Biomaterials (v.31, #18)

Mesoporous carbide-derived carbon for cytokine removal from blood plasma by Saujanya Yachamaneni; Gleb Yushin; Sun-Hwa Yeon; Yury Gogotsi; Carol Howell; Susan Sandeman; Gary Phillips; Sergey Mikhalovsky (4789-4794).
Porous carbons can be used for purification of bio-fluids due to their excellent biocompatibility with blood. Since the ability to adsorb a range of inflammatory cytokines within the shortest possible time is crucial to stop the progression of sepsis, the improvement of the adsorption rate is a key factor to achieving efficient removal of cytokines. Here, we demonstrate the effect of synthesis temperatures (from 600 °C to 1200 °C), carbon particle sizes (from below 35 μm to 300 μm), and annealing conditions (Ar, NH3, H2, Cl2, and vacuum annealing) that determine the surface chemistry, on the ability of carbide-derived carbons (CDCs) to remove cytokines TNF-α, IL-6, and IL-1β from blood plasma. Optimization of CDC processing and structure leads to up to two orders of magnitude increase in the adsorption rate. Mesoporous CDCs that were produced at 800 °C from Ti2AlC with the precursor particle size of <35 μm and annealed in NH3, displayed complete removal of large molecules of TNF-α in less than an hour, with >85% and >95% TNF-α removal in 5 and 30 min, respectively. This is a very significant improvement compared to the previously published results for CDC (90% TNF-α removal after 1 h) and activated carbons. Smaller interleukin IL-6 and IL-1β molecules can be completely removed within 5 min. These differences in adsorption rates show that carbons with controlled porosity can also be used for separation of protein molecules.
Keywords: Carbide derived-carbon; Protein adsorption; Cytokine; MAX phase; Mesoporous carbon; Sepsis;

The effect of heat- or ultra violet ozone-treatment of titanium on complement deposition from human blood plasma by Paula Linderbäck; Necati Harmankaya; Agneta Askendal; Sami Areva; Jukka Lausmaa; Pentti Tengvall (4795-4801).
Titanium (Ti) is a well known metallic biomaterial extensively used in dental, orthopaedic-, and occasionally also in blood contacting applications. It integrates well to bone and soft tissues, and is shown upon blood plasma contact to activate the intrinsic pathway of coagulation and bind complement factor 3b. The material properties depend largely on those of the nm-thick dense layer of TiO2 that becomes rapidly formed upon contact with air and water. The spontaneously formed amorphous Ti-oxide has a pzc∼5–6 and its water solubility is at the order of 1–2 micromolar. It is often subjected to chemical- and heat treatments in order to increase the anatase- and rutile crystallinity, to modify the surface topography and to decrease the water solubility. In this work, we prepared sol–gel derived titanium and smooth PVD titanium surfaces, and analysed their oxide and protein deposition properties in human blood plasma before and after annealing at 100–500 °C or upon UVO-treatment for up to 96 hours. The blood plasma results show that complement deposition vanished irreversibly after heat treatment at 250–300 °C for 30 minutes or after UVO exposure for 24 hours or longer. XPS and infrared spectroscopy indicated change of surface water/hydroxyl binding upon the heat- and UVO treatments, and increased Ti oxidation. XRD analysis confirmed an increased crystallinity and both control (untreated) and annealed smooth titanium displayed low XRD-signals indicating some nanocrystallinity, with predominantly anatase phase. The current results show that the behaviour of titanium dioxide in blood contact can be controlled through relatively simple means, such as mild heating and illumination in UV-light, which both likely irreversibly change the stoichiometry and structure of the outmost layers of titanium dioxide and its OH/H2O binding characteristics.
Keywords: Titanium; Titanium oxide; Sol–gel; Blood plasma; Protein adsorption; Complement;

Collagen fibers in the vertebrate tissue are responsible for its tensile strength. A disruption in the morphological or mechanical properties of collagen fibers is bound to impact tensile strength and contractility of tissues and affect several cellular processes. We had recently established that binding of discoidin domain receptor (DDR2) with collagen type I results in disruption of the native structure and morphology of collagen fibers. In this study we investigate if DDR2 affects the mechanical properties of collagen fibers. We used an analytical approach to determine the persistence length (PL) of collagen fibers from transmission electron microscope images of immobilized collagen. Fluctuations in the curvature of collagen fibers formed in-vitro (with or without recombinant DDR2) were analyzed to ascertain their PL. The PL values and fiber-diameter measurements were utilized to estimate Young's Modulus (E) of collagen fibers. Our results show that DDR2 significantly reduced PL and E of collagen fibers. We further found that PL for native collagen fibers increases as a function of collagen concentration with little dependence on fiber diameter. These results signify a physiological role of DDR2 in modulating extracellular matrix stiffness, which may be of relevance for tissue engineering and medical implants especially in diseases where DDR2 is upregulated.
Keywords: Collagen; ECM (extracellular matrix); Mechanical properties; Transmission electron microscopy (TEM); Recombinant protein;

Design and synthesis of a potent peptide containing both specific and non-specific cell-adhesion motifs by Yuxiao Lai; Cao Xie; Zheng Zhang; Weiyue Lu; Jiandong Ding (4809-4817).
This article reports a potent chemical to promote cell adhesion on a substrate by combination of both moieties for specific and non-specific adhesion. The cyclic (–RGDfK–) (R: arginine, G: glycine, D: aspartic acid, f: d-phenylalanine, K: lysine) is employed to trigger specific cell adhesion, and a linear tripeptide KKK is introduced to enhance early non-specific cell adhesion. A series of cyclic and linear peptides with different charges were synthesized and then functionalized with thiol end-group. All the peptides were immobilized on gold layers, which were later passivated by bovine serum albumin. The coverage of NIH/3T3 fibroblast cells on the substrate modified by the linker containing both cyclic (–RGDfK–) and linear KKK is, surprisingly, significantly better than the summation using one of them, which reveals the strong cooperativity of specific and non-specific cell adhesions. The resultant cell adhesion on the substrates modified by appropriate linkers was much better than on tissue-culture plates. The cooperativity principle and the design strategy of the combined linker might be helpful for fundamental research of cell–material or cell–extracellular matrix interactions, and for modification of new biomaterials in regenerative medicine and targeted drug delivery.
Keywords: Surface modification; Cell adhesion; RGD peptide; Cell spreading;

