Biomaterials (v.28, #36)

Skeletal muscle has been recognized as an essential source of progenitor or satellite cells, which are primarily responsible for muscle regeneration. Recently, muscle has also been identified as a valuable source of postnatal stem cells that appear to be distinct from satellite cells and possess the ability to differentiate into other cell lineages. These cells, named muscle-derived stem cells, possess a high myogenic capacity and effectively regenerate both skeletal and cardiac muscle. Remarkably, when genetically modified ex vivo to express growth factors, these cells can differentiate into osteogenic and chondrogenic lineages and have been shown to promote the repair of bone and cartilage. Muscle stem cell-based regenerative therapy and tissue engineering using ex vivo gene therapy, are promising approaches for the treatment of various musculoskeletal, cardiovascular, and urological disorders.
Keywords: Muscle; Stem cell; Gene therapy; Bone tissue engineering; Cartilage tissue engineering;

Thermoplastic biodegradable polyurethanes: The effect of chain extender structure on properties and in-vitro degradation by Lisa Tatai; Tim G. Moore; Raju Adhikari; François Malherbe; Ranjith Jayasekara; Ian Griffiths; Pathiraja A. Gunatillake (5407-5417).
Biodegradable polyurethanes are typically prepared from polyester polyols, aliphatic diisocyanates and chain extenders. We have developed a degradable chain extender (DCE) based on dl-lactic acid and ethylene glycol to accelerate hard segment degradation. Three series of polyurethane elastomers were synthesised to investigate the effect of incorporating DCE on synthesis, mechanical and thermal properties and in-vitro degradation. Polyurethane soft segments were based on poly(ε-caprolactone) (PCL) polyol. The hard segment was based on either ethyl lysine diisocyanate or hexamethylene diisocyanate in combination with ethylene glycol or DCE. Polyurethanes were characterised by gel permeation chromatography, tensile testing (Instron) and differential scanning calorimetry. Polymer degradation in-vitro (phosphate buffered saline) was tested by measuring mass loss, change in molecular weight and amine concentration in degradation products at three different time points over a 1 year period. Incorporation of DCE did not affect thermal or mechanical properties but had an influence on the in-vitro degradation. All polyurethanes exhibited considerable molecular weight decrease over the test period, and DCE-based polyurethanes showed the highest mass loss. The presence of the DCE and the initial molecular weight of the polyurethane are the key factors responsible for high mass losses. Differential scanning calorimetry, amine group analysis and the observation that mass loss was directly proportional to hard segment weight percentage, strongly supported that the polyurethane hard segment is the most susceptible segment to degradation in these polyurethanes. The PCL-based soft segment appears to undergo little or no degradation under these test conditions.
Keywords: Polyurethane; In-vitro degradation; Biodegradation; Thermoplastic polyurethanes; Poly(ester urethane); Degradable chain extenders;

The effect of hydrofluoric acid treatment of TiO2 grit blasted titanium implants on adherent osteoblast gene expression in vitro and in vivo by Juanli Guo; Ricardo J. Padilla; Wallace Ambrose; Ingeborg J. De Kok; Lyndon F. Cooper (5418-5425).
It is widely accepted that implant surface factors affect the quality of the bone-to-implant interface. Recent additional treatments superimposed on moderately rough cpTitanium surface provide further enhancement of bone-to-implant contact. The aim of this study was to compare osteoinductive and bone-specific gene expression in cells adherent to titanium dioxide-grit blasted (TiO2) versus TiO2 grit blasted and HF treated (TiO2/HF) cpTitanium implant surfaces. MC3T3-E1 cells were grown in osteogenic supplements on the titanium disk surfaces for 1–14 days. Real-time PCR was used to measure RUNX-2, Osterix, and bone sialoprotein (BSP) mRNA levels. Implants were placed in rat tibia and, following harvesting at 1–7 days after placement, real-time PCR was used to measure RUNX-2, alkaline phosphatase (ALP), and BSP mRNA levels in implant adherent cells. In cell culture, RUNX-2 and Osterix levels were significantly increased (p<0.05) on the TiO2/HF surfaces as compared to the TiO2 and smooth surfaces through the cultural period, while BSP expression was elevated on both TiO2 and TiO2/HF surfaces when compared to a machined surface control. In cells adherent to implants retrieved from rat tibia, RUNX-2 mRNA levels were 2-fold and 8-fold greater on the TiO2/HF surfaces at 1–3 and 7 days following implantation. This was paralleled by significantly greater levels of ALP at 3 and 7 days and BSP mRNA at 7 days following implantation. As a marker of osteoinduction, the increased levels of RUNX-2 in cells adherent to the TiO2/HF surfaces suggest that the additional HF treatment of the TiO2 grit blasted surface results in surface properties that support adherent cell osteoinduction. In vivo assessments of implant adherent cell phenotypes provide further insight into the mechanisms affecting alloplast–tissue interactions.
Keywords: Endosseous dental implant; Surface nanotechnology; Gene expression; Osteogenesis; Runx-2;

