Biomaterials (v.30, #26)

Nanodiamond (ND) is carbon nanomaterial developing for biological applications in recent years. In this study, we investigated the location and distribution of 100 nm carboxylated ND particles in cell division and differentiation. ND particles were taken into cells by macropinocytosis and clathrin-mediated endocytosis pathways. However, the cell growth ability was not altered by endocytic ND particles after long-term cell culture for 10 days in both A549 lung cancer cells and 3T3-L1 embryonic fibroblasts. ND particles were equal separating into two daughter cells of cell division approximately. Finally, the cell retained a single ND's cluster in cytoplasm after sub-cultured for several generations. Interestingly, ND's clusters were carried inside of cell but without inducing damages after long-term cell culture. Moreover, ND particles did not interfere with the gene or protein expressions on the regulation of cell cycle progression and adipogenic differentiation. Together, these findings provide that endocytic ND particles are non-cytotoxic in cell division and differentiation, which can be applied for the labeling and tracking of cancer and stem cells.
Keywords: Nanodiamond; Cancer cell; Stem cell; Endocytosis; Mitosis; Differentiation;

Differentiation and activity of human preosteoclasts on chitosan enriched calcium phosphate cement by Nathalie Rochet; Thierry Balaguer; Florian Boukhechba; Jean-Pierre Laugier; Danielle Quincey; Stéphane Goncalves; Georges F. Carle (4260-4267).
Chitosan associated to various scaffolds has been shown to promote growth and mineral rich matrix deposition by osteoblasts in vitro, whereas its influence on osteoclast differentiation, which plays also a central role in bone remodeling, has never been described. The purpose of this study was to investigate the differentiation and activity of human preosteoclastic cells on calcium phosphate cement containing 2% chitosan (Cementek®/chitosan) compared to the Cementek® alone. Human primary osteoclast precursors were cultured directly on both biomaterials in the presence of rhM-CSF and rhRANK-L for 7 days. Using LIVE/DEAD fluorescent assay, tartrate-resistant acid phosphatase staining, scanning electron microscopy and quantitative RT-PCR, we demonstrated that incorporation of chitosan to Cementek® does not affect the proliferation and adhesion of preosteoclasts but inhibits the formation of TRACP positive cells and prevents the osteoclastic resorption of the composite biomaterial compared to Cementek® alone. This inhibitory effect of chitosan on osteoclast resorption activity should have important implications on bone formation and bone remodeling after in vivo implantation. Indeed, based on the positive results obtained in vivo by several investigators, one can suggest that this property of chitosan can be beneficial for bone regeneration.
Keywords: Chitosan; Calcium phosphate cement; Osteoclast; SEM; Acid phosphatase; Gene expression;

The effect of UV-photofunctionalization on the time-related bioactivity of titanium and chromium–cobalt alloys by Wael Att; Norio Hori; Fuminori Iwasa; Masahiro Yamada; Takeshi Ueno; Takahiro Ogawa (4268-4276).
This study examined the possible changes in the bioactivity of titanium surfaces during their aging and investigated the effect of ultraviolet (UV) light treatment during the age-related change of titanium bioactivity. Rat bone marrow-derived osteoblastic cells were cultured on new titanium disks (immediately after either acid-etching, machining, or sandblasting), 4-week-old disks (stored after processing for 4 weeks in dark ambient conditions), and 4-week-old disks treated with UVA (peak wavelength of 365 nm) or UVC (peak wavelength of 250 nm). During incubation for 24 h, only 50% of the cells were attached to the 4-week-old surfaces as compared to the new surface. UVC treatment of the aged surface increased its cell attachment capacity to a level 50% higher than the new surfaces, whereas UVA treatment had no effect. Proliferation, alkaline phosphatase activity, and mineralization of cells were substantially lower on the 4-week-old surfaces than on the new surfaces, while they were higher on the UVC-treated 4-week-old surfaces as compared to the new surfaces. The age-related impaired bioactivity was found on all titanium topographies as well as on a chromium–cobalt alloy, and was associated with an increased percentage of surface carbon. Although both UVA and UVC treatment converted the 4-week-old titanium surfaces from hydrophobic to superhydrophilic, only UVC treatment effectively reduced the surface carbon to a level equivalent to the new surface. Thus, this study uncovered a time-dependent biological degradation of titanium and chromium–cobalt alloy, and its restoration enabled by UVC phototreatment, which surmounts the innate bioactivity of new surfaces, which is more closely linked to hydrocarbon removal than the induced superhydrophilicity.
Keywords: Titanium dioxide photocatalysis; Aging; Orthopedic and dental implant; Osseointegration; Superhydrophilicity;

