Biomaterials (v.29, #3)

The initial stages of the hydration process have been simulated on a single-Ca(I) terminated hydroxyapatite (0 0 0 1) surface in step-by-step fashion using periodic slab density functional theory (DFT). Adsorption configurations and energetic properties have been described at different H2O coverage. At low H2O coverage, oxygen prefers to form Ca―O bonds with surface Ca cations, but as coverage increases, H2O tends to loosely float on the already-formed water layer. The height of the first layer H2O relative to the surface is found to be 1.6 Å. The hydration process does not cause the decomposition of surface phosphate groups and hydroxyl channel, but does affect the energetics of subsequent Zn substitution and occupation on Ca(I) and Ca(II) sites. The Ca(II) vacancy site is found to be energetically more favorable for occupation due to less spatial constraint. This suggested mechanism of preferential occupation is different from previous attempts to explain the cation substitution site preference in bulk by ionic radius and electronegativity differences.
Keywords: DFT; Hydroxyapatite; Hydration; Ion exchange; Preferential site occupancy;

β-TCP bone graft substitutes in a bilateral rabbit tibial defect model by William R. Walsh; Frank Vizesi; Dean Michael; Jason Auld; Andy Langdown; Rema Oliver; Yan Yu; Hiroyuki Irie; Warwick Bruce (266-271).
The use of artificial bone graft substitutes has increased as the surgical applications widen and the availability of allograft bone decreases. The ideal graft substitute should reabsorb with time to allow and encourage new bone formation whilst maintaining its properties as an osteoconductive scaffold until it is no longer required. A potential disadvantage of some synthetic substitutes is their long dissolution time. Beta-tricalcium phosphates (β-TCPs) have some advantages when compared to hydroxyapatite (HA), when used as a filler, in that it is more rapidly reabsorbed.Three commercially available and clinically used β-TCP bone graft substitutes with the same chemistry (Vitoss, Osferion, Chronos) but with varying macro and microscopic characteristics were investigated using a bilateral tibial metaphyseal defect model in New Zealand white rabbits. When placed into tibial defects all three materials performed similarly in terms of mechanical properties of the healing defects. A decrease in properties was found at 12 weeks where implant resorption was nearly achieved while remodelling of the anteromedial cortex had yet to be completed. All materials were osteoconductive and supported new bone formation while implant resorption with time differed between materials. Vitoss resorbed faster than the other materials and is likely to differences in particle geometry, pore structure and interconnectivity.
Keywords: Bone; Bone graft substitutes; Tricalcium phosphate; Histology; Mechanical testing;

A thermoreversible hydrogel as a biosynthetic bandage for corneal wound repair by Chayanin Pratoomsoot; Hidetoshi Tanioka; Kuniko Hori; Satoshi Kawasaki; Shigeru Kinoshita; Patrick J. Tighe; Harminder Dua; Kevin M. Shakesheff; Felicity Rosamari A.J. Rose (272-281).
Ocular trauma and disorders that lead to corneal blindness account for over 2 million new cases of monocular blindness every year. A popular ocular surface reconstruction therapy, amniotic membrane transplantation, has been shown to aid corneal wound repair. However, the success rates of the procedure are variable. Here, we proposed to bioengineer a novel synthetic material that would serve as a biomimetic corneal bandage. The PLGA–PEG–PLGA triblock copolymer was synthesised via ring-opening polymerisation. Thermoreversible gelation behaviour was investigated at different polymer concentrations (23%, 30%, 35%, 40%, 45%, w/v) at temperatures ranging between 5 and 60 °C. Viscoelastic properties were studied in dynamic mechanical analysis with 1 °C/min temperature ramp. Cryo-SEM revealed a porous hydrogel with interconnecting networks. No adverse cytotoxicity was observed with an in vitro scratch-wound assay and in in vivo biocompatibility tests. We have demonstrated that the PLGA–PEG–PLGA hydrogel possessed a suitable gelling profile and, for the first time, the biocompatibility properties for this application as a potential bandage for corneal wound repair.
Keywords: Cornea; Biocompatibility; Thermoreversible; Hydrogel; Wound healing;