Poly(poly(ethyleneglycol) monomethacrylate) (P(PEGMA))-grafted magnetic nanoparticles (MNPs) were successfully prepared via a solvent-free atom transfer radical polymerization (ATRP) method. The macroinitiators were immobilized on the surface of 6.4±0.8 nm Fe3O4 nanoparticles via effective ligand exchange of oleic acid with 3-chloropropionic acid (CPA), which rendered the nanoparticles soluble in the PEGMA monomer. The so-obtained P(PEGMA)-grafted MNPs have a uniform hydrodynamic particle size of 36.0±1.2 nm. The successful grafting of P(PEGMA) on the MNP surface was ascertained from FTIR and XPS analyses. The uptake of the MNPs by macrophage cells is reduced by two-orders of magnitude to <2 pg Fe/cell after surface grafting with P(PEGMA). Furthermore, the morphology and viability of the macrophage cells cultured in a medium containing 0.2 mg/mL of P(PEGMA)-grafted MNPs were found similar to those of cells cultured without nanoparticles, indicating an absence of significant cytotoxicity effects. T 2-weighted magnetic resonance imaging (MRI) of P(PEGMA)-grafted MNPs showed that the magnetic resonance signal is enhanced significantly with increasing nanoparticle concentration in water. The R 1 and R 2 values per millimole Fe, and R 2/R 1 value of the P(PEGMA)-grafted MNPs were calculated to be 8.8 mm −1  s−1, 140 mm −1  s−1, and 16, respectively. These results indicate that the P(PEGMA)-grafted MNPs have great potential for application in MRI of specific biotargets.
Keywords: ATRP; Solvent-free; PEGMA; Magnetic nanoparticle; MRI;

The mechanism of cell-damaging reactive oxygen generation by colloidal fullerenes by Zoran Markovic; Biljana Todorovic-Markovic; Duska Kleut; Nadezda Nikolic; Sanja Vranjes-Djuric; Maja Misirkic; Ljubica Vucicevic; Kristina Janjetovic; Aleksandra Isakovic; Ljubica Harhaji; Branka Babic-Stojic; Miroslav Dramicanin; Vladimir Trajkovic (5437-5448).
Because of the ability to induce cell death in certain conditions, the fullerenes (C60) are potential anticancer and toxic agents. The colloidal suspension of crystalline C60 (nano-C60, nC60) is extremely toxic, but the mechanisms of its cytotoxicity are not completely understood. By combining experimental analysis and mathematical modelling, we investigate the requirements for the reactive oxygen species (ROS)-mediated cytotoxicity of different nC60 suspensions, prepared by solvent exchange method in tetrahydrofuran (THF/nC60) and ethanol (EtOH/nC60), or by extended mixing in water (aqu/nC60). With regard to their capacity to generate ROS and cause mitochondrial depolarization followed by necrotic cell death, the nC60 suspensions are ranked in the following order: THF/nC60>EtOH/nC60>aqu/nC60. Mathematical modelling of singlet oxygen (1O2) generation indicates that the 1O2-quenching power (THF/nC60<EtOH/nC60<aqu/nC60) of the solvent intercalated in the fullerene crystals determines their ability to produce ROS and cause cell damage. These data could have important implications for toxicology and biomedical application of colloidal fullerenes.
Keywords: Carbon; Nanoparticle; Cytotoxicity; Free radical; Modelling;