Induction of cartilage integration by a chondrocyte/collagen-scaffold implant by Moreica B. Pabbruwe; Ehsanollah Esfandiari; Wael Kafienah; John F. Tarlton; Anthony P. Hollander (4277-4286).
The integration of implanted cartilage is a major challenge for the success of tissue engineering protocols. We hypothesize that in order for effective cartilage integration to take place, matrix-free chondrocytes must be induced to migrate between the two tissue surfaces. A chondrocyte/collagen-scaffold implant system was developed as a method of delivering dividing cells at the interface between two cartilage surfaces. Chondrocytes were isolated from bovine nasal septum and seeded onto both surfaces of a collagen membrane to create the chondrocyte/collagen-scaffold implant. A model of two cartilage discs and the chondrocyte/collagen-scaffold sandwiched in between was used to effect integration in vitro. The resulting tissue was analysed histologically and biomechanically. The cartilage–implant–cartilage sandwich appeared macroscopically as one continuous piece of tissue at the end of 40 day cultures. Histological analysis showed tissue continuum across the cartilage–scaffold interface. The integration was dependent on both cells and scaffold. Fluorescent labeling of implanted chondrocytes demonstrated that these cells invade the surrounding mature tissue and drive a remodelling of the extracellular matrix. Using cell-free scaffolds we also demonstrated that some chondrocytes migrated from the natural cartilage into the collagen scaffold. Quantification of integration levels using a histomorphometric repair index showed that the chondrocyte/collagen-scaffold implant achieved the highest repair index compared to controls, reflected functionally through increased tensile strength. In conclusion, cartilage integration can be achieved using a chondrocyte/collagen-scaffold implant that permits controlled delivery of chondrocytes to both host and graft mature cartilage tissues. This approach has the potential to be used therapeutically for implantation of engineered tissue.
Keywords: Cartilage tissue engineering; Cartilage integration; Collagen biomaterial; Cartilage repair; Osteoarthritis;

The potential of mesenchymal stem cells (MSCs) as a viable cell source for cartilage repair hinges on the development of engineered scaffolds that support adequate cartilage tissue formation. Evolving networks (hydrogels with mesh sizes that change over time due to crosslink degradation) may provide the control needed to enhance overall tissue formation when compared to static scaffolds. In this study, MSCs were photoencapsulated in combinations of hydrolytically and enzymatically degradable hyaluronic acid (HA) hydrogels to investigate the tunability of these hydrogels and the influence of network evolution on neocartilage formation. In MSC-laden HA hydrogels, compressive mechanical properties increased when degradation complemented extracellular matrix deposition and decreased when degradation was too rapid. In addition, dynamic hydrogels that started at a higher wt% and decreased to a lower wt% were not equivalent to static hydrogels that started at the higher or lower wt%. Specifically, evolving 2 wt% hydrogels (2 wt% degrading to 1 wt%) expressed up-regulation of type II collagen and aggrecan, and exhibited increased glycosaminoglycan content over non-evolving 2 and 1 wt% hydrogels. Likewise, mechanical properties and size maintenance were superior in the dynamic system compared to the static 2 wt% and 1 wt% hydrogels, respectively. Thus, hydrogels with dynamic properties may improve engineered tissues and help translate tissue engineering technology to clinical application.
Keywords: Hyaluronic acid; Hydrogel; Mesenchymal stem cell; Degradation; Crosslinking;

Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture by Hiroshi Ijiri; Fasséli Coulibaly; Gento Nishimura; Daisuke Nakai; Elaine Chiu; Chiemi Takenaka; Keiko Ikeda; Hiroshi Nakazawa; Norio Hamada; Eiji Kotani; Peter Metcalf; Shin Kawamata; Hajime Mori (4297-4308).
Certain insect viruses produce stable infectious micro-crystals called polyhedra which function to protect the virus after the death of infected larvae. Polyhedra form within infected cells and contain numerous virus particles embedded in a crystalline lattice of the viral protein polyhedrin. We have previously demonstrated that the N-terminal 75 amino acids of the Bombx mori cypovirus (BmCPV) turret protein (VP3) can function as a polyhedrin recognition signal leading to the incorporation of foreign proteins into polyhedra. Foreign proteins tagged with the VP3 polyhedrin recognition signal were incorporated into polyhedra by co-expression with polyhedrin in insect cells. We have used this method to encapsulate a wide variety of foreign proteins into polyhedra. The atomic structure of BmCPV polyhedrin showed that the N-terminal H1 α-helix of polyhedrin plays a significant role in cross-linking and stabilizing polyhedra. Here we show that the polyhedrin H1-helix can also function as a polyhedrin recognition signal and can be used like the VP3 N-terminal sequence to target foreign proteins into polyhedra. In addition, the two targeting methods can be used together to produce polyhedra containing both EGFP and Discosoma sp. Red Fluorescent Protein (DsRed). The modified polyhedra were imaged using dual-wavelength confocal microscopy showing that the two foreign proteins are uniformly incorporated into polyhedra at similar levels. We have investigated the biological and physiological properties of fibroblast growth factor-2 (FGF-2), FGF-7 and epidermal growth factor (EGF) immobilized on polyhedra with either the H1 or the VP3 tag. Growth factors produced by both methods were functional, inducing the growth of fibroblast cells and keratinocytes. The results demonstrate the utility and flexibility of modified polyhedra for encapsulating and stabilizing bioactive proteins.
Keywords: Cypovirus; Polyhedra; FGF-2; FGF-7; EGF; Keratinocytes;

Three-dimensional reconstituted extracellular matrix scaffolds for tissue engineering by Karthikeyan Narayanan; Kwong-Joo Leck; Shujun Gao; Andrew C.A. Wan (4309-4317).
The extracellular matrix (ECM) is a rich meshwork of proteins and proteoglycans. Besides assuming a cell adhesive and structural support role, the ECM also helps to sequester and present growth factors to cells. ECM derived from tissues has been used as biological scaffolds for tissue engineering. In contrast, it has been difficult to employ ECM derived from cell lines as scaffolds due to its lack of form and structure. We have developed a mild, aqueous-based method for incorporating cell line derived ECM into biological scaffolds based on polyelectrolyte complexation, using the example of ECM from MC-3T3, a mouse preosteoblast cell line. A DNase step was incorporated in the ECM isolation procedure to further purify it of genetic material. Immunohistochemistry of fibers incorporated with MC-3T3 ECM reveal the presence of the ECM components, collagen type I, collagen type IV, fibronectin and heparan sulfate, on their surface. Reconstituted ECM scaffolds retained the cell-adhesion characteristics of the ECM, as demonstrated by ‘reseeding’ the ECM-secreting cell on the scaffolds. Human mesenchymal stem cells (hMSCs) were seeded onto the fibrous scaffolds incorporated with MC-3T3 ECM, and implanted subcutaneously into SCID mice. After 4 weeks of implantation, histological evidence showed that the hMSC seeded ECM scaffolds had induced bone formation at the ectopic site.
Keywords: Extracellular matrix; Scaffold; Stem cell; Osteogenesis;

Cell-responsive hydrogel for encapsulation of vascular cells by Thomas P. Kraehenbuehl; Lino S. Ferreira; Prisca Zammaretti; Jeffrey A. Hubbell; Robert Langer (4318-4324).
The in vitro potential of a synthetic matrix metalloproteinase (MMP)-responsive poly(ethylene glycol) (PEG)-based hydrogel as a bioactive co-encapsulation system for vascular cells and a small bioactive peptide, thymosin β4 (Tβ4), was examined. We show that the physical incorporation of Tβ4 in this bioactive matrix creates a three-dimensional (3D) environment conducive for human umbilical vein endothelial cell (HUVEC) adhesion, survival, migration and organization. Gels with entrapped Tβ4 increased the survival of HUVEC compared to gels without Tβ4, and significantly up-regulated the endothelial genes vascular endothelial-cadherin and angiopoietin-2, whereas von Willebrand factor was significantly down-regulated. Incorporation of Tβ4 significantly increased MMP-2 and MMP-9 secretion of encapsulated HUVEC. The gel acts as a controlled Tβ4-release system, as MMP-2 and MMP-9 enzymes trigger the release. In addition, Tβ4 facilitated HUVEC attachment and induced vascular-like network formation upon the PEG-hydrogels. These MMP-responsive PEG-hydrogels may thus serve as controlled co-encapsulation system of vascular cells and bioactive factors for in situ regeneration of ischemic tissues.
Keywords: Vascular tissue engineering; Biomimetic hydrogel; Matrix metalloproteinase (MMP); Human umbilical vein endothelial cells (HUVEC); Thymosin β4;