Nonvolatile buffer coating of titanium to prevent its biological aging and for drug delivery by Takeo Suzuki; Katsutoshi Kubo; Norio Hori; Masahiro Yamada; Norinaga Kojima; Yoshihiko Sugita; Hatsuhiko Maeda; Takahiro Ogawa (4818-4828).
The osseointegration capability of titanium decreases over time. This phenomenon, defined as biological aging of titanium, is associated with the disappearance of hydrophilicity and the progressive accumulation of hydrocarbons on titanium surfaces. The objective of this study was to examine whether coating of titanium surfaces with 4-(2-Hydroxylethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, a nonvolatile zwitterionic chemical buffering agent, could prevent the time-dependent degradation of the bioactivity of titanium. Commercially pure titanium samples, prepared as disks and cylinders, were acid-etched with H2SO4. A third of the samples were used for experiments immediately after processing (new surfaces), while another third were stored under dark ambient conditions for 3 months (3-month-old surfaces). The remaining third were coated with HEPES after acid-etching and were stored for 3 months (HEPES-coated 3-month-old surfaces). The 3-month-old surfaces were hydrophobic, while new and HEPES-coated 3-month-old surfaces were superhydrophilic. Protein adsorption and the number of osteoblasts attached during an initial culture period were substantially lower for 3-month-old surfaces than for new and HEPES-coated 3-month-old surfaces. Alkaline phosphatase activity and calcium deposition in osteoblast cultures were reduced by more than 50% on 3-month-old surfaces compared to new surfaces, whereas such degradation was not found on HEPES-coated 3-month-old surfaces. The strength of in vivo bone-implant integration for 3-month-old implants, evaluated by the push-in test, was 60% lower than that for new implants. The push-in value of HEPES-coated 3-month-old implants was equivalent to that of new implants. Coating titanium surfaces with HEPES containing an antioxidant amino acid derivative, N-acetyl cysteine (NAC), further enhanced osteoblast attachment to the surfaces, along with the increase level of intracellular glutathione reserves as a result of cellular uptake of NAC. These results suggest that HEPES coating of titanium surfaces maintained their superhydrophilicity for at least 3 months and resulted in a continuous retention of bioactivity and osteoconductivity similar to freshly prepared surfaces. This coating technology may be useful for preventing biological aging of titanium and delivering biological molecules for synergistic enhancement of bone-titanium integration.
Keywords: Bone-titanium integration; Hydrophilicity; Osseointegration; Implants; HEPES; N-acetyl cysteine (NAC);

The cytotoxicity of CdTe quantum dots and the relative contributions from released cadmium ions and nanoparticle properties by Yuanyuan Su; Mei Hu; Chunhai Fan; Yao He; Qingnuan Li; Wenxin Li; Lian-hui Wang; Pingping Shen; Qing Huang (4829-4834).
A systematic study was carried out on the relationship between the cytotoxicity of quantum dots (QDs) and free cadmium ions using CdCl2 solution with known amounts of Cd2+ as the control. We found that the CdTe QDs were more cytotoxic than CdCl2 solutions even when the intracellular Cd2+ concentrations were identical in HEK293 cells treated with them, implying the cytotoxicity of CdTe QDs cannot attributed solely to the toxic effect of free Cd2+. Moreover, we discovered that the cytotoxicity of QDs was based on the concentration of total QDs ingested by cells. Our data clearly showed that specific properties of nanopartices have an obvious influence on their cytotoxicity.
Keywords: Quantum dots; Cytotoxicity; Nanomaterials;

Cell contraction forces in scaffolds with varying pore size and cell density by Karolina A. Corin; Lorna J. Gibson (4835-4845).
The contractile behavior of cells is relevant in understanding wound healing and scar formation. In tissue engineering, inhibition of the cell contractile response is critical for the regeneration of physiologically normal tissue rather than scar tissue. Previous studies have measured the contractile response of cells in a variety of conditions (e.g. on two-dimensional solid substrates, on free-floating tissue engineering scaffolds and on scaffolds under some constraint in a cell force monitor). Tissue engineering scaffolds behave mechanically like open-cell elastomeric foams: between strains of about 10 and 90%, cells progressively buckle struts in the scaffold. The contractile force required for an individual cell to buckle a strut within a scaffold has been estimated based on the strut dimensions (radius, r, and length, l) and the strut modulus, E s. Since the buckling force varies, according to Euler's law, with r 4/l 2, and the relative density of the scaffold varies as (r/l)2, the cell contractile force associated with strut buckling is expected to vary with the square of the pore size for scaffolds of constant relative density. As the cell density increases, the force per cell to achieve a given strain in the scaffold is expected to decrease. Here we model the contractile response of fibroblasts by analyzing the response of a single tetrakaidecahedron to forces applied to individual struts (simulating cell contractile forces) using finite element analysis. We model tetrakaidecahedra of different strut lengths, corresponding to different scaffold pore sizes, and of varying numbers of loaded struts, corresponding to varying cell densities. We compare our numerical model with the results of free-floating contraction experiments of normal human dermal fibroblasts (NHDF) in collagen-GAG scaffolds of varying pore size and with varying cell densities.
Keywords: Collagen-GAG scaffolds; Contraction; Pore size; Cell density;

