Biomaterials (v.28, #17)

In vitro lifetime of dental ceramics under cyclic loading in water by A.R. Studart; F. Filser; P. Kocher; L.J. Gauckler (2695-2705).
All-ceramic dental restorations exhibit enhanced esthetics and biocompatibility as compared to traditional metal-based prosthesis. However, long-term fatigue and subcritical crack growth in the presence of water and cyclic loading can decrease the strength of ceramic components over time. We investigated the cyclic fatigue in water of three dental materials currently used as frameworks in all-ceramic restorations: a 3 mol%-yttria partially stabilized zirconia (3Y-TZP, Cercon, Degudent GmbH), a Al2O3–ZrO2–Glass composite (Inceram-Zirconia, Vita Zahnfabrik GmbH) and a Li2O·2SiO2 glass ceramic (Empress 2, Ivoclar Vivadent AG). Fatigue and fast fracture tests were performed to determine the Weibull distribution of lifetime and initial mechanical strength for each framework component. In spite of its noticeable susceptibility to fatigue in water, the 3Y-TZP material was found to be particularly suitable for the preparation of posterior all-ceramic bridges due to its high initial mechanical strength. Guidelines are provided for the selection of materials and the design of all-ceramic posterior bridges exhibiting lifetime longer than 20 years under severe wet and cyclic loading conditions.
Keywords: Dental restorative material; Fatigue; Mechanical properties; Ceramic structure; Zirconia;

Bioactive hydrogel scaffolds for controllable vascular differentiation of human embryonic stem cells by Lino S. Ferreira; Sharon Gerecht; Jason Fuller; Hester F. Shieh; Gordana Vunjak-Novakovic; Robert Langer (2706-2717).
We propose a new methodology to enhance the vascular differentiation of human embryonic stem cells (hESCs) by encapsulation in a bioactive hydrogel. hESCs were encapsulated in a dextran-based hydrogel with or without immobilized regulatory factors: a tethered RGD peptide and microencapsulated VEGF165. The fraction of cells expressing vascular endothelial growth factor (VEGF) receptor KDR/Flk-1, a vascular marker, increased up to 20-fold, as compared to spontaneously differentiated embryoid bodies (EBs). The percentage of encapsulated cells in hydrogels with regulatory factors expressing ectodermal markers including nestin or endodermal markers including α-fetoprotein decreased 2- or 3-fold, respectively, as compared to EBs. When the cells were removed from these networks and cultured in media conditions conducive for further vascular differentiation, the number of vascular cells was higher than the number obtained through EBs, using the same media conditions. Functionalized dextran-based hydrogels could thus enable derivation of vascular cells in large quantities, particularly endothelial cells, for potential application in tissue engineering and regenerative medicine.
Keywords: Stem cell; Hydrogel; Cell encapsulation; Dextran; Scaffold; Endothelial cells;

Analysis of progenitor cell–scaffold combinations by in vivo non-invasive photonic imaging by Irene Román; Marta Vilalta; Julio Rodriguez; Annette M. Matthies; Samer Srouji; Erella Livne; Jeffrey A. Hubbell; Nuria Rubio; Jerónimo Blanco (2718-2728).
Recent developments in stem cell research have promoted a flourishing of new biomaterials and scaffolds for tissue repair. However, there is a scarcity of procedures to monitor the performance of scaffold-stem cell combinations implanted in live animals, avoiding the inherent artefacts associated with in vitro assay conditions. We report the implementation of a procedure based on the use of the luciferase gene as a cell proliferation tracer to monitor, by in vivo non-invasive imaging, the performance of stem cell–biomaterial combinations used for tissue regeneration. In a model system using immunodepressed mice we show preference of a mouse embryonic mesenchymal cell line (C3H/10T1/2) for specific implantation sites and biomaterials during a prolonged in vivo growth period (3 months). Moreover, we analyzed the safety of implanted cells using a sensitive luminometric procedure and showed that the implanted cells did not spread to other organs. Our results demonstrate the utility of this simple and resource-saving procedure in the development and screening of biomaterials for tissue engineering.
Keywords: In vivo imaging; Cell proliferation; Scaffold; Luciferase; Enhanced green fluorescent protein (EGFP);

The three-dimensional (3D)-engineered tissues composed of only cells and extracellular matrices (ECM) were constructed by the hydrogel template approach. The disulfide-crosslinked poly(γ-glutamic acid) hydrogels were prepared as a template hydrogel. These template hydrogels were easily decomposed under physiological conditions using reductants such as cysteine, glutathione and dithiothreitol by cleavage of disulfide crosslinkage to thiol groups. The decomposed polymers are soluble in cell culture medium. The cleaving of disulfide bond was determined by UV–vis and FT-IR spectroscopies. We successfully prepared the 3D-engineered tissues (thickness/diameter, 2 mm/1 cm) composed of mouse L929 fibroblast cells and ECM by the decomposition of only the template hydrogel with cysteine after 10 days 3D-cell culture on/in the template hydrogel. The size and thickness of the 3D-engineered tissues was completely transferred from the template hydrogel. The cultured L929 cells viability in the obtained engineered tissues was confirmed by a culture test, WST-1 method and LIVE/DEAD staining assay. The engineered tissue was self-standing and highly dense composite of the cultured cells and collagen produced by the cells. This hydrogel template approach may be useful as a new class of soft-tissue engineering technology to substitute a synthetic polymer scaffold to the ECM scaffold produced from the cultured cells.
Keywords: Cell adhesion; Biodegradation; Hydrogel; Polyamino acid; Scaffold;

