Biomaterials (v.28, #26)

Cellular lifespan and regenerative medicine by Thomas Petersen; Laura Niklason (3751-3756).
Tissue engineering is a promising approach to aid in the treatment of a wide range of clinical disorders by developing replacement tissues for damaged or diseased organs. Such approaches, however, will require large and functional cell populations in order to produce a tissue that can replicate in vivo function. Most adult cells have limited replicative potential that limits their use in tissue engineering applications. Thus, cell populations with expanded lifespan or increased replicative potential are of interest. Stem cell-derived populations may allow the creation of large cell populations that have increased replicative potential over adult differentiated cells. In addition, ectopic human telomerase reverse transcriptase expression and induced B-cell lymphoma 2 (bcl-2) expression can allow adult cells to proliferate more extensively than unaltered cells. However, concerns for malignant transformation exist with telomerase and bcl-2 approaches. The current states of research in these areas are reviewed as they relate to tissue engineering and the cellular lifespan.
Keywords: Telomerase; bcl-2; ALT; Cellular lifespan;

Systematic review of the chemical composition of contemporary dental adhesives by Kirsten L. Van Landuyt; Johan Snauwaert; Jan De Munck; Marleen Peumans; Yasuhiro Yoshida; André Poitevin; Eduardo Coutinho; Kazuomi Suzuki; Paul Lambrechts; Bart Van Meerbeek (3757-3785).
Dental adhesives are designed to bond composite resins to enamel and dentin. Their chemical formulation determines to a large extent their adhesive performance in clinic. Irrespective of the number of bottles, an adhesive system typically contains resin monomers, curing initiators, inhibitors or stabilizers, solvents and sometimes inorganic filler. Each one of these components has a specific function.The aim of this article is to systematically review the ingredients commonly used in current dental adhesives as well as the properties of these ingredients. This paper includes an extensive table with the chemical formulation of contemporary dental adhesives.
Keywords: Dental adhesive; Chemical composition; Resin; Initiator; Inhibitor; Filler;

Strong, macroporous, and in situ-setting calcium phosphate cement-layered structures by Hockin H.K. Xu; Elena F. Burguera; Lisa E. Carey (3786-3796).
Calcium phosphate cement (CPC) is highly promising for clinical uses due to its in situ-setting ability, excellent osteoconductivity and bone-replacement capability. However, the low strength limits its use to non-load-bearing applications. The objectives of this study were to develop a layered CPC structure by combining a macroporous CPC layer with a strong CPC layer, and to investigate the effects of porosity and layer thickness ratios. The rationale was for the macroporous layer to accept tissue ingrowth, while the fiber-reinforced strong layer would provide the needed early-strength. A biopolymer chitosan was incorporated to strengthen both layers. Flexural strength, S (mean±sd; n=6) of CPC-scaffold decreased from (9.7±1.2) to (1.8±0.3) MPa (p<0.05), when the porosity increased from 44.6% to 66.2%. However, with a strong-layer reinforcement, S increased to (25.2±6.7) and (10.0±1.4) MPa, respectively, at these two porosities. These strengths matched/exceeded the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. Relationships were established between S and the ratio of strong layer thickness/specimen thickness, a/h:S=(17.6 a/h+3.2) MPa. The scaffold contained macropores with a macropore length (mean±sd; n=147) of (183±73) μm, suitable for cell infiltration and tissue ingrowth. Nano-sized hydroxyapatite crystals were observed to form the scaffold matrix of CPC with chitosan. In summary, a layered CPC implant, combining a macroporous CPC with a strong CPC, was developed. Mechanical strength and macroporosity are conflicting requirements. However, the novel functionally graded CPC enabled a relatively high strength and macroporosity to be simultaneously achieved. Such an in situ-hardening nano-apatite may be useful in moderate stress-bearing applications, with macroporosity to enhance tissue ingrowth and implant resorption.
Keywords: Calcium phosphate cement; Hydroxyapatite; Strength; Macroporosity; Layered structure; Bone repair;