Neovascularization induced by porous collagen scaffold implanted on intact and cryoinjured rat hearts by Andrea Callegari; Sveva Bollini; Laura Iop; Angela Chiavegato; Gianluca Torregrossa; Michela Pozzobon; Gino Gerosa; Paolo De Coppi; Nicola Elvassore; Saverio Sartore (5449-5461).
The potential of collagen scaffolds for promoting angiogenesis/arteriogenesis was studied in vivo by implantation on healthy or cryoinjured left ventricles of rats up to 60 days post-injury. Blood vessels content and extra-vascular cell infiltration were evaluated within the collagen scaffold, the cryoinjured areas, and the “border zones” of the myocardium facing the cryoinjured zones. The collagen cardiac patches were almost completely absorbed in 60 days and became populated by new arterioles and capillaries in both intact and cryoinjured heart (arterioles in cryoinjured vs. intact zones were about 2,3-fold higher; capillaries in cryoinjured vs. intact zones were 1.7-fold higher). Collagen cardiac patches exerted a “trophic” effect on the organizing granulation tissue that emerged from the wound-healing process, increasing vessel density of 2.7-fold for arterioles and 4-fold for capillaries. Interstitial cells in collagen cardiac patches rarely (<1%) expressed cardiogenic stem cells markers such as Sca-1- or MDR1, whereas markers of neural crest cells GFAP+/nestin+ cells ranged from 3/30% to 30/70% in collagen cardiac patches placed on intact vs. cryoinjured heart, respectively. Myofibroblasts and cardiomyocytes (CM) were absent but macrophages populated the collagen scaffolds even after 60 days from implantation. Western blotting of collagen cardiac patches after implantation on intact/cryoinjured hearts confirmed that markers of endothelial and smooth muscle cells, but not of CM, were expressed. The porous collagen scaffold was able to elicit a powerful angiogenetic and arteriogenetic response in the intact and cryoinjured hearts, representing an ideal tool for therapeutic angio-arteriogenesis and a potentially useful substrate for stem cell seeding.
Keywords: Scaffold; Collagen; Angiogenesis; Neovascolarization; Heart; Cryoinjury;

Cartilage repair using an in vitro generated scaffold-free tissue-engineered construct derived from porcine synovial mesenchymal stem cells by Wataru Ando; Kosuke Tateishi; David A. Hart; Daisuke Katakai; Yoshinari Tanaka; Ken Nakata; Jun Hashimoto; Hiromichi Fujie; Konsei Shino; Hideki Yoshikawa; Norimasa Nakamura (5462-5470).
The objective was to in vitro generate a mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC) to facilitate in vivo repair in a porcine chondral defect model. Porcine synovial MSCs were cultured in monolayer at high density and were subsequently detached from the substratum. The cell/matrix complex spontaneously contracted to develop a basic TEC. Immunohistochemical analysis showed that the basic TEC contained collagen I and III, fibronectin, and vitronectin. The basic TEC exhibited stable adhesion to the surface of a porcine cartilage matrix in an explant culture system. The TEC cultured in chondrogenic media exhibited elevated expression of glycosaminoglycan and chondrogenic marker genes. The TEC were implanted in vivo into chondral defects in the medial femoral condyle of 4-month-old pigs, followed by sacrifice after 6 months. Implantation of a TEC into chondral defects initiated repair with a chondrogenic-like tissue, as well as secure biological integration to the adjacent cartilage. Histologically, the repair tissue stained positively with Safranin O and for collagen II. Biomechanical evaluation revealed that repair tissue exhibited mechanical properties similar to those of normal porcine cartilage in static compression and friction tests. This technology is a unique and promising method for stem cell-based cartilage repair.
Keywords: Cartilage tissue engineering; Mesenchymal stem cell; Animal model; Cell culture; Connective tissue; ECM (extracellular matrix);