A wide variety of different cell types have been shown to respond to nanofabricated growth surfaces via the process of contact guidance, however little is known about the intracellular mechanisms that control these events. In the present study we have identified the multi-functional signalling adaptor protein, RACK1, as a novel negative regulator of contact guidance on custom-engineered nanometric grooves. We found that over-expression of RACK1 in human breast cancer cells leads to a pro-adherent morphology characterised by the formation of stress fibres and focal adhesions. Enforced expression of RACK1 also limits the response of cells to contact guidance on nanometric grooves. In contrast, ablation of RACK1 protein with specific anti-sense oligonucleotides led to a dramatic enhancement of bi-directional extension of cells on nanometrically deep grooved surfaces, with a corresponding loss of focal adhesions and stress fibres. RACK1 therefore exerts a tonic inhibitory effect on cell contact guidance, while positively promoting an adhesive phenotype. This is the first example of an intracellular signalling molecule involved in the regulation of cell contact guidance on nanometric growth surfaces.
Keywords: Nanotopography; Contact guidance; Cell spreading; Cell morphology; Signal transducing mediator; Antisense;

Synthetic sandwich culture of 3D hepatocyte monolayer by Yanan Du; Rongbin Han; Feng Wen; Susanne Ng San San; Lei Xia; Thorsten Wohland; Hwa Liang Leo; Hanry Yu (290-301).
The sandwich culture of hepatocytes, between double layers of extra-cellular matrix (ECM), is a well-established in vitro model for re-establishing hepatic polarity and maintaining differentiated functions. Applications of the ECM-based sandwich culture are limited by the mass transfer barriers induced by the top gelled ECM layer, complex molecular composition of ECM with batch-to-batch variation and uncontrollable coating of the ECM double layers. We have addressed these limitations of the ECM-based sandwich culture by developing an ‘ECM-free’ synthetic sandwich culture, which is constructed by sandwiching a 3D hepatocyte monolayer between a glycine-arginine-glycine-aspatic acid-serine (GRGDS)-modified polyethylene terephthalate (PET) track-etched membrane (top support) and a galactosylated PET film (bottom substratum). The bioactive top support and bottom substratum in the synthetic sandwich culture substituted for the functionalities of the ECM in the ECM-based sandwich culture with further improvement in mass transfer and optimal material properties. The 3D hepatocyte monolayer in the synthetic sandwich culture exhibited a similar process of hepatic polarity formation, better cell–cell interaction and improved differentiated functions over 14-day culture compared to the hepatocytes in collagen sandwich culture. The novel 3D hepatocyte monolayer sandwich culture using bioactive synthetic materials may readily replace the ECM-based sandwich culture for liver tissue engineering applications, such as drug metabolism/toxicity testing and hepatocyte-based bioreactors.
Keywords: Sandwich culture; Hepatocyte; Synthetic materials; Polarity; RGD peptide; Galactosylation;

Assessment of stem cell/biomaterial combinations for stem cell-based tissue engineering by Sabine Neuss; Christian Apel; Patricia Buttler; Bernd Denecke; Anandhan Dhanasingh; Xiaolei Ding; Dirk Grafahrend; Andreas Groger; Karsten Hemmrich; Alexander Herr; Willi Jahnen-Dechent; Svetlana Mastitskaya; Alberto Perez-Bouza; Stephanie Rosewick; Jochen Salber; Michael Wöltje; Martin Zenke (302-313).
Biomaterials are used in tissue engineering with the aim to repair or reconstruct tissues and organs. Frequently, the identification and development of biomaterials is an iterative process with biomaterials being designed and then individually tested for their properties in combination with one specific cell type. However, recent efforts have been devoted to systematic, combinatorial and parallel approaches to identify biomaterials, suitable for specific applications. Embryonic and adult stem cells represent an ideal cell source for tissue engineering. Since stem cells can be readily isolated, expanded and transplanted, their application in cell-based therapies has become a major focus of research. Biomaterials can potentially influence e.g. stem cell proliferation and differentiation in both, positive or negative ways and biomaterial characteristics have been applied to repel or attract stem cells in a niche-like microenvironment. Our consortium has now established a grid-based platform to investigate stem cell/biomaterial interactions. So far, we have assessed 140 combinations of seven different stem cell types and 19 different polymers performing systematic screening assays to analyse parameters such as morphology, vitality, cytotoxicity, apoptosis, and proliferation. We thus can suggest and advise for and against special combinations for stem cell-based tissue engineering.
Keywords: Mesenchymal stem cells; Preadipocytes; Endothelial progenitor cells; Dental pulp stem cells; Hematopoietic stem cells; Embryonic stem cells;