Magnetic control of vascular network formation with magnetically labeled endothelial progenitor cells by C. Wilhelm; L. Bal; P. Smirnov; I. Galy-Fauroux; O. Clément; F. Gazeau; J. Emmerich (3797-3806).
We describe the applications of new cellular magnetic labeling method to endothelial progenitor cells (EPC), which have therapeutic potential for revascularization. Via their negative surface charges, anionic magnetic nanoparticles adsorb non-specifically to the EPC plasma membrane, thereby triggering efficient spontaneous endocytosis. The label is non-toxic and does not affect the cells’ proliferative capacity. The expression of major membrane proteins involved in neovascularisation is preserved. Labeled cells continue to differentiate in vitro and to form tubular structures in Matrigel (an in vitro model of neovascularization). This process was followed in situ by using high-resolution MRI. Finally, we show that magnetic forces can be used to move magnetically labeled EPC in vitro and to modify their organization in Matrigel both in vitro an in vivo. Magnetic cell targeting opens up new possibilities for vascular tissue engineering and for delivering localized cell-based therapies.
Keywords: Endothelial progenitor cells; Vascular networks; Iron oxide nanoparticles; Biocompatibility; Magnetic resonance imaging (MRI); Magnetic targeting;

Immunological responses in mice to full-thickness corneal grafts engineered from porcine collagen by Lei Liu; Lucia Kuffová; May Griffith; Zexu Dang; Elizabeth Muckersie; Yuwen Liu; Christopher R. McLaughlin; John V. Forrester (3807-3814).
Tissue-engineered (TE) corneas were fabricated from porcine collagen cross-linked with 1-ethyl-3-(3-dimethyl aminoproplyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and were transplanted into BALB/c mice orthotopically using a full-thickness penetrating keratoplasty (PKP) procedure. The biocompatibility was evaluated by assessing both local and systemic immune responses. Myeloid cells including granulocytes and macrophages were the main infiltrating cells in recipient cornea and in retro-TE corneal membrane which developed 7–10 days post surgery. Sodium citrate was found to be effective in reducing fibrin accumulation in anterior chamber post grafting at early time points, but it did not prevent formation of the retro-TE corneal membrane. No significant T cell activation was observed in the submandibular draining lymph nodes (SMDLN) by flow cytometry. Anti-porcine type I collagen IgG antibodies were detected in the serum of grafted mice from 2 weeks post grafting and the concentration of antibodies increased with time. Overall, porcine collagen-EDC/NHS TE corneas were tolerated well in murine recipients, causing mainly a self-limiting local innate immune response and a low-grade humoral response with little evidence of sustained T cell activation. Retro-TE corneal membrane formation was the main complication and barrier to clarity.
Keywords: Porcine collagen; Tissue-engineered cornea; Keratoplasty; Immunology;

To engineer implantable liver tissues, we designed a novel scaffold with a three-dimensional (3D) branching and joining flow-channel network comprising multiple tetrahedral units (4-mm edge length). For the fabrication of this network, biodegradable polycaprolactone (PCL) and 80% (w/w) NaCl salt particles serving as porogen were thoroughly mixed and applied in a selective laser sintering (SLS) process, a technique adapted to rapid prototyping. We thus obtained a scaffold that had high (89%) porosity with a pore size of 100–200 μm and 3D flow channels. To evaluate its biocompatibility, human hepatoma Hep G2 cells were seeded into the scaffold using avidin–biotin (AB) binding and cultured in a perfusion system for 9 days. The results demonstrated that such 3D flow channels are essential to the cells’ growth and function. In addition, the AB binding-based seeding remarkably improved the overall performance of the cell-loaded scaffolds. The fabrication of a much finer scaffold, having a 500 cm3 scale, based on the same design and the use of human hepatocyte progenitors, may, in the near future, lead to the development of an implantable liver tissue equivalent for use in humans.
Keywords: Avidin–biotin binding system; Polycaprolactone; Perfusion culture; Selective laser sintering; Implantable liver;