The use of biotin–avidin binding to facilitate biomodification of thermoresponsive culture surfaces by Masanori Nishi; Jun Kobayashi; Sebastian Pechmann; Masayuki Yamato; Yoshikatsu Akiyama; Akihiko Kikuchi; Katsumi Uchida; Marcus Textor; Hirofumi Yajima; Teruo Okano (5471-5476).
Here, we report biomodification of temperature-responsive culture surfaces with biotinylated biomolecules utilizing streptavidin and biotinylation of the surfaces. Poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) was covalently grafted onto tissue culture polystyrene (TCPS) dishes. Biotinylated Arg-Gly-Asp-Ser (RGDS) peptides with different spacer lengths (biotin-conjugated G n RGDS (n=1, 6, 12, 16)) were examined. Human umbilical vein endothelial cells (HUVECs) adhered and were well spread on G12RGDS-immobilized surfaces in the absence of serum at 37 °C, while much less cell adhesion was observed with the other peptides. Adhered HUVECs were detached on reducing temperature to 20 °C, or on adding free RGDS peptide. Interestingly, cell detachment was accelerated by applying both these techniques. Consequently, by optimizing the spacer length, biomolecules can be functionally immobilized onto thermoresponsive surfaces via the affinity binding between avidin and biotin.
Keywords: Temperature-responsive; Cell culture; Biofunctionalization; RGDS;

Repair of cranial bone defects with adipose derived stem cells and coral scaffold in a canine model by Lei Cui; Bo Liu; Guangpeng Liu; Wenjie Zhang; Lian Cen; Jian Sun; Shuo Yin; Wei Liu; Yilin Cao (5477-5486).
Adipose derived stem cells (ASCs) with osteogenic differentiation potential have been documented as an alternative cell source for bone regeneration. However, most of previous in vivo studies were carried out on small animals along with relatively short-term follow-up. In this study, we investigated the feasibility of using ASCs and coral scaffolds to repair a cranial bone defect in a canine model, and followed up the outcome for up to 6 month. Autologous ASCs isolated from canine subcutaneous fat were expanded, osteogenically induced, and seeded on coral scaffolds. Bilateral full-thickness defects (20 mm×20 mm) of parietal bone were created. The defects were either repaired with ASC-coral constructs (experimental group) or with coral alone (control group). Three-dimensional CT scan showed that new bones were formed in the experimental group at 12 weeks post-implantation, while coral scaffolds were partially degraded in the control group. By radiographic analysis at 24 weeks post-transplantation, it was shown that an average of 84.19±6.45% of each defect volume had been repaired in experimental side, while the control side had only 25.04±18.82% of its volume filled. Histological examination revealed that the defect was repaired by typical bone tissue in experimental side, while only minimal bone formation with fibrous connection was observed in the control group. The successful repair of critical-sized bone defects via the current approach substantiates the potentiality of using ASCs with coral scaffolds for bone regeneration.
Keywords: Bone engineering; Adipose derived stem cells; Coral;

Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system by Efrem Curcio; Simona Salerno; Giuseppe Barbieri; Loredana De Bartolo; Enrico Drioli; Augustinus Bader (5487-5497).
Isolated hepatocytes in spheroid configuration exhibit a high degree of cell–cell contacts, which are important in the maintenance of viability and liver specific functions. In the absence of a vascular network, the cells in a large spheroid size experience mass transfer limitations of metabolites and oxygen in the core of aggregates. In this paper transport phenomena related to the diffusion and reaction of oxygen, glucose and lactate are mathematically described and experimentally verified for hepatocyte spheroids cultured in a rotating-wall polystyrene system (RWPS) not permeable for gases and in a rotating-wall membrane system (RWMS) with oxygen-permeable membrane. The concentration profiles of glucose, oxygen and lactate in the hepatocyte spheroids were estimated for different diameters of aggregates by solving the mass transfer equations for simultaneous diffusion and reaction, by finite element method. Simulation results evidenced that, for aggregates with size lower than 300 μm cultured in both RWPS and RWMS systems, the concentration profiles of glucose and lactate towards the core of spheroids (effective diffusion coefficients in the order of 10−11  m2/s) are not significantly affected by the metabolic rate (c.a 10−6  μg/mm3/s for glucose and about one order of magnitude less for lactate). On the contrary, the transport of oxygen (diffusion coefficient: 3.4×10−10  m2/s, reaction rate: 1.5×10−5  μg/mm3/s) is critically affected by the size of the multicellular spheroids and significant gradients in oxygen concentration may develop in spheroids. Aggregates with a size greater than 200 μm suffer severe oxygen limitation in the most part of its size attaining the lowest partial pressure in the centre. The improved viability predicted by the model culturing hepatocyte spheroids in the RWMS, characterized by a higher O2 permeability with respect to RWPS, was experimentally confirmed. The results demonstrated that the mathematical model used in this study represents a useful support to experimental procedures in order to obtain hepatocyte spheroids with optimal size.
Keywords: Hepatocytes; Spheroids; Rotating wall membrane system; Mass transfer; Metabolic reactions;