Polypyrrole-coated electrospun PLGA nanofibers for neural tissue applications by Jae Y. Lee; Chris A. Bashur; Aaron S. Goldstein; Christine E. Schmidt (4325-4335).
Electrospinning is a promising approach to create nanofiber structures that are capable of supporting adhesion and guiding extension of neurons for nerve regeneration. Concurrently, electrical stimulation of neurons in the absence of topographical features also has been shown to guide axonal extension. Therefore, the goal of this study was to form electrically conductive nanofiber structures and to examine the combined effect of nanofiber structures and electrical stimulation. Conductive meshes were produced by growing polypyrrole (PPy) on random and aligned electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers, as confirmed by scanning electron micrographs and X-ray photon spectroscopy. PPy–PLGA electrospun meshes supported the growth and differentiation of rat pheochromocytoma 12 (PC12) cells and hippocampal neurons comparable to non-coated PLGA control meshes, suggesting that PPy–PLGA may be suitable as conductive nanofibers for neuronal tissue scaffolds. Electrical stimulation studies showed that PC12 cells, stimulated with a potential of 10 mV/cm on PPy–PLGA scaffolds, exhibited 40–50% longer neurites and 40–90% more neurite formation compared to unstimulated cells on the same scaffolds. In addition, stimulation of the cells on aligned PPy–PLGA fibers resulted in longer neurites and more neurite-bearing cells than stimulation on random PPy–PLGA fibers, suggesting a combined effect of electrical stimulation and topographical guidance and the potential use of these scaffolds for neural tissue applications.
Keywords: Polypyrrole; Nanofibers; Nerve tissue engineering; Electrical stimulation; PC12 cells; Hippocampal neurons;

Multifunctional protein-encapsulated polycaprolactone scaffolds: Fabrication and in vitro assessment for tissue engineering by Seher Ozkan; Dilhan M. Kalyon; Xiaojun Yu; Craig A. McKelvey; Michael Lowinger (4336-4347).
Here we demonstrate the use of a twin screw extrusion/spiral winding (TSESW) process to generate protein-encapsulated tissue engineering scaffolds. Bovine serum albumin (BSA) was distributed into PCL matrix using both wet and hot melt extrusion methods. The encapsulation efficiency and the time-dependent release rate, as well as the tertiary structure of BSA (via circular dichroism), were investigated as a function of processing method and conditions. Within the relatively narrow processing window of this demonstration study it was determined that the wet extrusion method gave rise to greater stability of the BSA on the basis of circular dichroism data. The rate of proliferation of human fetal osteoblast (hFOB) cells and the rate of mineral deposition were found to be greater for wet extruded scaffolds, presumably due to the important differences in surface topographies (smoother scaffold surfaces upon wet extrusion). Overall, these findings suggest that the twin screw extrusion/spiral winding (TSESW) process offers significant advantages and flexibility in generating a wide variety of non-cytotoxic tissue engineering scaffolds with controllable distributions of porosity, physical and chemical properties and protein concentrations that can be tailored for the specific requirements of each tissue engineering application.
Keywords: Scaffold; Tissue engineering; Sustained release; Extrusion; Protein delivery;