The role of ERK signaling in protein hydrogel remodeling by vascular smooth muscle cells by Helen Hong; Caitlyn M. McCullough; Jan P. Stegemann (3824-3833).
Collagen type I and fibrin hydrogels have been used for cell-based therapies and tissue engineering. These matrices can be broken down and remodeled by cells, but the effects that these proteins have on cell function are not completely understood. We examined activation of the extracellular signal-regulated kinase (ERK) signaling pathway by vascular smooth muscle cells (VSMC) in response to 2D and 3D matrices of type I collagen, fibrin, or a 1:1 composite mixture of these proteins. After 3 days of culture, ERK phosphorylation, osteopontin secretion, and MMP-2 activation were all markedly increased in 3D matrices, compared with 2D substrates. A strong positive correlation existed between these protein markers of the synthetic phenotype and phosphorylated ERK levels, and this relationship persisted across matrix geometries and compositions. Cell proliferation in 3D matrices was inversely correlated to ERK activation, while on 2D substrates a modest positive correlation was observed. Pharmacologic inhibition of ERK signaling confirmed that this pathway was involved in the observed phenotype shifts. This study suggests that contextual activation of the ERK pathway results in different effects on cell phenotype, depending on the geometry and composition of the ECM. These findings add to our understanding of cell function and remodeling in protein-based hydrogel biomaterials.
Keywords: Collagen; Fibrin; Smooth muscle cells; ERK; Osteopontin; MMP-2;

Adipose tissue engineering with naturally derived scaffolds and adipose-derived stem cells by Lauren Flynn; Glenn D. Prestwich; John L. Semple; Kimberly A. Woodhouse (3834-3842).
A tissue-engineered adipose substitute would have numerous applications in plastic and reconstructive surgery. This work involves the characterization of the in vitro cellular response of primary human adipose-derived stem cells (ASC) to three dimensional, naturally derived scaffolds. To establish a more thorough understanding of the influence of the scaffold environment on ASC, we have designed several different soft tissue scaffolds composed of decellularized human placenta and crosslinked hyaluronan (XLHA). The cellular organization within the scaffolds was characterized using confocal microscopy. Adipogenic differentiation was induced and the ASC response was characterized in terms of glycerol-3-phosphate dehydrogenase (GPDH) activity and intracellular lipid accumulation. The results indicate that the scaffold environment impacts the ASC response and that the adipogenic differentiation of the ASC was augmented in the non-adhesive XLHA gels.
Keywords: Adipose tissue engineering; Scaffold; Extracellular matrix; Hyaluronan; Stem cells;

Kinetic analysis of nanoparticulate polyelectrolyte complex interactions with endothelial cells by Sean M. Hartig; Rachel R. Greene; Gianluca Carlesso; James N. Higginbotham; Wasif N. Khan; Ales Prokop; Jeffrey M. Davidson (3843-3855).
A non-toxic, nanoparticulate polyelectrolyte complex (PEC) drug delivery system was formulated to maintain suitable physicochemical properties at physiological pH. Toxicity, binding, and internalization were evaluated in relevant microvascular endothelial cells. PEC were non-toxic, as indicated by cell proliferation studies and propidium iodide staining. Inhibitor studies revealed that PEC were bound, in part, via heparan sulfate proteoglycans and internalized through macropinocytosis. A novel, flow cytometric, Scatchard protocol was established and showed that PEC, in the absence of surface modification, bind cells non-specifically with positive cooperativity, as seen by graphical transformations.
Keywords: Polyelectrolyte complexes; Flow cytometry; Scatchard plots; Nanoparticles; Drug delivery;

Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering by Martin Ehrbar; Simone C. Rizzi; Ruslan Hlushchuk; Valentin Djonov; Andreas H. Zisch; Jeffrey A. Hubbell; Franz E. Weber; Matthias P. Lutolf (3856-3866).
The molecular engineering of cell-instructive artificial extracellular matrices is a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration. This work reports on a novel method to produce such smart biomaterials by recapitulating the crosslinking chemistry and the biomolecular characteristics of the biopolymer fibrin in a synthetic analog. We use activated coagulation transglutaminase factor XIIIa for site-specific coupling of cell adhesion ligands and engineered growth factor proteins to multiarm poly(ethylene glycol) macromers that simultaneously form proteolytically sensitive hydrogel networks in the same enzyme-catalyzed reaction. Growth factor proteins are quantitatively incorporated and released upon cell-derived proteolytic degradation of the gels. Primary stromal cells can invade and proteolytically remodel these networks both in an in vitro and in vivo setting. The synthetic ease and potential to engineer their physicochemical and bioactive characteristics makes these hybrid networks true alternatives for fibrin as provisional drug delivery platforms in tissue engineering.
Keywords: ECM; Fibrin; Hydrogel; Biomimetic material; Polyethylene glycol; Growth factor;

Transition behavior in fatigue of human dentin: Structure and anisotropy by D. Arola; J. Reid; M.E. Cox; D. Bajaj; N. Sundaram; E. Romberg (3867-3875).
The influence of tubule orientation on the transition from fatigue to fatigue crack growth in human dentin was examined. Compact tension (CT) and rectangular beam specimens were prepared from the coronal dentin of molars with three unique tubule orientations (i.e., 0°, 45° and 90°). The CT specimens (N=25) were used to characterize fatigue crack initiation and steady-state cyclic extension, whereas the rectangular beams (N=132) were subjected to 4-pt flexure and used in quantifying the stress-life fatigue response. The transition behavior was analyzed using both the Kitagawa–Takahashi and El Haddad approaches. Results showed that both the fatigue crack growth and stress-life responses were dependent on the tubule orientation. The average Paris Law exponent for crack growth perpendicular (90°) to the tubules (m=13.3±1.1) was significantly greater (p<0.05) than that for crack growth oblique (45°) to the tubules (m=11.5±1.87). Similarly, the fatigue strength of dentin with 90° tubule orientation was significantly lower (p<0.05) than that for the other two orientations, regardless of the range of cyclic stress. The apparent endurance strengths of specimens with 0° (44 MPa) and 45° (53 MPa) orientations were nearly twice that of the 90° (24 MPa) orientation. Based on these results, human dentin exhibits the largest degree of anisotropy within the stress-life regime and the transition from fatigue to fatigue crack growth occurs under the lowest cyclic stress range when the tubules are aligned with the cyclic normal stress (90° orientation).
Keywords: Anisotropy; Dentin; Fatigue; Fracture; Tubules;

Collagen, fibrin and albumin are popular proteins for making biological scaffolds for tissue engineering because of their biocompatibility, biodegradability, and availability. A major drawback of biological protein-based biomaterials is the limited control over their physical and biodegradation properties. Our laboratory has been developing new protein-based biomaterials with tunable properties without the use of cytotoxic protein cross-linking techniques. We describe the formation and assembly of photopolymerizable biomimetic hydrogel scaffolds made from protein–polymer conjugates of poly(ethylene glycol) (PEG) and collagen, fibrin or albumin. The conjugation of PEG to these proteins (PEGylation) was verified by SDS-PAGE and the polymerization reaction into a hydrogel network was confirmed by shear rheometry. The differences in rheology and swelling characteristics of the three hydrogel materials underscore the importance of the molecular relationship between the PEG and the protein constituent in this protein–polymer arrangement. The biofunctionality of the PEGylated collagen and fibrinogen hydrogels sustained both cell adhesion and proteolytic degradation that enabled 3-D cell spreading and migration within the hydrogel network. PEG–albumin hydrogels exhibited poor cell spreading and migration by virtue of the fact that the albumin backbone lacks any known cell adhesion sites. Despite differences in the biological and structural composition of the PEGylated fibrinogen and collagen hydrogels, the rate of cellular migration within each material was not significantly different.
Keywords: Albumin; Fibrin; Collagen; Poly(ethylene glycol); Scaffold; Smooth muscle cells;