The effect of the dosage of NT-3/chitosan carriers on the proliferation and differentiation of neural stem cells by Zhaoyang Yang; Hongmei Duan; Linhong Mo; Hui Qiao; Xiaoguang Li (4846-4854).
This study aimed to determine the optimal dosage range of NT-3 in the soluble form or loaded with chitosan carriers by using NT-3/chitosan carriers to support the survival and proliferation of neural stem cells (NSCs) and induce them to differentiate into desired phenotypes. NSCs were co-cultured with chitosan carriers loaded with different doses of NT-3. As the control, NSCs were cultured in the defined medium, into which were added different doses of NT-3 in the soluble form every day. The ELISA kit was used to study the NT-3 releasing kinetics, which showed that, in the initial co-culture stage from 1 h to 14 weeks, the chitosan carriers loaded with different doses of NT-3 released NT-3 stably and constantly. The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was conducted to measure the cell viability, and the immunocytochemical methods were adopted to quantitatively analyze the phenotypes differentiating from the NSCs. Compared to the 100 ng NT-3 daily addition group (1400 ng over 14 days), the 25 ng, 50 ng and 200 ng NT-3 daily addition group showed dramatically shorter processes length and much lower differentiation percentage from NSCs into neurons. By contrast, the NT-3 (25 ng)-chitosan carriers group had not only higher cell viability, but also similar processes length and differentiation percentage from NSCs into neurons to the 100 ng NT-3 daily addition group. The method developed in this study significantly reduced the NT-3 amount required to support the survival, proliferation and differentiation of NSCs in vitro. Meanwhile, the chitosan carriers used here provided an ideal 3-dimensional scaffold for the adhesion, migration, proliferation and differentiation of NSC and the differentiated cells. Therefore, this method may open a new field for the large-scaled culture and amplification of NSCs in vitro to replace the lost neural cells, meanwhile lower the consumption of neurotrophic factors in the cell transplantation therapy of brain and spinal injury.
Keywords: Chitosan carriers; NT-3; Neural stem cells; Cell differentiation; Neuron;

A tissue-engineered trachea derived from a framed collagen scaffold, gingival fibroblasts and adipose-derived stem cells by Ken Kobayashi; Teruhisa Suzuki; Yukio Nomoto; Yasuhiro Tada; Masao Miyake; Akihiro Hazama; Ikuo Wada; Tatsuo Nakamura; Koichi Omori (4855-4863).
In some types of tracheal disease, tracheal resection is required. For patients with tracheal resection, artificial grafts, made from collagen sponge with a spiral polypropylene stent and mesh, have been clinically used by our group. However, epithelial regeneration was confirmed to be slow. In the present study, we investigated the potential of gingival fibroblasts (GFBs) and adipose-derived stem cells (ASCs) as autologous transplanted cells in combination with artificial graft for tracheal epithelial regeneration. In in vitro co-culturing with tracheal epithelial cells, GFBs stimulated epithelial cell differentiation and reconstruction of a pseudostratified epithelium. ASCs stimulated epithelial cell proliferation and reconstruction of a multi-layered epithelium. Subsequently, we prepared three kinds of bioengineered scaffolds from GFBs and/or ASCs and implanted them into rat tracheal defects. The bioengineered scaffolds containing GFBs were covered with tracheal epithelial cells after 1 week, and highly ciliated epithelium was formed after 2 weeks of transplantation. The bioengineered scaffold containing ASCs induced thick epithelium, and then pseudostratified epithelium containing goblet cells was formed. Furthermore, the application of both GFBs and ASCs had synergistic effects on tracheal epithelial regeneration, suggesting that bioengineered scaffolds containing GFBs and ASCs are useful for hastening tracheal epithelial regeneration.
Keywords: Autologous cell; Collagen; Epithelium; Fibroblast; Polypropylene; Scaffold;

Hydrogels are increasingly used as a cell encapsulation and transplantation device. The successful use of a hydrogel greatly relies on an ability to control hydrogel stiffness which affects structural integrity and regulates cellular phenotypes. However, conventional strategies to increase the gel stiffness lead to decrease in the gel permeability and subsequently deteriorate the viability of cells encapsulated in a gel matrix. This study presents a strategy to decouple the inversed dependency of permeability on the stiffness of a hydrogel by chemically cross-linking methacrylic alginate with poly(ethylene glycol) dimethacrylate (PEGDA). As expected, gel stiffness represented by elastic modulus was tuned over one order of magnitude with the concentration of methacrylic alginate and the degree of substitution of methacrylic groups. In contrast, swelling ratio of the hydrogel indicative of gel permeability was minimally changed because of multiple hydrophilic groups of alginate, similar to function of proteoglycans in a natural extracellular matrix. Furthermore, viability of neural cells encapsulated in a hydrogel of PEGDA and methacrylic alginate rather increased with hydrogel stiffness. Overall, the results of this study demonstrate an advanced biomaterial design paradigm which allows one to culture cells in a 3D matrix of varying rigidity. This study will therefore greatly expedite the use of a hydrogel system in both fundamental studies and clinical settings of cell therapies.
Keywords: Poly(ethylene glycol); Methacrylic alginate; Hydrogel; Cell encapsulation; Swelling ratio; Elastic modulus;