Long-term neurite orientation on astrocyte monolayers aligned by microtopography by Annette Sørensen; Tijna Alekseeva; Kashyap Katechia; Mary Robertson; Mathis O. Riehle; Susan C. Barnett (5498-5508).
After spinal cord injury neuronal connections are not easily re-established. Success has been hampered by the lack of orientation of neurites inside scar tissue and a lack of neurites crossing out of the site of injury. Oriented scaffolds in biodegradable polymers could be an excellent way to support both the orientation of neurites within the injury site as well as aiding their crossing out of the lesion. To establish the validity of using grooved micro-topography in polycaprolactone in combination with glia we have studied the long-term (3 weeks) orientation of neuronal cells on monolayers of astrocytes on the top of grooved topographies of various dimensions. We find that neurites are significantly aligned by groove/ridge type topographies which are “buried” under a monolayer of astrocytes for up to 3 weeks. This alignment is significantly lower than that of neurites growing directly on the topography, but these neurons do not survive on the poly-l-lysine coated polymer for more than a week. The alignment of neurites on the astrocyte layer to the underlying topography decreases over time, and with groove width. Topographies with 12.5 or 25 μm lateral dimension appear optimal for the long-term alignment and can support myelination. We have shown for the first time that micro-topography can act through an overlaid astrocyte layer and results in aligned neurites in long-term culture and that these can be myelinated by endogenous oligodendrocytes.
Keywords: Astrocytes; Nerve guide; Myelination; Topography;

The effect of endogenous overexpression of hyaluronan synthases on material, morphological, and biochemical properties of uncrosslinked collagen biomaterials by David D. Allison; Natalia Vasco; Kathleen R. Braun; Thomas N. Wight; K. Jane Grande-Allen (5509-5517).
Hyaluronan is an essential component of the native extracellular matrix that has often been added exogenously to biomaterials. The role of endogenously produced hyaluronan on soft tensile tissue mechanics, however, has been largely overlooked. To investigate this aspect of hyaluronan using a cell-mediated approach, cells overexpressing the hyaluronan synthases (has), namely has-1, has-2, has-3 or the empty vector control LXSN, were seeded within collagen gel scaffolds. The resulting engineered tissues were grown under static tension for 6 weeks. Following 6 weeks of culture, the samples were characterized to assess collagen gel contraction, matrix organization, production of hyaluronan, and tissue material properties. The engineered tissues containing cells transfected to overexpress one of the has isozymes had significantly increased retention of hyaluronan within the scaffold; elevated hyaluronan secretion into the culture medium (all but has-2); reduced contraction; reduced collagen density; and significantly altered material properties compared to the LXSN controls. These results indicate that the cell-mediated endogenous overproduction of hyaluronan within biomaterials alters their material, morphological and biochemical characteristics. This investigation, the first to examine the role of endogenously produced hyaluronan in engineered tissue mechanics, suggests that overproduction of hyaluronan in soft connective tissues can transform their biological and biomechanical functionality.
Keywords: Cell culture; Collagen; Connective tissue; Hyaluronic acid/hyaluronan; ECM (extracellular matrix); Glycosaminoglycan;