The effect of enzymatically degradable poly(ethylene glycol) hydrogels on smooth muscle cell phenotype by Catharina Adelöw; Tatiana Segura; Jeffrey A. Hubbell; Peter Frey (314-326).
The formation of scar tissue due to dedifferentiation of smooth muscle cells (SMCs) is one of the major issues faced when engineering bladder tissue. Furthermore, cell sources for regenerating the SMC layer are also limiting. Here we explore if human mesenchymal stem cells (MCSs), cultured in enzymatically degradable poly(ethylene glycol) (PEG) hydrogel scaffolds can be differentiated into SMC-like cells. We explored the degree to which a less synthetic SMC phenotype can be achieved when primary human SMCs are cultured within these scaffolds, It was observed that when both MSCs and SMCs are cultured in the PEG hydrogel scaffolds, but not on traditional tissue culture plastic, they up-regulate markers associated with the less synthetic SMC phenotype, decreased expression of α 5 integrin and THY-1, and increased expression of α-smooth muscle actin (αSMA) and myosin. Furthermore, we show that MSCs and SMCs cultured in the PEG hydrogels are able to proliferate and express matrix metalloproteinases for up to 21 d in culture, the duration of the study. This study addresses the importance of the cellular microenvironment on cell fate, and proposes synthetic instructive biomaterials as a means to direct cell differentiation and circumvent scar tissue formation during bladder reconstruction.
Keywords: Hydrogel; PEG; ECM; Bladder; Mesenchymal stem cell; Smooth muscle cell;

Construction and characterization of a thrombin-resistant designer FGF-based collagen binding domain angiogen by Luke P. Brewster; Cicely Washington; Eric M. Brey; Andrew Gassman; Anu Subramanian; Jen Calceterra; William Wolf; Connie L. Hall; William H. Velander; Wilson H. Burgess; Howard P. Greisler (327-336).
Humans demonstrate limited spontaneous endothelialization of prosthetic bypass grafts. However the local application of growth factors to prosthetic grafts or to injured blood vessels can provide an immediate effect on endothelialization. Novel chimeric proteins combining potent angiogens with extracellular matrix binding domains may localize to exposed matrices and provide sustained activity to promote endothelial regeneration after vascular interventions. We have ligated a thrombin-resistant mutant of fibroblast growth factor (FGF)-1 (R136K) with a collagen binding domain (CBD) in order to direct this growth factor to sites of exposed vascular collagen or selected bioengineered scaffolds. While FGF-1 and R136K are readily attracted to a variety of matrix proteins, R136K-CBD demonstrated selective and avid binding to collagen ∼4x that of FGF-1 or R136K alone (P<0.05). The molecular stability of R136K-CBD was superior to FGF-1 and R136K. Its chemotactic activity was superior to R136K and FGF-1 (11±1% vs. 6±2% and 4±1%; P<0.01). Its angiogenic activity was similar to R136K and significantly greater than control by day 2 (P<0.01). After day 3, FGF-1-treated endothelial cell's (EC) sprouts had regressed back to levels insignificant compared to the control group (P=0.17), while both R136K and R136K-CBD continued to demonstrate greater sprout lengthening as compared to control (P<0.0002). The mitogenic activity of all growth factors was greater than control groups (20% PBS); in all comparisons (P<0.0001). This dual functioning angiogen provides proof of concept for the application of designer angiogens to matrix binding proteins to intelligently promote endothelial regeneration of selected matrices.
Keywords: Angiogenesis; Collagen; Endothelial cell; Endothelialization; Fibroblast growth factor; Growth factors;