Mesenchymal stem cell seeded knitted silk sling for the treatment of stress urinary incontinence by Xiao Hui Zou; Yun Long Zhi; Xiao Chen; Hang Mei Jin; Lin Lin Wang; Yang Zi Jiang; Zi Yin; Hong Wei Ouyang (4872-4879).
Stress urinary incontinence remains a worldwide problem affecting patients of all ages. Implantation of suburethral sling is the cornerstone treatment. Current slings have inherent disadvantages. This study aims to develop a tissue engineered sling with bone marrow derived mesenchymal stem cell seeded degradable silk scaffold. The mesenchymal stem cells were obtained from Sprague–Dawley rats and were characterized in vitro. Layered cell sheets were formed after two weeks of culture and were labeled with carboxyfluorescein diacetate. Forty female rats were divided into four groups: Group A (n  = 5) had sham operation; other three groups underwent bilateral proximal sciatic nerve transection and were confirmed with stress urinary incontinence by the leak-point pressure measurement at 4 weeks after operation. Then, Group B (n  = 5) had no sling placed; Group C (n  = 15) was treated with a silk sling; and Group D (n  = 15) was treated with the tissue engineered sling. Histology and the leak-point pressure measurements were done at 4 and 12 weeks after the sling implantation while collagen content and mechanical testing were done at 12 weeks. The results showed that Group B had a significantly lower leak-point pressure (24.0 ± 4.2 cmH2O) at 4 weeks (P  < 0.05), while Group C (38.0 ± 3.3 cmH2O) and Group D (36.3 ± 3.1 cmH2O) almost reached to the normal level shown by Group A (41.6 ± 3.8 cmH2O) (p  > 0.05). At 12 weeks, tissue engineered sling of group D has higher collagen content (70.84 ± 14.49 μg/mg) and failure force (2.436 ± 0.192 N) when compared those of Group C (38.94 ± 7.05 μg/mg and 1.521 ± 0.087 N) (p  < 0.05). Both the silk sling and tissue engineered sling showed convincing functional effects for the treatment of stress urinary incontinence in a rat model. And the better ligament-like tissue formation in the tissue engineered sling suggested potential long-term function.
Keywords: Bone marrow derived mesenchymal stem cell; Silk sling; Stress urinary incontinence; Leak-point pressure;

A defined long-term in vitro tissue engineered model of neuromuscular junctions by Mainak Das; John W. Rumsey; Neelima Bhargava; Maria Stancescu; James J. Hickman (4880-4888).
Neuromuscular junction (NMJ) formation, occurring between motoneurons and skeletal muscle, is a complex multistep process involving a variety of signaling molecules and pathways. In vitro motoneuron-muscle co-cultures are powerful tools to study the role of different growth factors, hormones and cellular structures involved in NMJ formation. In this study, a serum-free culture system utilizing defined temporal growth factor application and a non-biological substrate resulted in the formation of robust NMJs. The system resulted in long-term survival of the co-culture and selective expression of neonatal myosin heavy chain, a marker of myotube maturation. NMJ formation was verified by colocalization of dense clusters of acetylcholine receptors visualized using alpha-bungarotoxin and synaptophysin containing vesicles present in motoneuron axonal terminals. This model will find applications in basic NMJ research and tissue engineering applications such as bio-hybrid device development for limb prosthesis and regenerative medicine as well as for high-throughput drug and toxin screening applications.
Keywords: In vitro test; Muscle; Nerve tissue engineering; Neural cell; Surface modification; Self assembly;

The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension by Monika L. Bayer; Chin-Yan C. Yeung; Karl E. Kadler; Klaus Qvortrup; Keith Baar; René B. Svensson; S. Peter Magnusson; Michael Krogsgaard; Manuel Koch; Michael Kjaer (4889-4897).
Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts to initiate collagen fibrillogenesis when cultured in fixed-length fibrin gels. Fibroblasts were dissected from semitendinosus and gracilis tendons from healthy humans and cultured in 3D linear fibrin gels. The fibroblasts synthesized an extracellular matrix of parallel collagen fibrils that were aligned along the axis of tension. The fibrils had a homogeneous narrow diameter that was similar to collagen fibrils occurring in embryonic tendon. Immunostaining showed colocalization of collagen type I with collagen III, XII and XIV. A fibronectin network was formed in parallel with the collagen, and fibroblasts stained positive for integrin α5. Finally, the presence of cell extensions into the extracellular space with membrane-enclosed fibrils in fibripositors indicated characteristics of embryonic tendon. We conclude that mature human tendon fibroblasts retain an intrinsic capability to perform collagen fibrillogenesis similar to that of developing tendon, which implies that the hormonal/mechanical milieu, rather than intrinsic cellular function, inhibits regenerative potential in mature tendon.
Keywords: Collagen; Extracellular matrix; Fibroblast; Tendon;

Dipeptide-based polyphosphazene and polyester blends for bone tissue engineering by Meng Deng; Lakshmi S. Nair; Syam P. Nukavarapu; Tao Jiang; William A. Kanner; Xudong Li; Sangamesh G. Kumbar; Arlin L. Weikel; Nicholas R. Krogman; Harry R. Allcock; Cato T. Laurencin (4898-4908).
Polyphosphazene–polyester blends are attractive materials for bone tissue engineering applications due to their controllable degradation pattern with non-toxic and neutral pH degradation products. In our ongoing quest for an ideal completely miscible polyphosphazene–polyester blend system, we report synthesis and characterization of a mixed-substituent biodegradable polyphosphazene poly[(glycine ethyl glycinato)1(phenyl phenoxy)1phosphazene] (PNGEG/PhPh) and its blends with a polyester. Two dipeptide-based blends namely 25:75 (Matrix1) and 50:50 (Matrix2) were produced at two different weight ratios of PNGEG/PhPh to poly(lactic acid–glycolic acid) (PLAGA). Blend miscibility was confirmed by differential scanning calorimetry, Fourier transform infrared spectroscopy, and scanning electron microscopy. Both blends resulted in higher tensile modulus and strength than the polyester. The blends showed a degradation rate in the order of Matrix2 < Matrix1 < PLAGA in phosphate buffered saline at 37 °C over 12 weeks. Significantly higher pH values of degradation media were observed for blends compared to PLAGA confirming the neutralization of PLAGA acidic degradation by polyphosphazene hydrolysis products. The blend components PLAGA and polyphosphazene exhibited a similar degradation pattern as characterized by the molecular weight loss. Furthermore, blends demonstrated significantly higher osteoblast growth rates compared to PLAGA while maintaining osteoblast phenotype over a 21-day culture. Both blends demonstrated improved biocompatibility in a rat subcutaneous implantation model compared to PLAGA over 12 weeks.
Keywords: Polyphosphazenes; Poly(lactic acid–glycolic acid); Polymeric blends; Biodegradation; Biocompatibility; Bone tissue engineering;