Scaffolds based on degradable alginate hydrogels and poly(lactide-co-glycolide) microspheres for stem cell culture by Randolph S. Ashton; Akhilesh Banerjee; Supriya Punyani; David V. Schaffer; Ravi S. Kane (5518-5525).
We describe a method for creating alginate hydrogels with adjustable degradation rates that can be used as scaffolds for stem cells. Alginate hydrogels have been widely tested as three-dimensional constructs for cell culture, cell carriers for implantation, and in tissue regeneration applications; however, alginate hydrogel implants can take months to disappear from implantation sites because mammals do not produce endogenous alginases. By incorporating poly(lactide-co-glycolide) (PLGA) microspheres loaded with alginate lyase into alginate hydrogels, we demonstrate that alginate hydrogels can be enzymatically degraded in a controlled and tunable fashion. We demonstrate that neural progenitor cells (NPCs) can be cultured and expanded in vitro in this degradable alginate hydrogel system. Moreover, we observe a significant increase in the expansion rate of NPCs cultured in degrading alginate hydrogels versus NPCs cultured in standard, i.e. non-degrading, alginate hydrogels. Degradable alginate hydrogels encapsulating stem cells may be widely applied to develop novel therapies for tissue regeneration.
Keywords: Alginate; Stem cells; Hydrogel; Degradation; Controlled release;

Development and evaluation of silk fibroin-based nerve grafts used for peripheral nerve regeneration by Yumin Yang; Fei Ding; Jian Wu; Wen Hu; Wei Liu; Jie Liu; Xiaosong Gu (5526-5535).
Silk fibroin (SF), derived from natural silk long used as a textile material, has recently become an important biomaterial for tissue engineering applications. We have previously reported on good in vitro biocompatibility of SF fibers with peripheral nerve tissues and cells. In the present study, we developed a novel biomimetic design of the SF-based nerve graft (SF graft) which was composed of a SF-nerve guidance conduit (NGC) inserted with oriented SF filaments. The SF-NGC prepared via well-established procedures exhibits an eggshell-like microstructure that is responsible for its superior mechanical and permeable properties beneficial to nerve regeneration. The SF graft was used for bridge implantation across a 10-mm long sciatic nerve defect in rats, and the outcome of peripheral nerve repair at 6 months post-implantation was evaluated by a combination of electrophysiological assessment, FluoroGold retrograde tracing and histological investigation. The examined functional and morphological parameters show that SF grafts could promote peripheral nerve regeneration with effects approaching those elicited by nerve autografts which are generally considered as the gold standard for treating large peripheral nerve defects, thus raising a potential possibility of using these newly developed nerve grafts as a promising alternative to nerve autografts.
Keywords: Silk fibroin; Nerve graft; Nerve guidance conduit; Biomimetic design; Rat sciatic nerve defect; Peripheral nerve regeneration;

The pro-angiogenic characteristics of a cross-linked gelatin matrix by Lars Dreesmann; Michael Ahlers; Burkhard Schlosshauer (5536-5543).
To overcome limitations on regeneration in the nervous system and other organs caused by insufficient blood supply, we have developed a gelatin sponge material which stimulates blood vessel formation, i.e. angiogenesis. Controlled chemical cross-linking was employed to slow down enzymatic degradation of the gelatin matrix. Four different in vitro assays using L929 fibroblasts and purified endothelial cells indicated that the sponge material did not release toxic components, but provided a permissive substratum for cell attachment, cell migration and pronounced cell proliferation, all of which are crucial for the formation of vasculature. Two in vivo models were employed to directly monitor the pro-angiogenic impact of the sponge material. Implantation of gelatin sponges onto the chorioallantoic membrane of fertilized chicken eggs induced robust attraction of endothelial cells and formation of blood vessels. Angiogenesis inside gelatin implants occurred more than 200 times faster than in a commercial collagen sponge. Similarly, after subcutaneous implantation of tube-like sponges into mice, an increasing immigration of cells and subsequent formation of functional vasculature became evident. Immunocytochemistry revealed no fibronection accumulation and no scarring. In summary, our matrix based on cross-linked gelatin promises to be a valuable component of future implants, improving neuronal and non-neuronal regeneration by concomitant pro-angiogenic stimulation.
Keywords: Angiogenesis; Gelatin; Cell culture; Endothelial cells; Implant;