Miscibility and in vitro osteocompatibility of biodegradable blends of poly[(ethyl alanato) (p-phenyl phenoxy) phosphazene] and poly(lactic acid-glycolic acid) by Meng Deng; Lakshmi S. Nair; Syam P. Nukavarapu; Sangamesh G. Kumbar; Tao Jiang; Nicholas R. Krogman; Anurima Singh; Harry R. Allcock; Cato T. Laurencin (337-349).
Previously we demonstrated the ability of ethyl glycinato substituted polyphosphazenes to neutralize the acidic degradation products and control the degradation rate of poly(lactic acid-glycolic acid) (PLAGA) by blending. In this study, blends of high strength poly[(50% ethyl alanato) (50% p-phenyl phenoxy) phosphazene] (PNEA50PhPh50) and 85:15 PLAGA were prepared using a mutual solvent approach. Three different solvents, methylene chloride (MC), chloroform (CF) and tetrahydrofuran (THF) were studied to investigate solvent effects on blend miscibility. Three different blends were then fabricated at various weight ratios namely 25:75 (BLEND25), 50:50 (BLEND50), and 75:25 (BLEND75) using THF as the mutual solvent. The miscibility of the blends was evaluated by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR). Among these, BLEND25 was miscible while BLEND50 and BLEND75 were partially miscible. Furthermore, BLEND25 formed apatite layers on its surface as evidenced in a biomimetic study performed. These novel blends showed cell adhesion and proliferation comparable to PLAGA. However, the PNEA50PhPh50 component in the blends was able to increase the phenotypic expression and mineralized matrix synthesis of the primary rat osteoblasts (PRO) in vitro. Blends of high strength PNEA50PhPh50 and 85:15 PLAGA are promising biomaterials for a variety of musculoskeletal applications.
Keywords: Polyphosphazenes; Poly(lactic acid-glycolic acid); Polymer blends; Osteocompatibility; Bone tissue engineering;

Antibiotic-loaded poly-ε-caprolactone and porous β-tricalcium phosphate composite for treating osteomyelitis by Takahiro Miyai; Atsuo Ito; Gaku Tamazawa; Tomonori Matsuno; Yu Sogo; Chiho Nakamura; Atsushi Yamazaki; Tazuko Satoh (350-358).
A composite of poly-ε-caprolactone (PCL) loaded with gatifloxacine (GFLX), an antibiotic, and a β-tricalcium phosphate (βTCP) porous ceramic body was prepared by a solvent-free process in which no toxic solvent was used. GFLX mostly retained its bactericidal property after the processing. The composite of GFLX-loaded PCL and βTCP ceramic released GFLX for 4 weeks in Hanks’ balanced solution, and had sustained bactericidal activity against Streptococcus milleri and Bacteroides fragilis for at least 1 week. The composite of the GFLX-loaded PCL and βTCP ceramic was implanted in an osteomyelitis lesion induced by S. milleri and B. fragilis in the rabbit mandible. The osteomyelitis lesion expanded in the mesial–distal direction when no composite was implanted or when the lesion was treated with debridement only. The composite of GFLX-loaded PCL and βTCP showed efficacy in controlling infection at the bone defect formed by debridement, and supported bone tissue reconstruction at the bone defect. Twelve and 50 weeks after the implantation, the inflammation even disappeared. New bone formation was observed on the surface of the composite after 4 weeks. After 50 weeks, ingrowth of bone tissues with vascular channels was observed along the PCL and βTCP interface, which indicated degradation of PCL and/or βTCP ceramic at the ceramic/polymer interface followed by replacement by bone tissues. The GFLX concentrations in the serum and soft tissues were very low. Therefore, the composite of GFLX-loaded PCL and βTCP ceramic would help arrest osteomyelitis when it is used in addition to intravenous antibiotic administration, and help new bone formation and osteoconduction.
Keywords: Polycaprolactone; Drug delivery; Calcium phosphate; Antimicrobial; Osteoconduction; Animal model;