Fabrication of cell microintegrated blood vessel constructs through electrohydrodynamic atomization by John J. Stankus; Lorenzo Soletti; Kazuro Fujimoto; Yi Hong; David A. Vorp; William R. Wagner (2738-2746).
Biodegradable synthetic matrices that resemble the size scale, architecture and mechanical properties of the native extracellular matrix (ECM) can be fabricated through electrospinning. Tubular conduits may also be fabricated with properties appropriate for vascular tissue engineering. Achieving substantial cellular infiltration within the electrospun matrix in vitro remains time consuming and challenging. This difficulty was overcome by electrospraying smooth muscle cells (SMCs) concurrently with electrospinning of a biodegradable, elastomeric poly(ester urethane) urea (PEUU) small-diameter conduit. Constructs were cultured statically or in spinner flasks. Hematoxylin and eosin (H&E) staining demonstrated qualitatively uniform SMCs integration radially and circumferentially within the conduit after initial static culture. In comparison with static culture, samples cultured in spinner flasks indicated 2.4 times more viable cells present from MTT and significantly larger numbers of SMCs spread within the electrospun fiber networks by H&E image analysis. Conduits were strong and flexible with mechanical behaviors that mimicked those of native arteries, including static compliance of 1.6±0.5×10−3  mmHg−1, dynamic compliance of 8.7±1.8×10−4  mmHg−1, burst strengths of 1750±220 mmHg, and suture retention. This method to rapidly and efficiently integrate cells into a strong, compliant biodegradable tubular matrix represents a significant achievement as a tissue engineering approach for blood vessel replacement.
Keywords: Blood vessel; Electrospray; Electrospinning; Elastomer; Polyurethane; Smooth muscle cell;

Effects of trabecular calcium phosphate scaffolds on stress signaling in osteoblast precursor cells by Mark R. Appleford; Sunho Oh; Judith A. Cole; David L. Carnes; Myunghyun Lee; Joel D. Bumgardner; Warren O. Haggard; Joo L. Ong (2747-2753).
The objective of this research was to investigate stress-signaling patterns in response to two-dimensional (2-D) and three-dimensional (3-D) calcium phosphate (CP) materials using human embryonic palatal mesenchyme cells (HEPM, CRL-1486, ATCC, Manassas, VA), an osteoblast precursor cell line. Control discs and scaffolds were fabricated from hydroxyapatite and β tri-CP ceramics. Phospho-specific antibody cell-based ELISA technique was utilized on members of the mitogen-activated protein kinase cascade including; the extracellular signal-regulated kinases (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and the anti-apoptosis mediator protein kinase B (AKT). Quantification of these signals was evaluated during the early attachment phase of osteoblast precursor cells. In this study, it was observed that 3-D CP scaffolds significantly activated the stress mediators p38 and JNK but not ERK1/2. This signal trend was matched with an up-regulation in AKT, suggesting the ability of cells to manage high stress signals in response to 3-D CP architecture and that 3-D CP scaffolds are necessary for studies simulating a natural trabecular bone organization. The absence of these signals in 2-D CP surfaces indicated the importance of local architecture conditions on cell stress response. It was concluded from this study that osteoblast precursor cells cultured in 3-D CP scaffolds experience greater stress-signaling patterns when compared to 2-D CP surfaces.
Keywords: Scaffolds; Cell signaling; Calcium phosphates; Histomorphometry;

Protein complexed with chondroitin sulfate in poly(lactide-co-glycolide) microspheres by Eun Seong Lee; Keun-Hong Park; Dongmin Kang; In Suh Park; Hyo Young Min; Don Haeng Lee; Sungwon Kim; Jong Ho Kim; Kun Na (2754-2762).
Chondroitin sulfate (CsA) is an acidic mucopolysaccharide, which is able to form ionic complexes with positively charged proteins. In this study, a protein–CsA complex was constructed to nano-sized particles. Zeta potential measurements revealed that a CsA-to-protein fraction of greater than 0.1 results in a neutralization of the positive charge on lysozyme (Lys). Based on this preliminary study, we have prepared poly(lactide-co-glycolide) (PLGA) microspheres harboring Lys/CsA complexes via the multi-emulsion method. Protein stability in the PLGA microspheres was preserved during both microsphere preparation and protein release. The profiles of Lys release from the PLGA microspheres evidenced nearly zero-order kinetics, depending on the quantity of CsA. An in vivo fluorescent image of experimental mouse tissue showed that the PLGA microspheres with the Lys/CsA complex had released the entirety of their Lys without no residual amount after 23 days, but microspheres without the complex harbored a great deal of residual Lys, which is attributable to its degradation by acidic PLGA degradates. The tissue reaction evidenced by the PLGA microspheres stabilized with CsA showed minimal foreign body reaction and little configuration of immune cells including neutrophils and macrophages, but the reactions of the PLGA microspheres without CsA were characterized by a relatively elevated inflammation. These results show that CsA is a viable candidate for long-acting micro-particular protein delivery.
Keywords: Chondroitin sulfate; Ionic complex; Protein stabilization; Poly(lactide-co-glycolide) microsphere;