Regulation of angiogenesis during osseointegration by titanium surface microstructure and energy by Andrew L. Raines; Rene Olivares-Navarrete; Marco Wieland; David L. Cochran; Zvi Schwartz; Barbara D. Boyan (4909-4917).
Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the α2β1 integrin. However, clinical success of implanted materials is dependent not only upon osseointegration but also on neovascularization in the peri-implant bone. Here we tested the hypothesis that Ti surface microtopography and energy interact via α2β1 signaling to regulate the expression of angiogenic growth factors. Primary human osteoblasts (HOB), MG63 cells and MG63 cells silenced for α2 integrin were cultured on Ti disks with different surface microtopographies and energies. Secreted levels of vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), and angiopoietin-1 (Ang-1) were measured. VEGF-A increased 170% and 250% in MG63 cultures, and 178% and 435% in HOB cultures on SLA and modSLA substrates, respectively. In MG63 cultures, FGF-2 levels increased 20 and 40-fold while EGF increased 4 and 6-fold on SLA and modSLA surfaces. These factors were undetectable in HOB cultures. Ang-1 levels were unchanged on all surfaces.Media from modSLA MG63 cultures induced more rapid differentiation of endothelial cells and this effect was inhibited by anti-VEGF-A antibodies. Treatment of MG63 cells with 1α,25(OH)2D3 enhanced levels of VEGF-A on SLA and modSLA.Silencing the α2 integrin subunit increased VEGF-A levels and decreased FGF-2 levels. These results show that Ti surface microtopography and energy modulate secretion of angiogenic growth factors by osteoblasts and that this regulation is mediated at least partially via α2β1 integrin signaling.
Keywords: Titanium; Microstructure; Surface energy; Osteoblast; Angiogenesis; VEGF;

Modularly assembled porous cell-laden hydrogels by Bo Liu; Yang Liu; Andrew K. Lewis; Wei Shen (4918-4925).
Porous cell-laden hydrogels have been modularly assembled to address the challenges in 3-dimensional tissue engineering. Microgels photolithographically fabricated from solutions of poly(ethylene glycol) diacrylate are assembled into large porous constructs in the presence of a polypeptide-based, physically bonded cross-linker. The assembly occurs through a physiologically permissive Michael-type addition reaction between the acrylate groups on the surface of the microgels and the thiol groups on the cross-linker. The constructs assembled from star-shaped microgels exhibit higher porosity, permeability, and pore interconnectivity than those formed from circle- and square-shaped microgels. The correlation between the properties of assembled constructs and the morphological features of microgels suggests the possibility for bottom-up modulation of the construct properties. The high pore interconnectivity revealed on the level of individual microgels suggests that these constructs are suitable for tissue engineering. Cells remain viable inside centimeter-sized constructs when cultured under perfusion. These constructs have the combined advantages of preformed porous scaffolds and in situ forming hydrogels in allowing enhanced mass transfer, uniform cell seeding, and protection of cells from excessive, non-physiological shear stress. Large constructs can be assembled in one step and have no limitations in size. This method will provide opportunities to create large 3-dimensional tissue engineered products.
Keywords: Scaffold; Hydrogel; Microencapsulation; Self assembly;

Regulating in vivo calcification of alginate microbeads by Christopher S.D. Lee; Hunter R. Moyer; Rolando A. Gittens I.; Joseph K. Williams; Adele L. Boskey; Barbara D. Boyan; Zvi Schwartz (4926-4934).
Alginate calcification has been previously reported clinically and during animal implantation; however no study has investigated the mechanism, extensively characterized the mineral, or evaluated multiple methods to regulate or eliminate mineralization. In the present study, alginate calcification was first studied in vitro: calcium-crosslinked alginate beads sequestered surrounding phosphate while forming traces of hydroxyapatite. Calcification in vivo was then examined in nude mice using alginate microbeads with and without adipose stem cells (ASCs). Variables included the delivery method, site of delivery, sex of the animal, time in vivo, crosslinking solution, and method of storage prior to delivery. Calcium-crosslinked alginate microbeads mineralized when injected subcutaneously or implanted intramuscularly after 1–6 months. More extensive analysis with histology, microCT, FTIR, XRD, and EDS showed calcium phosphate deposits throughout the microbeads with surface mineralization that closely matched hydroxyapatite found in bone. Incorporating 25 mm bisphosphonate reduced alginate calcification whereas using barium chloride eliminated mineralization. Buffering the crosslinking solution with HEPES at pH 7.3 while washing and storing samples in basal media prior to implantation also eliminated calcification in vivo. This study shows that alginate processing prior to implantation can significantly influence bulk hydroxyapatite formation and presents a method to regulate alginate calcification.
Keywords: Calcification; Alginate; Microencapsulation; Adipose stem cell microbeads; Bone tissue engineering;

Several studies have validated successful mandibular reconstruction with prefabricated tissue-engineered bone flaps and recombinant human bone morphogenetic protein-2 (rhBMP-2) implanted in situ. Whether rhBMP-2 applied with the prefabrication technique enables faster ossification of mandibular defects than rhBMP-2 applied in situ is unknown. We aimed to compare mandibular reconstruction with prefabricated, vascularized tissue-engineered bone flaps with rhBMP-2 and rhBMP-2 applied in situ in primates (Rhesus monkey). We also compared the use of the carriers demineralized freeze-dried bone allograft (DFDBA) and coralline hydroxyapatite (CHA) for applying rhBMP-2. After computed tomography of the monkey head, custom meshes were made, loaded with rhBMP-2-incorporated DFDBA or CHA, and implanted in the latissimus dorsi muscle. Meanwhile, contralateral segmental mandibular defects were created, and custom meshes loaded with DFDBA, CHA, or rhBMP-2-incooperated DFDBA and CHA were implanted in situ. Thirteen weeks later, the bone flaps with rhBMP-2-incorporated DFDBA or CHA were transferred to repair segmental mandibular defects. The meshes loaded with DFDBA or CHA alone showed no bone regeneration 13 weeks after implantation in latissimus dorsi muscle. Radiography, angiography and histological analysis were used to evaluate the repair and vascularization of the implant. Segmental mandibular defects were successfully restored with prefabricated bone flaps and rhBMP-2-incorporated CHA in situ, but other segmental mandibular defects remained with rhBMP-2-incorporated DFDBA, DFDBA and CHA in situ. Moreover, mandibles reconstructed with rhBMP-2-incorporated CHA bone flaps revealed more regenerated and homogeneous bone formation than did other reconstructions. The study suggested that the prefabrication technique induced better mandibular reconstruction and bone regeneration in quantity and quality.
Keywords: Mandibular reconstruction; Prefabricated vascularized bone flaps; rhBMP-2; Demineralized freeze-dried bone allograft; Coralline hydroxyapatite;