Numerous experimental studies have attempted to determine the optimal properties for a scaffold for use in bone tissue engineering but, as yet, no computational or theoretical approach has been developed that suggests how best to combine the various design parameters, e.g. scaffold porosity, Young's modulus, and dissolution rate. Previous research has shown that bone regeneration during fracture healing and osteochondral defect repair can be simulated using mechanoregulation algorithms based on computing strain and/or fluid flow in the regenerating tissue. In this paper a fully three-dimensional approach is used for computer simulation of tissue differentiation and bone regeneration in a regular scaffold as a function of porosity, Young's modulus, and dissolution rate—and this is done under both low and high loading conditions. The mechanoregulation algorithm employed determines tissue differentiation both in terms of the prevailing biophysical stimulus and number of precursor cells, where cell number is computed based on a three-dimensional random-walk approach. The simulations predict that all three design variables have a critical effect on the amount of bone regenerated, but not in an intuitive way: in a low load environment, a higher porosity and higher stiffness but a medium dissolution rate gives the greatest amount of bone whereas in a high load environment the dissolution rate should be lower otherwise the scaffold will collapse—at lower initial porosities however, higher dissolution rates can be sustained. Besides showing that scaffolds may be optimised to suit the site-specific loading requirements, the results open up a new approach for computational simulations in tissue engineering.
Keywords: Scaffolds; Tissue engineering; Systems biology; Computational simulation; Stem cell fate;

Nanoparticulate carrier containing water-insoluble iodinated oil as a multifunctional contrast agent for computed tomography imaging by Won Ho Kong; Won Jae Lee; Zheng Yun Cui; Ki Hyun Bae; Tae Gwan Park; Jin Hoon Kim; Keunchil Park; Soo Won Seo (5555-5561).
Contrast-enhanced computed tomography (CT) imaging is a valuable and routine strategy for the clinical diagnosis of various diseases. However, all current CT contrast agents are liquids, so they flow through the blood vessels and disappear very quickly by extravasation. If it were possible to make a blood-compatible particulate contrast agent, we could highlight a particular tissue by either passive or active targeting. In this work, Pluronic F127 and a naturally iodinated compound, Lipiodol, were used to form radiopaque nanoreservoir structures. The resultant nanoparticles have a stable structure at high concentrations, sufficient X-ray absorption, a safety profile similar to or better than that of Iopromide, and a longer circulation time than commercial iodinated preparations. The utility of the resultant radiopaque nanoparticles as a contrast agent was tested using micro-SPECT/CT imaging in vivo. Together with the very good solubility of hydrophobic drugs (e.g., Taxol) in Lipiodol, these results suggest the possibility that these particulate structures and their bioconjugates could become functional CT contrast agents that could deliver therapeutic agents to a particular tissue.
Keywords: Contrast agent; Computed tomography; Hydrophobic drug; Pluronic; Lipiodol;

Chemotherapeutic implants via subcritical CO2 modification by Heather M. Powell; Olukemi Ayodeji; Taryn L. Summerfield; David M. Powell; Douglas A. Kniss; David L. Tomasko; John J. Lannutti (5562-5569).
Polymer-based biomaterials have a broad range of current applications in medicine. Many implants generate a favorable biomedical outcome solely by providing short-term mechanical stability that allows healing of the surrounding tissues. An example is polymeric reconstructive resorbable plates having initial strengths sufficient to stabilize bone segments while allowing the osteosynthesis needed to restore original function following tumor resection. Simultaneous, localized delivery of the widely employed chemotherapeutic paclitaxel following tumor removal presents a particularly desirable goal in this context. By using compressed/subcritical CO2 at moderate pressures (as opposed to the more familiar supercritical pressures) to embed paclitaxel in clinically utilized reconstructive plating, the form of the implant can be preserved while adding an inherently localized chemotherapeutic function. In vitro tests demonstrate the efficacy of the embedded paclitaxel against adherent MCF-7 breast cancer cells within the immediate area of the polylactic acid (PLA). CO2 can be utilized to add dual structural-chemotherapeutic function to polymeric surfaces without a change in form. The ability to ‘piggyback’ chemotherapeutic function into nearly any polymeric surface should find widespread utility.
Keywords: Chemotherapy; Controlled drug release; In vitro test; Polylactic acid; Degradation;