Improved functions of human hepatocytes on NH3 plasma-grafted PEEK-WC–PU membranes by Simona Salerno; Antonella Piscioneri; Stefania Laera; Sabrina Morelli; Pietro Favia; Augustinus Bader; Enrico Drioli; Loredana De Bartolo (4348-4356).
PEEK-WC–PU membranes were modified with an NH3 glow discharge process to graft N-containing functional groups at their surface in order to improve the maintenance of human hepatocytes. Native and modified membrane surfaces were characterized with XPS, ToF-SIMS and WCA measurements. We have investigated morphological behaviour and specific functions of primary human hepatocytes on native and modified PEEK-WC–PU membranes in a small-scale gas-permeable bioreactor. N-containing groups grafted at the surface of the membranes improved the initial steps of adhesion and the maintenance of phenotype and differentiated functions of cells. Confocal microscopy of cell morphology evidenced human hepatocytes exhibiting a polygonal shape and organizing a 3D structure. The presence of CK19 positive cells, a marker of biliary duct epithelium, was also found on native and modified membranes. Liver specific functions, investigated in terms of urea production, albumin synthesis and diazepam biotransformation, were maintained at high levels up to 19 days, particularly on surface-modified membranes.
Keywords: Human hepatocytes; NH3 plasma; Liver specific functions; Cell morphology; Membranes;

Synthesis, characterization and therapeutic efficacy of a biodegradable, thermoresponsive hydrogel designed for application in chronic infarcted myocardium by Kazuro L. Fujimoto; Zuwei Ma; Devin M. Nelson; Ryotaro Hashizume; Jianjun Guan; Kimimasa Tobita; William R. Wagner (4357-4368).
Injection of a bulking material into the ventricular wall has been proposed as a therapy to prevent progressive adverse remodeling due to high wall stresses that develop after myocardial infarction. Our objective was to design, synthesize and characterize a biodegradable, thermoresponsive hydrogel for this application based on copolymerization of N-isopropylacrylamide (NIPAAm), acrylic acid (AAc) and hydroxyethyl methacrylate-poly(trimethylene carbonate) (HEMAPTMC). By evaluating a range of monomer ratios, poly(NIPAAm-co-AAc-co-HEMAPTMC) at a feed ratio of 86/4/10 was shown to be ideal since it formed a hydrogel at 37 °C, and gradually became soluble over a 5 month period in vitro through hydrolytic cleavage of the PTMC residues. HEMAPTMC, copolymer and degradation product chemical structures were verified by NMR. No degradation product cytotoxicity was observed in vitro. In a rat chronic infarction model, the infarcted left ventricular (LV) wall was injected with the hydrogel or phosphate buffered saline (PBS). In the PBS group, LV cavity area increased and contractility decreased at 8 wk (p  < 0.05 versus pre-injection), while in the hydrogel group both parameters were preserved during this period. Tissue ingrowth was observed in the hydrogel injected area and a thicker LV wall and higher capillary density were found for the hydrogel versus PBS group. Smooth muscle cells with contractile phenotype were also identified in the hydrogel injected LV wall. The designed poly(NIPAAm-co-AAc-co-HEMAPTMC) hydrogel of this report may thus offer an attractive biomaterial-centered treatment option for ischemic cardiomyopathy.
Keywords: Thermally responsive material; Hydrogel; Biodegradation; Cytotoxicity; Cardiac tissue engineering; Smooth muscle cell;

The in vivo bone formation by mesenchymal stem cells in zein scaffolds by Jinwen Tu; Huajie Wang; Huiwu Li; Kerong Dai; Jinye Wang; Xiaoling Zhang (4369-4376).
In our previous study, a three-dimensional zein porous scaffold was prepared. This scaffold showed proper mechanical properties, good biocompatibility, and controllable biodegradation. In addition, it allowed blood vessels to form inside in vivo. In the current study, we prepared the complexes of zein scaffolds and rabbit MSCs, and investigated ectopic bone formation in nude mice. Furthermore, we implanted them into the radius defects of rabbits and assessed whether they could be helpful in the repair of critical-sized bone defects. The results showed that the complexes of zein scaffolds and rabbit MSCs could undergo ectopic bone formation in the thigh muscle pouches of nude mice. More importantly, the complexes could lead to the repair of critical-sized radius defects in rabbits accompanied with blood vessels' formation, which clearly demonstrates promise for the treatment of bone defects through tissue engineering.
Keywords: Zein scaffold; MSCs; Ectopic bone formation; Bone tissue engineering;