A pH-sensitive molecularly imprinted nanospheres/hydrogel composite as a coating for implantable biosensors by Chunyan Wang; Alireza Javadi; Mehdi Ghaffari; Shaoqin Gong (4944-4951).
A pH-sensitive molecularly imprinted polymer (MIP) nanospheres/hydrogel composite exhibiting controlled release of dexamethasone-21 phosphate disodium (DXP) was developed as a potential coating for implantable biosensors to improve their biocompatibility. The molecularly imprinted pH-sensitive nanospheres were prepared by UV-initiated precipitation polymerization using DXP as the template molecule. The DXP loading and release experiments showed that the MIP nanospheres exhibited a higher loading level and slower release rate than non-imprinted polymer (NIP) nanospheres due to the interaction of DXP with the DXP-imprinted cavities within the MIP nanospheres. Furthermore, the MIP nanospheres exhibited a faster DXP release rate at a lower pH value within the pH range tested (i.e., 6.0–7.4), which is desirable for suppressing inflammation because inflammation induces an acidic microenvironment. In contrast, the NIP nanospheres did not show a notable pH-responsive DXP release behavior. The hydrogel poly(2-hydroxyethyl methacrylate (HEMA) -N-vinyl-2-pyrrolidinone (NVP) -2-methacryloyloxyethyl phosphorylcholine (MPC)) was prepared by UV polymerization. The MIP nanospheres were successfully incorporated into the hydrogel. The equilibrium water content and swelling kinetics of the MIP nanospheres/hydrogel composite were similar to those of pure hydrogel. The MIP nanospheres/hydrogel composite exhibited a much better controlled DXP release profile than the pure hydrogel. This pH-sensitive MIP nanospheres/hydrogel composite designed as a coating for implantable biosensors can potentially suppress the inflammation response of the implanted biosensors efficiently thereby effectively improving their lifetime.
Keywords: Molecularly imprinted nanospheres; Hydrogel; Controlled release; Composite;

Surface modification is an important part of the fabrication of nanoparticles that have specific properties and functions. Here we describe the development of a functional polypeptide and a simple available technology for surface modification of nanoparticles. A NS polypeptide, which is 100 amino acids composed of repeated units of four asparagine and one serine residue (NS), as a molecule for nanoparticle surface modification was designed. Modification of the surface of a magnetic nanoparticle with the NS polypeptide results in reduction of particle–particle and particle–cell interactions. When NS polypeptide is used in single fusion protein as a linker to display protein G on nanoparticles, the nanoparticle acquires the capacity to specifically bind target cells and to avoid nonspecific adsorption of non-target cells. This technology, incorporating a functional polypeptide, may represent a completely new strategy for surface modification of nanoparticles for use in a variety of cell-associated applications.
Keywords: Surface modification; Nanoparticle; Peptide; Adsorption;

Imaging and inhibition of multi-drug resistance in cancer cells via specific association with negatively charged CdTe quantum dots by Yanyan Zhou; Lixin Shi; Qingning Li; Hui Jiang; Gang Lv; Juan Zhao; Chunhui Wu; Matthias Selke; Xuemei Wang (4958-4963).
Photoluminescent semiconductor quantum dots (QDs) have received significant attention in biological and biomedical fields because of their attractive properties. In this contribution, we have explored and evaluated the utilization of water-soluble nanocrystal CdTe quantum dots (QDs) capped with negatively charged 3-mercapitalpropionic acid (MPA)-QDs to enhance the drug uptake into the target cancer cells and the efficiency of the biomarker and cancer treatments, by using the cytotoxicity evaluation, total internal reflection fluorescence microscopy, electrochemistry and UV–Vis absorption spectroscopy. Our results illustrate that the MPA-CdTe QDs could effectively facilitate the interaction of anticancer agent daunorubicin (DNR) with leukemia cells and the efficiency of biolabeling in cancer cells. Therefore, the present study affords a new potential method for simultaneous cellular inhibition and imaging of cancer cells.
Keywords: Leukemia; Drug resistance; Cadmium telluride; Daunorubicin; Bio-imaging; Drug delivery;

Perfluorodecalin/[InGaP/ZnS quantum dots] nanoemulsions as 19F MR/optical imaging nanoprobes for the labeling of phagocytic and nonphagocytic immune cells by Yong Taik Lim; Mi Young Cho; Ji-Hyun Kang; Young-Woock Noh; Jee-Hyun Cho; Kwan Soo Hong; Jin Woong Chung; Bong Hyun Chung (4964-4971).
Multimodal imaging contrast agents with unique magnetic resonance (MR) and optical imaging capabilities have great potentials in the diagnosis and therapy of disease. Using a rational materials design approach, the bimodal imaging contrast agent, perfluorodecalin (PFD)/[InGaP/ZnS quantum dots (QDs)] composite nanoemulsions is developed in this study. 19F molecules in the PFD/[InGaP/ZnS QDs] nanoemulsions provide a 19F-based MR imaging capability, while fluorescent QDs dispersed in PFD nanodroplets provide an optical imaging modality. This study also demonstrates that these bimodal imaging contrast agents can be delivered easily into both phagocytic and nonphagocytic immune cells. Internalization of multifunctional PFD/[InGaP/ZnS QDs] nanoemulsions into immunotherapeutic cells permits the labeled cells to be imaged by both magnetic resonance and fluorescence imaging with little effect on cell viability and function. The results of our study highlight the potential of PFD/[InGaP/ZnS QDs] nanoemulsion as a bimodal imaging nanoprobe for molecular imaging in immune cell-based cancer therapies.
Keywords: Nanoparticle; Quantum dots; Perfluorocarbon; Intracellular delivery; Immune cell; Cell imaging;