Ceramide is a bioactive sphingolipid-derived second messenger that has been demonstrated to induce apoptosis and cell cycle arrest in various cancer cell culture systems. Although in vitro tumor cell culture models have illuminated the potential therapeutic utility of a cell-permeable analog of ceramide, C6, in vivo delivery is impeded by the extreme hydrophobicity and physical-chemical properties of this bioactive lipid. Previously, we have demonstrated that the incorporation of C6 into pegylated liposomal vesicles is an effective anti-cancer drug delivery strategy in vitro and in vivo. Here, we report the utilization of a novel multi-functional polymeric drug delivery system designed to therapeutically target C6 to solid tumor tissue. This delivery system is a hydrolytically degradable and temperature-sensitive linear-dendritic nanoparticle with a lower critical solution temperature (LCST) of 30 °C. C6 was effectively loaded into the nanoparticles, and released continuously for at least 1 month in vitro, measured by mass spectroscopy. The preferential uptake of fluorescein isothiocyanate-labeled linear-dendritic nanoparticles into human MDA-MB-231 breast adenocarcinoma cells at temperature above the LCST (37 °C) was confirmed by confocal microscopy and quantified by flow cytometry. The accumulation of NBD-C6 into MDA-MB-231 cells was highly enhanced by the thermoresponsive linear-dendritic nanoparticles, but not by non-thermoresponsive liposome and PEG-dendritic polymer, at temperature above the LCST (37 °C). The linear-dendritic nanoparticles alone were not toxic, but their complexes with C6 caused significant growth inhibition and apoptosis to MDA-MB-231 cells at 37 °C. The designed thermoresponsive and biodegradable linear-dendritic nanoparticles have great potential for thermally targeted and sustained release of C6 for the treatment of solid tumors with hyperthermia.
Keywords: Linear-dendritic nanoparticles; Thermoresponsive; Biodegradable; Ceramide; Cancer; Drug delivery;

Stem cells have the potential to be differentiated to a specific cell type through genetic manipulation and therefore, represent a new and versatile source of cell replacement in regenerative medicine. However, conventional ways of gene transfer to these progenitor cells, suffer from a number of disadvantages particularly involving safety and efficacy issues. We have recently reported on the development of a bio-functionalized DNA carrier of carbonate apatite by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high-affinity interactions with embryonic stem cell surface and accelerated transgene delivery for subsequent expression. Here, we show the molecular basis of synthesizing highly functional composite particles utilizing DNA, cell-adhesive proteins and inorganic crystals, and finally establish a superior transfection system for a mouse stem cell line having potential applications in cell-based therapy.
Keywords: Carbonate apatite; Nano-crystals; Cell-adhesive proteins; Fibronectin; E-cadherin; Endocytosis;

Systemically administered ligand-coated nanoparticles have been proved to recognize biological targets in-vivo. This can provide breakthrough solutions for the early detection, imaging and cure of diseases. In cardiovascular applications, nanoparticles have been targeted directly to the diseased vasculature, and such delivery approach is becoming increasingly popular even in cancer research, supported by the growing body of evidences on the biological differences between normal and tumor vasculature. This work focuses on the optimal design of nanoparticles for vascular targeting throughout mathematical modeling. Such nanoparticles should be engineered so as to recognize specifically and adhere firmly to the diseased vessel walls withstanding the hydrodynamic dislodging forces and control uptake by the endothelial cells. A stochastic approach for predicting the adhesion strength of nanoparticles to a cell layer under flow has been coupled to a mathematical model for the receptor-mediated endocytosis of nanoparticles. The main geometrical, biophysical and biological parameters governing both events have been identified and their relative importance highlighted. Three different states for the particle/cell system have been predicted, namely no adhesion, adhesion with no endocytosis and adhesion with endocytosis, based upon the geometrical and biophysical properties of the particle and the biological conditions at the site of adhesion. Design maps have been generated to be used as a preliminary reference for choosing the properties of the nanoparticle as a function of physiological parameters, as the wall shear stress and the receptors surface density, at the site of desired adhesion within the target vasculature.
Keywords: Nanoparticles; Vascular targeting; Adhesion; Endocytosis; Drug delivery;