Electrophysiological characterization of embryonic hippocampal neurons cultured in a 3D collagen hydrogel by Tao Xu; Peter Molnar; Cassie Gregory; Mainak Das; Thomas Boland; James J. Hickman (4377-4383).
Rat embryonic hippocampal neurons were cultured in (1) 3D collagen hydrogels as ‘entrapped’ evenly distributed cells, (2) at the interface of two collagen layers (sandwich model), and (3) on the surface of collagen coated coverslips (2D model). In the ‘entrapment’ model the neuronal processes grew out of the plane of the cell body and extended into the collagen matrix, in contrast to the sandwich model where the cells and their processes rarely left the plane in which they were seeded. Hippocampal neurons ‘entrapped’ in the 3D collagen gel grew the same number, but shorter, processes and exhibited improved survival compared to neurons cultured in the 2D model. There was no difference in the electrophysiological properties of the neurons cultured in the 3D compared to the 2D model except in the resting membrane potential and in the duration of the after-hyperpolarization. Spontaneous postsynaptic currents were recorded in 14- and 21-day-old 3D cultures evidencing functional synapse formation. Our results indicate that the physiological characteristics of 3D neuronal cultures are similar to traditional 2D cultures. However, functional 3D networks of hippocampal neurons will be necessary for multi-level circuit formation, which could be essential for understanding the basis of physiological learning and memory.
Keywords: Memory; Nerve tissue engineering; Neuronal network; Collagen; Electrophysiology; Three dimensional;

Mass preparation of size-controlled mouse embryonic stem cell aggregates and induction of cardiac differentiation by cell patterning method by Daisuke Sasaki; Tatsuya Shimizu; Shinako Masuda; Jun Kobayashi; Kazuyoshi Itoga; Yukiko Tsuda; Jun K. Yamashita; Masayuki Yamato; Teruo Okano (4384-4389).
Embryonic stem cells (ESCs) are promising cell sources for cell-based therapy. It has been established that the formation of ESC aggregates promotes their differentiation into the derivatives of all three germ layers. ESC aggregates are generally prepared via the formation of suspended spherical aggregates called embryoid bodies (EBs). Because the differentiation efficiency depends on the size of EBs, it becomes one of the research topics how to prepare size-controlled EBs in a scalable manner for reproducible and high-throughput experiments. Here, we have developed a novel culture method that enables simple mass preparation of size-controlled ESC aggregates on a culture surface instead of floating EBs. We developed a maskless photolithography device that enabled rapid fabrication of micropatterned surfaces. Utilizing this device, we fabricated the culture substrates the surfaces of which comprised arrays of cell-adhesive circular micro-domains (100–400 μm in diameter) and the rest of non-cell-adhesive domains. We seeded mouse ESCs on this substrate and prepared size-controlled ESC aggregates on the micro-domains. We analyzed cardiac differentiation in the ESC aggregates and found that the optimal diameter of micro-domains was 200 μm. The present method is useful for the simple and reproducible mass preparation of ESC-derived differentiated cells and high-throughput assays.
Keywords: Embryonic stem cell; Embryoid body; Cell patterning; Photolithography; Cardiomyocyte; Differentiation;

Stimulatory effect of hydrothermally synthesized biodegradable hydroxyapatite granules on osteogenesis and direct association with osteoclasts by Yoshinori Gonda; Koji Ioku; Yasuaki Shibata; Takatoshi Okuda; Giichiro Kawachi; Masanobu Kamitakahara; Hisashi Murayama; Katsumi Hideshima; Shimeru Kamihira; Ikuho Yonezawa; Hisashi Kurosawa; Tohru Ikeda (4390-4400).
Calcium-deficient hydroxyapatite (HA) granules with a unique spherical shape were prepared using an applied hydrothermal method. Spherical stoichiometric HA granules were also prepared by normal sintering and both granules were used for implantation into rat tibiae to compare the biological responses to each implant. Twelve and 24 weeks after implantation, the volume of calcium-deficient HA granules was significantly less than that of stoichiometric HA granules, and the biodegradability of calcium-deficient HA granules was confirmed. The larger number of osteoclasts, larger osteoblast surface and larger bone volume in the implanted area of calcium-deficient HA than those of stoichiometric HA suggested that osteoclastic resorption of calcium-deficient HA affected osteogenesis in that area. To analyze the direct contribution of osteoclasts to osteogenesis, C2C12 multipotent myoblastic cells, which have the potential to differentiate into osteoblasts in the presence of bone morphogenetic protein 2, were cultured with supernatants of osteoclasts cultured on calcium-deficient HA, stoichiometric HA, β-tricalcium phosphate disks or plastic dishes, or bone marrow macrophages cultured on plastic dishes. Supernatants of osteoclasts but not bone marrow macrophages stimulated the expression of Runx2 and osteocalcin in C2C12 cells in concert with bone morphogenetic protein 2. The expression of alkaline phosphatase was stimulated with supernatants of osteoclasts cultured on ceramic disks. These results suggested that osteoclasts produced certain soluble factors which stimulated osteoblastic differentiation and they were thought to be associated with the induction of a larger osteoblast surface and bone volume in the animals implanted with calcium-deficient HA granules.
Keywords: Hydroxyapatite; Bone graft; Cell culture; Osteoblast; Osteoclast;