Doubly amphiphilic poly(2-oxazoline)s as high-capacity delivery systems for hydrophobic drugs by Robert Luxenhofer; Anita Schulz; Caroline Roques; Shu Li; Tatiana K. Bronich; Elena V. Batrakova; Rainer Jordan; Alexander V. Kabanov (4972-4979).
Solubilization of highly hydrophobic drugs with carriers that are non-toxic, non-immunogenic and well-defined remains a major obstacle in pharmaceutical sciences. Well-defined amphiphilic di- and triblock copolymers based on poly(2-oxazolines) were prepared and used for the solubilization of Paclitaxel (PTX) and other water-insoluble drugs. Probing the polymer micelles in water with the fluorescence probe pyrene, an unusual high polar microenvironment of the probe was observed. This coincides with an extraordinary large loading capacity for PTX of 45 wt.% active drug in the formulation as well as high water solubility of the resulting formulation. Physicochemical properties of the formulations, ease of preparation and stability upon lyophilization, low toxicity and immunogenicity suggest that poly(2-oxazoline)s are promising candidates for the delivery of highly challenging drugs. Furthermore, we demonstrate that PTX is fully active and provides superior tumor inhibition as compared to the commercial micellar formulation.
Keywords: Drug delivery; Solvatochromism; Polymer micelles; Polyoxazolines; Polymerization;

Injectable PLGA based colloidal gels for zero-order dexamethasone release in cranial defects by Qun Wang; Jinxi Wang; Qinghua Lu; Michael Scott Detamore; Cory Berkland (4980-4986).
Bone fillers have emerged as an alternative to the invasive surgery often required to repair skeletal defects. Achieving controlled release from these materials is desired for accelerating healing. Here, oppositely-charged Poly (d,l-lactic-co-glycolic acid) (PLGA) nanoparticles were used to create a cohesive colloidal gel as an injectable drug-loaded filler to promote healing in bone defects. The colloid self-assembled through electrostatic forces resulting in a stable 3-D network that may be extruded or molded to the desired shape. The colloidal gel demonstrated shear-thinning behavior due to the disruption of interparticle interactions as the applied shear force was increased. Once the external force was removed, the cohesive property of the colloidal gel was recovered. Similar reversibility and shear-thinning behavior were also observed in colloidal gels loaded with dexamethasone. Near zero-order dexamethasone release was observed over two months when the drug was encapsulated in PLGA nanoparticles and simply blending the drug with the colloidal gel showed similar kinetics for one month. Surgical placement was facilitated by the pseudoplastic material properties and in vivo observations demonstrated that the PLGA colloidal gels stimulated osteoconductive bone formation in rat cranial bone defects.
Keywords: PLGA; Colloidal gel; Drug delivery; Bone defect;

Surface functionalization of polyketal microparticles with nitrilotriacetic acid–nickel complexes for efficient protein capture and delivery by Jay C. Sy; Edward A. Phelps; Andrés J. García; Niren Murthy; Michael E. Davis (4987-4994).
Microparticle drug delivery systems have been used for over 20 years to deliver a variety of drugs and therapeutics. However, effective microencapsulation of proteins has been limited by low encapsulation efficiencies, large required amounts of protein, and risk of protein denaturation. In this work, we have adapted a widely used immobilized metal affinity protein purification strategy to non-covalently attach proteins to the surface of microparticles. Polyketal microparticles were surface modified with nitrilotriacetic acid–nickel complexes which have a high affinity for sequential histidine tags on proteins. We demonstrate that this high affinity interaction can efficiently capture proteins from dilute solutions with little risk of protein denaturation. Proteins that bound to the Ni–NTA complex retain activity and can diffuse away from the microparticles to activate cells from a distance. In addition, this surface modification can also be used for microparticle targeting by tethering cell-specific ligands to the surface of the particles, using VE-Cadherin and endothelial cells as a model. In summary, we show that immobilized metal affinity strategies have the potential to improve targeting and protein delivery via degradable polymer microparticles.
Keywords: Affinity; Drug delivery; Growth factors; Microsphere; Nickel; Surface modification;

Multifunctional doxorubicin loaded superparamagnetic iron oxide nanoparticles for chemotherapy and magnetic resonance imaging in liver cancer by Jin Hee Maeng; Don-Haeng Lee; Kyung Hee Jung; You-Han Bae; In-Suh Park; Seok Jeong; Yong-Sun Jeon; Chang-Koo Shim; Wooyoung Kim; Jungahn Kim; Jeongmi Lee; Yoon-Mi Lee; Ji-Hee Kim; Won-Hong Kim; Soon-Sun Hong (4995-5006).
To develop a drug delivery system with enhanced efficacy and minimized adverse effects, we synthesized a novel polymeric nanoparticles, (YCC-DOX) composed of poly (ethylene oxide)-trimellitic anhydride chloride-folate (PEO-TMA-FA), doxorubicin (DOX) and superparamagnetic iron oxide (Fe3O4) and folate. The efficacy of the nanoparticles was evaluated in rats and rabbits with liver cancer, in comparison with free-DOX (FD) and a commercial liposome drug, DOXIL®. YCC-DOX showed the anticancer efficacy and specifically targeted folate receptor (FR)-expressing tumors, thereby increasing the bioavailability and efficacy of DOX. The relative tumor volume of the YCC-DOX group was decreased two- and four-fold compared with the FD and DOXIL® groups in the rat and rabbit models, respectively. Furthermore, YCC-DOX showed higher MRI sensitivity comparable to a conventional MRI contrast agent (Resovist®), even in its lower iron content. In the immunohistochemical analysis, YCC-DOX group showed the lower expression of CD34 and Ki-67, markers of angiogenesis and cell proliferation, respectively, while apoptotic cells were significantly rich in the YCC-DOX group in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. These results indicate that YCC-DOX is a promising candidate for treating liver cancer and monitoring the progress of the cancer using MRI.
Keywords: Drug delivery; Doxorubicin; Liver cancer; Magnetic resonance imaging; Nanoparticles;