In this study, a heparin-conjugated poly(l-lactic-co-glycolic acid) (HP-PLGA) scaffold was developed for the sustained delivery of bone morphogenetic protein-2 (BMP-2), and then used to address the hypothesis that BMP-2 delivered from this scaffold could enhance ectopic bone formation. We found the amount of heparin conjugated to the PLGA scaffolds could be increased up to 3.2-fold by using scaffolds made from star-shaped PLGA, as compared to scaffolds made from linear PLGA, and that the release of BMP-2 from the HP-PLGA scaffold was sustained for at least 14 days in vitro. The BMP-2 released from the HP-PLGA scaffold stimulated an increase in alkaline phosphatase (ALP) activity of osteoblasts for 14 days in vitro, suggesting that the HP-PLGA scaffold delivery system releases BMP-2 in a bioactive form for a prolonged period. By contrast, BMP-2 release from unmodified (no heparin) PLGA scaffolds induced a transient increase in ALP activity for the first 3 days and a decrease thereafter. In vivo bone formation studies showed the BMP-2-loaded HP-PLGA scaffolds induced bone formation to a much greater extent than did either BMP-2-loaded unmodified PLGA scaffolds or unloaded (no BMP-2) HP-PLGA scaffolds, with 9-fold greater bone formation area and 4-fold greater calcium content in the BMP-2-loaded HP-PLGA scaffold group compared to the BMP-2-loaded unmodified PLGA scaffold group. Collectively, these results demonstrate that the HP-PLGA delivery system is capable of potentiating the osteogenic efficacy of BMP-2, and underscore its importance as a possible bone regeneration strategy.
Keywords: Bone morphogenetic protein; Bone regeneration; Poly(l-lactide-co-glycolide); Scaffold; Sustained delivery;

The effect on bone regeneration of a liposomal vector to deliver BMP-2 gene to bone grafts in peri-implant bone defects by J. Park; R. Lutz; E. Felszeghy; J. Wiltfang; E. Nkenke; F.W. Neukam; K.A. Schlegel (2772-2782).
Successful bone–implant osseointegration in large peri-implant bone defects is often difficult, even through autologous bone grafting. Recently, cell-mediated regional gene therapy was introduced to deliver potent morphogens or growth factors in regenerative medicine. We applied liposomal vectors carrying bone morphogenetic protein (BMP)-2 cDNA directly into freshly created peri-implant bone defects on pig calvariae, with or without autologous bone graft. The BMP-2 gene was efficiently introduced into immigrating cells and trabecular cells lining the marginal bone surrounding the bony defect. After 1 week, abundant BMP-2 protein was detected throughout the peri-implant bone defect by immunohistochemistry. At 4 weeks, BMP-producing cells were still present in the defect and peri-implant area, which significantly enhanced new bone formation, compared with the control groups. Interestingly within a week of BMP-2 gene delivery with bone grafts, most osteoblastic cells lining the grafted bone chips also produced BMP-2. Particulated bone was immediately reorganized into newly formed trabecular bone. Grafted bone without BMP-2 gene delivery was still scattered and new bone matrix formation was not detected until 4 weeks after bone grafting. In conclusion, direct application of the BMP-2 gene using a liposomal vector enhanced bone regeneration in a bony defect and gene delivery combined with bone graft could induce a rapid osseointegration of the bone–implant interface at earlier stage.
Keywords: Bone morphogenetic protein (BMP); Gene transfer; Liposome; Bone graft; Dental implant; De novo bone formation;

Nanostructured magnetizable materials that switch cells between life and death by Thomas R. Polte; Mengyan Shen; John Karavitis; Martin Montoya; Jay Pendse; Shannon Xia; Eric Mazur; Donald E. Ingber (2783-2790).
Development of biochips containing living cells for biodetection, drug screening and tissue engineering applications is limited by a lack of reconfigurable material interfaces and actuators. Here we describe a new class of nanostructured magnetizable materials created with a femtosecond laser surface etching technique that function as multiplexed magnetic field gradient concentrators. When combined with magnetic microbeads coated with cell adhesion ligands, these materials form microarrays of ‘virtual’ adhesive islands that can support cell attachment, resist cell traction forces and maintain cell viability. A cell death (apoptosis) response can then be actuated on command by removing the applied magnetic field, thereby causing cell retraction, rounding and detachment. This simple technology may be used to create reconfigurable interfaces that allow users to selectively discard contaminated or exhausted cellular sensor elements, and to replace them with new living cellular components for continued operation in future biomedical microdevices and biodetectors.
Keywords: Magnetic particles; Magnetic gradient concentrator; Culture substrate; Apoptosis; Mechanical force; Cell shape;