PHB/PHBHHx scaffolds and human adipose-derived stem cells for cartilage tissue engineering by Chuan Ye; Ping Hu; Min-Xian Ma; Yang Xiang; Ri-Guang Liu; Xian-Wen Shang (4401-4406).
The goal of this study was to investigate the potential of polyhydroxybutyrate (PHB)/poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) (PHB/PHBHHx) to produce neocartilage upon seeding with differentiated human adipose-derived stem cells (hASCs). hASCs were grown on a three-dimensional PHB/PHBHHx scaffold in vitro with or without chondrogenic media for 14 days. Scanning electron microscopy showed that differentiated cells produced abundant extracellular matrices with increasing culture time. No cytotoxicity was observed by the live/dead cell viability assay. GAG and total collagen content in the differentiated cells increased significantly with in vitro culture time. After 14 days of in vitro culture, the differentiated cells grown on the (PHB/PHBHHx) scaffold (differentiated cells/(PHB/PHBHHx)) were implanted into the subcutaneous layer nude mice for 12 or 24 weeks, non-differentiated cells/(PHB/PHBHHx) were implanted as the control group. The differentiated cells/(PHB/PHBHHx) implants formed cartilage-like tissue after 24 weeks of implantation, and stained positive for collagen type II, safranin O, and toluidine blue. In addition, typical cartilage lacuna was observed, and there were no remnants of PHB/PHBHHx. Collagen type II was detected by Western blot at 12 and 24 weeks of implantation. In the control group, no cartilage formation was observed. This study demonstrated that PHB/PHBHHx is a suitable material for cartilage tissue engineering.
Keywords: PHB; PHBHHx; Stem cell; Cartilage tissue engineering; Scaffold; Differentiation;

Crosstalk between osteoblasts and endothelial cells co-cultured on a polycaprolactone–starch scaffold and the in vitro development of vascularization by Marina I. Santos; Ronald E. Unger; Rui A. Sousa; Rui L. Reis; C. James Kirkpatrick (4407-4415).
The reconstruction of bone defects based on cell-seeded constructs requires a functional microvasculature that meets the metabolic demands of the engineered tissue. Therefore, strategies that augment neovascularization need to be identified. We propose an in vitro strategy consisting of the simultaneous culture of osteoblasts and endothelial cells on a starch-based scaffold for the formation of pre-vascular structures, with the final aim of accelerating the establishment of a vascular bed in the implanted construct. Human dermal microvascular endothelial cells (HDMECs) were co-cultured with human osteoblasts (hOBs) on a 3D starch-based scaffold and after 21 days of culture HDMEC aligned and organized into microcapillary-like structures. These vascular-like structures evolved from a cord-like configuration to a more complex branched morphology, had a lumen and stained in the perivascular region for type IV collagen. Genetic profiling of 84 osteogenesis-related genes was performed on co-culture vs. monoculture. Osteoblasts in co-culture showed a significant up-regulation of type I collagen and immunohistochemistry revealed that the scaffold was filled with a dense matrix stained for type I collagen. In direct contact with HDMEC hOBs secreted higher amounts of VEGF in relation to monoculture and the highest peak in the release profile correlated with the formation of microcapillary-like structures. The heterotypic communication between the two cell types was also assured by direct cell–cell contact as shown by the expression of the gap junction connexin 43. In summary, by making use of heterotypic cellular crosstalk this co-culture system is a strategy to form vascular-like structures in vitro on a 3D scaffold.
Keywords: Co-culture; Vascularization; Bone; Tissue engineering; Polymer;