Transport and biodistribution of dendrimers across human fetal membranes: Implications for intravaginal administration of dendrimer-drug conjugates by Anupa R. Menjoge; Raghavendra S. Navath; Abbas Asad; Sujatha Kannan; Chong J. Kim; Roberto Romero; Rangaramanujam M. Kannan (5007-5021).
Dendrimers are emerging as promising topical antimicrobial agents, and as targeted nanoscale drug delivery vehicles. Topical intravaginal antimicrobial agents are prescribed to treat the ascending genital infections in pregnant women. The fetal membranes separate the extra-amniotic space and fetus. The purpose of the study is to determine if the dendrimers can be selectively used for local intravaginal application to pregnant women without crossing the membranes into the fetus. In the present study, the transport and permeability of PAMAM (poly (amidoamine)) dendrimers, across human fetal membrane (using a side by side diffusion chamber), and its biodistribution (using immunofluorescence) are evaluated ex-vivo. Transport across human fetal membranes (from the maternal side) was evaluated using Fluorescein (FITC), an established transplacental marker (positive control, size ∼400 Da) and fluorophore-tagged G4-PAMAM dendrimers (∼16 kDa). The fluorophore-tagged G4-PAMAM dendrimers were synthesized and characterized using 1H NMR, MALDI TOF MS and HPLC analysis. Transfer was measured across the intact fetal membrane (chorioamnion), and the separated chorion and amnion layers. Over a 5 h period, the dendrimer transport across all the three membranes was less than <3%, whereas the transport of FITC was relatively fast with as much as 49% transport across the amnion. The permeability of FITC (7.9 × 10−7 cm2/s) through the chorioamnion was 7-fold higher than that of the dendrimer (5.8 × 10−8 cm2/s). The biodistribution showed that the dendrimers were largely present in interstitial spaces in the decidual stromal cells and the chorionic trophoblast cells (in 2.5–4 h) and surprisingly, to a smaller extent internalized in nuclei of trophoblast cells and nuclei and cytoplasm of stromal cells. Passive diffusion and paracellular transport appear to be the major route for dendrimer transport. The overall findings further suggest that entry of drugs conjugated to dendrimers would be restricted across the human fetal membranes when administered topically by intravaginal route, suggesting new ways of selectively delivering therapeutics to the mother without affecting the fetus.
Keywords: Dendrimers; Transplacental transport; Membrane permeability; Chorioamnion; Biodistribution; Topical intravaginal; Nanotoxicology;

Polymer live-cell array for real-time kinetic imaging of immune cells by Naomi Zurgil; Elena Afrimzon; Assaf Deutsch; Yaniv Namer; Yana Shafran; Maria Sobolev; Yishay Tauber; Orit Ravid-Hermesh; Mordechai Deutsch (5022-5029).
Direct quantitative experimental investigations of the function of lymphocytes and other immune cells are challenging due to the cell mobility and the complexity of intercellular communications. In order to facilitate such investigations, an in vitro system is required that is noninvasive and provides kinetic data on cellular responses to challenges such as drug treatments. The present work reports the development of a disposable, inexpensive polymer-made device, the Polymer Live Cell Array (PLCA), for real-time, kinetic analysis of immune cells. The PLCA proved to be optically and biologically compatible, thus individual immune cells can be observed and treated independently without being tethered. The cells share a common space which facilitates cellular communications via secreted molecules or via direct intercellular interactions. These properties facilitate real-time, non-intrusive, repeated measurements of immune cells under multiple experimental treatments.
Keywords: In vitro single-cell analysis; Lymphocyte imaging; Polymer live cell array; Biocompatibility; Kinetic individual cell measurement; Cell retaining technology;

Exploiting the superior protein resistance of polymer brushes to control single cell adhesion and polarisation at the micron scale by Julien E. Gautrot; Britta Trappmann; Fabian Oceguera-Yanez; John Connelly; Ximin He; Fiona M. Watt; Wilhelm T.S. Huck (5030-5041).
The control of the cell microenvironment on model patterned substrates allows the systematic study of cell biology in well defined conditions, potentially using automated systems. The extreme protein resistance of poly(oligo(ethylene glycol methacrylate)) (POEGMA) brushes is exploited to achieve high fidelity patterning of single cells. These coatings can be patterned by soft lithography on large areas (a microscope slide) and scale (substrates were typically prepared in batches of 200). The present protocol relies on the adsorption of extra-cellular matrix (ECM) proteins on unprotected areas using simple incubation and washing steps. The stability of POEGMA brushes, as examined via ellipsometry and SPR, is found to be excellent, both during storage and cell culture. The impact of substrate treatment, brush thickness and incubation protocol on ECM deposition, both for ultra-thin gold and glass substrates, is investigated via fluorescence microscopy and AFM. Optimised conditions result in high quality ECM patterns at the micron scale, even on glass substrates, that are suitable for controlling cell spreading and polarisation. These patterns are compatible with state-of-the-art technologies (fluorescence microscopy, FRET) used for live cell imaging. This technology, combined with single cell analysis methods, provides a platform for exploring the mechanisms that regulate cell behaviour.
Keywords: Polymer brush; Extra-cellular matrix; Patterning; Single cell; Cell polarisation;