Regeneration of ovine articular cartilage defects by cell-free polymer-based implants by Christoph Erggelet; Katja Neumann; Michaela Endres; Kathrin Haberstroh; Michael Sittinger; Christian Kaps (5570-5580).
The aim of our study was the evaluation of a cell-free cartilage implant that allows the recruitment of mesenchymal stem and progenitor cells by chemo-attractants and subsequent guidance of the progenitors to form cartilage repair tissue after microfracture. Chemotactic activity of human serum on human mesenchymal progenitors was tested in 96-well chemotaxis assays and chondrogenic differentiation was assessed by gene expression profiling after stimulating progenitors with hyaluronan in high-density cultures. Autologous serum and hyaluronan were combined with polyglycolic acid (PGA) scaffolds and were implanted into full-thickness articular cartilage defects of the sheep pre-treated with microfracture. Defects treated with microfracture served as controls. Human serum was a potent chemo-attractant and efficiently recruited mesenchymal progenitors. Chondrogenic differentiation of progenitors upon stimulation with hyaluronan was shown by the induction of typical chondrogenic marker genes like type II collagen and aggrecan. Three months after implantation of the cell-free implant, histological analysis documented the formation of a cartilaginous repair tissue. Controls treated with microfracture showed no formation of repair tissue. The cell-free cartilage implant consisting of autologous serum, hyaluronan and PGA utilizes the migration and differentiation potential of mesenchymal progenitors for cartilage regeneration and is well suited for the treatment of cartilage defects after microfracture.
Keywords: Cartilage regeneration; Microfracture; Cell-free implant; Stem cells; Chemotaxis;

Hydrolysable core-crosslinked thermosensitive polymeric micelles: Synthesis, characterisation and in vivo studies by Cristianne J. Rijcken; Cor J. Snel; Raymond M. Schiffelers; Cornelus F. van Nostrum; Wim E. Hennink (5581-5593).
In this study, core-crosslinked (CCL) biodegradable thermosensitive micelles based on mPEG5000 and N-(2-hydroxyethyl)methacrylamide)-oligolactates (mPEG-b-p(HEMAm-Lac n )) were synthesised and their properties investigated. Rapidly heating aqueous solutions of partially methacrylated block copolymers to above their critical micelle temperature (CMT), followed by illumination in presence of a photoinitiator yielded almost monodisperse CCL micelles with a size of 68±7 nm. Either below the CMT or after addition of sodium dodecyl sulphate, the non-crosslinked (NCL) micelles rapidly disintegrated whereas the CCL micelles kept their integrity. NCL micelles fell apart after 5 h in pH 7.4 at 37 °C as a result of the hydrolysis of lactate side chains, whereas the CCL micelles had a much higher stability and only degraded after cleavage of the ester bonds in the crosslinks. The circulation kinetics and biodistribution of CCL micelles were considerably better than those of NCL micelles, i.e., 58% of the injected dose (ID) of CCL versus 6% of NCL micelles was recovered in the circulation 4 h post-injection. Furthermore, the liver uptake of the CCL micelles (10% ID) was much lower than that of the NCL micelles (24% ID) 4 h after administration, while tumour accumulation was almost 6 times higher for the CCL micelles. Likely, NCL micelles dissociated after i.v. administration and/or were opsonised and captured by macrophages while the dense PEG shell of CCL micelles made them less prone towards opsonisation. The excellent physical stability of these degradable CCL micelles and very favourable biodistribution profile renders them very suitable for drug targeting purposes.
Keywords: Self-assembly; Micelle; Photopolymerisation; Crosslinking; Biodegradation; Poly(ethylene glycol);

Biospecific anchoring and spatially confined germination of bacterial spores in non-biofouling microwells by Kyung-Bok Lee; Young Hwan Jung; Zee-Won Lee; Soohyun Kim; Insung S. Choi (5594-5600).
In this paper, we report a simple method for spatially confining Bacillus subtilis (BS) spores into semi-three dimensional, non-biofouling microwells by using biospecific (such as biotin–streptavidin) interactions. Non-biofouling poly(ethylene glycol) (PEG)-based microwells were fabricated by employing a process of capillary molding on a glass slide. The biospecific interactions between biotinylated BS spores and streptavidin led to the selective deposition of BS spores onto the bottom of the microwells of which presented streptavidin. The viability of the patterned spores was confirmed by the induction of germination. Bacterial spores were found to maintain extreme robustness until they were exposed to favorable conditions. This work suggests that the use of bacterial spore-based sensors would increase the shelf-life (such as long-term storage and stability) of cell-based sensors.
Keywords: Spore; Vegetative cells; Microwells; Biospecific interactions; Patterning;