This study developed a transplantable platform based on cationic hydrogels to deliver antisense oligodeoxynucleotides (ASOs) targeting the mRNA of TNF-α. Cationic agarose (c-agarose) was obtained by conjugating ethylenediamine to agarose via an N,N′-carbonyldiimidazole (CDI)-activation method. ASO–c-agarose system was constructed by mixing ASO in cationic agarose gel of proper concentration and gelation temperature. In vivo assessment of ASO distribution suggested that the system specifically target to spleen, wherein the c-agarose-delivered ASO had a concentration remarkably 50-fold higher than that of the naked ASO. The distribution of c-agarose-delivered ASO was scarcely detectable in liver and kidney. Next, three types of animal models were setup to evaluate the therapeutic efficacies of ASO–Gel, including the adjuvant-induced arthritis (AA), carrageen/lipopolysaccharide (LPS)-induced arthritis (CLA) and collagen-induced arthritis (CIA) models. The effects of ASO–c-agarose in alleviating inflammation and tissue destruction were evidenced in more than 90% of the testing animals, with decrease of main inflammatory cytokines, lightening of joint swelling and tissue damage, as well as increase in their body weights. All these findings suggest that this highly operable devise for the conveyance of antisense nucleotides together with its spleen-targeting property, could become a useful means of antisense-based therapeutics against rheumatoid arthritis and other diseases.
Keywords: Rheumatoid arthritis; TNF-α; Spleen-specific; Cationic hydrogel;

The development of a gene vector electrostatically assembled with a polysaccharide capsule by Tomoaki Kurosaki; Takashi Kitahara; Shigeru Kawakami; Koyo Nishida; Junzo Nakamura; Mugen Teshima; Hiroo Nakagawa; Yukinobu Kodama; Hideto To; Hitoshi Sasaki (4427-4434).
The purpose of this study was to develop a gene vector electrostatically assembled with a polysaccharide capsule. We used pDNA/polyethylenimine (PEI) complexes as efficient non-viral vectors. The pDNA/PEI complex was electrostatically encapsulated with various polysaccharides such as fucoidan, λ-carrageenan, xanthan gum, alginic acid, hyaluronic acid, and chondroitin sulfate (CS). The pDNA/PEI complex was shown as nanoparticles with positive ζ-potential, although the ternary complexes encapsulated with polysaccharides were shown as nanoparticles with negative ζ-potential. The pDNA/PEI complex showed high agglutination activity and cytotoxicity, although the ternary complexes encapsulated with polysaccharides had no agglutination activities and lower cytotoxicities. The pDNA/PEI complex showed high uptake and high transgene efficiency in B16-F10 cells. On the other hand, most of the ternary complexes show little uptake and gene expression. The ternary complex encapsulated by CS, however, showed comparable transgene efficiency to the pDNA/PEI complex. The uptake and gene expression of the ternary complex encapsulated by CS were significantly inhibited by hypothermia and the addition of CS, suggesting that the ternary complex was taken by CS-specific receptor-mediated energy-dependent process.
Keywords: Self assembly; Gene transfer; Surface modification; DNA; Chondroitin sulfate;

Computational design of drainage systems for vascularized scaffolds by James G. Truslow; Gavrielle M. Price; Joe Tien (4435-4443).
This computational study analyzes how to design a drainage system for porous scaffolds so that the scaffolds can be vascularized and perfused without collapse of the vessel lumens. We postulate that vascular transmural pressure – the difference between lumenal and interstitial pressures – must exceed a threshold value to avoid collapse. Model geometries consisted of hexagonal arrays of open channels in an isotropic scaffold, in which a small subset of channels was selected for drainage. Fluid flow through the vessels and drainage channel, across the vascular wall, and through the scaffold were governed by Navier–Stokes equations, Starling's Law of Filtration, and Darcy's Law, respectively. We found that each drainage channel could maintain a threshold transmural pressure only in nearby vessels, with a radius-of-action dependent on vascular geometry and the hydraulic properties of the vascular wall and scaffold. We illustrate how these results can be applied to microvascular tissue engineering, and suggest that scaffolds be designed with both perfusion and drainage in mind.
Keywords: Microvascular tissue engineering; Drainage; Collapse; Scaffold; Perfusion; Computational model;