérec; Christophe Di Giorgio; Pierre Vierling; Jacques Defaye; Carmen Ortiz Mellet; José M. García Fernández (7263-7273).
Fully homogeneous facial amphiphiles consisting in a cyclodextrin (CD) platform onto which a polycationic cluster and a multi-tail hydrophobic moiety have been installed (polycationic amphiphilic CDs; paCDs) self-organized in the presence of plasmid DNA to form nanometric complexes (CDplexes) which exhibit broad-range transfection capabilities. We hypothesized that biorecognizable moieties located at the hydrophilic rim in the CD scaffold would be exposed at the surface of the corresponding nanoparticles after DNA-promoted aggregation, endowing the system with molecular recognition abilities towards cell receptors. This concept has been demonstrated by developing an efficient synthetic strategy for the preparation of multivalent polycationic glyco-amphiphilic CDs (pGaCDs). Self-assembled nanoparticles obtained from mannosylated pGaCDs and pDNA (average hydrodynamic diameter 80 nm) have been shown to be specifically recognized by mannose-specific lectins, including concanavalin A (Con A) and the human macrophage mannose receptor (MMR). Further macrophage adhesion studies indicated that unspecific binding, probably due to electrostatic interactions with negatively charged cell membrane components, can also operate. The relative specific versus non-specific internalization is dependent on the pGaCD:pDNA proportion, being optimal at a protonable nitrogen/phosphate (N/P) ratio of 5. The resulting GlycoCDplexes were shown to specifically mediate transfection in Raw 264.7 (murine macrophage) cells expressing the mannose-fucose receptor in vitro. FACS experiments confirmed that transfection using these nanoparticles is mannose-dependent, supporting the potential of the approach towards vectorized gene delivery.► Monodisperse polycationic amphiphilic cyclodextrins (paCDs) efficiently complex pDNA. ► Mannosyl-paCD:pDNA nanocomplexes (Man-CDplexes) are recognized by specific lectins. ► Man-CDplexes adhere to macrophages via the macrophage mannose receptor (MMR). ► MMR-mediated and non-specific internalization take place in RAW 264.7 cells. ► Selective transfection of macrophages was achieved by adjusting the N/P ratio.
Keywords: Carbohydrates; Cyclodextrins; Lectins; Nonviral gene delivery; Self-assembled nanoparticles;