Biomaterials (v.33, #13)

The effect of oxidation on the degradation of photocrosslinkable alginate hydrogels by Oju Jeon; Daniel S. Alt; Shaoly M. Ahmed; Eben Alsberg (3503-3514).
Recently, we reported on a new photocrosslinkable alginate-based hydrogel, which has controllable physical and cell adhesive properties. The macromer solution containing cells can be injected in a minimally invasive manner into a defect site and crosslinked while maintaining high cell viability. The number of hydrolyzable ester bonds in the formed crosslinks may be controlled by altering the degree of methacrylation on the alginate polymer backbone. However, the degradation rate of the hydrogels has been found to be slower in vivo than in vitro. The purpose of this study was to develop photocrosslinked alginate hydrogels with an increased range of biodegradation rates for more rapid in vivo biodegradation in regenerative medicine and bioactive factor delivery applications. Therefore, we oxidized alginate prior to methacrylation to change the uronate residue conformations to an open-chain adduct, which makes it more vulnerable to hydrolysis. Here, we demonstrate that the swelling behavior, degradation profiles, and storage moduli of photocrosslinked hydrogels formed from oxidized, methacrylated alginates (OMAs) are tunable by varying the degree of alginate oxidation. The OMA macromers and photocrosslinked OMA hydrogels exhibited cytocompatibility when cultured with human bone marrow-derived mesenchymal stem cells (hBMMSCs). In addition, hMSCs derived from bone marrow or adipose tissue photoencapsulated within these hydrogels remained viable, and their proliferation rate was a function of alginate oxidation level and initial hydrogel weight fraction. Oxidation permits a wider range of photocrosslinked OMA hydrogels physical properties, which may enhance these therapeutic materials’ utility in tissue engineering and other biomedical applications.
Keywords: Biodegradation; Biomaterials; Mesenchymal stem cells; Tissue engineering;

Regulation of the differentiation of mesenchymal stem cells in vitro and osteogenesis in vivo by microenvironmental modification of titanium alloy surfaces by Yan Hu; Kaiyong Cai; Zhong Luo; Yuan Zhang; Liqi Li; Min Lai; Yanhua Hou; Yuran Huang; Jinghua Li; Xingwei Ding; Bin Zhang; K.L. Paul Sung (3515-3528).
To mimic the extracellular microenvironment of bone, a bioactive multilayered structure of gelatin/chitosan pair, containing bone morphogenetic protein 2(BMP2) and fibronectin (FN), was constructed onto Ti6Al4V surface via a layer-by-layer assembly technique. The successful fabrication of multilayered structure was confirmed by contact angle measurement, field emission scanning electron microscopy (FE-SEM) and confocal laser scanning microscopy (CLSM), respectively. Bioactive BMP2 released in a sustained manner along with the degradation of multilayered structure. MSCs grown onto the multilayer coated TC4 substrates displayed significantly higher (p < 0.01 or p < 0.05) production levels of alkaline phosphatase (ALP), mineralization and genes expressions of runt related transcription factor 2 (Runx2), osterix, osteocalcin (OC), osteopontin (OPN), ALP and collagen type (Col) compared to the controls after culture for 7 days and 21 days, respectively. More importantly, MicroCT analysis and histological observations demonstrated that the multilayer coated Ti6Al4V implants in vivo promoted the bone density and new bone formation around them after implantation for 4 weeks and 12 weeks, respectively. The results indicated that Ti6Al4V coated with biofunctional multilayers was beneficial for osteogenesis and integration of implant/bone. The study therefore presents an alternative to fabricate bio-functionalized Ti6Al4V-based implants for potential application in orthopedics field.
Keywords: Ti6Al4V alloy; BMP2; Layer-by-layer assembly technique; Extracellular microenvironment; Mesenchymal stem cells; Osteogenesis;

A dual-layer silk fibroin scaffold for reconstructing the human corneal limbus by Laura J. Bray; Karina A. George; Dietmar W. Hutmacher; Traian V. Chirila; Damien G. Harkin (3529-3538).
Membranes prepared from Bombyx mori silk fibroin have shown potential as a substrate for human limbal epithelial (L-EC) and stromal cell cultivation. Here we present fibroin as a dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. We have compared the growth and phenotype of L-EC on non-porous versus porous fibroin membranes. Furthermore, we have compared the growth of limbal mesenchymal stromal cells (L-MSC) in either serum-supplemented medium or the MesenCult-XF® culture system within fibroin fibrous mats. The co-culture of L-EC and L-MSC in fibroin dual-layer constructs was also examined. L-EC on porous membranes displayed a squamous monolayer; in contrast, L-EC on non-porous fibroin appeared cuboidal and stratified. Both constructs maintained evidence of corneal phenotype (cytokeratin 3/12) and distribution of ΔNp63+ progenitor cells. L-MSC cultivated within fibroin fibrous mats in serum-supplemented medium contained less than 64% of cells expressing the characteristic MSC phenotype of CD73+CD90+CD105+ after two weeks, compared with over 81% in MesenCult-XF® medium. Dual-layer fibroin scaffolds consisting of L-EC and L-MSC maintained a similar phenotype as on the separate layers. These results support the feasibility of a 3D engineered limbus constructed from B. mori silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration.
Keywords: Silk fibroin; Cornea; Limbus; Epithelium; Mesenchymal stromal cells;

Biologic scaffolds composed of central nervous system extracellular matrix by Peter M. Crapo; Christopher J. Medberry; Janet E. Reing; Stephen Tottey; Yolandi van der Merwe; Kristen E. Jones; Stephen F. Badylak (3539-3547).
Acellular biologic scaffolds are commonly used to facilitate the constructive remodeling of three of the four traditional tissue types: connective, epithelial, and muscle tissues. However, the application of extracellular matrix (ECM) scaffolds to neural tissue has been limited, particularly in the central nervous system (CNS) where intrinsic regenerative potential is low. The ability of decellularized liver, lung, muscle, and other tissues to support tissue-specific cell phenotype and function suggests that CNS-derived biologic scaffolds may help to overcome barriers to mammalian CNS repair. A method was developed to create CNS ECM scaffolds from porcine optic nerve, spinal cord, and brain, with decellularization verified against established criteria. CNS ECM scaffolds retained neurosupportive proteins and growth factors and, when tested with the PC12 cell line in vitro, were cytocompatible and stimulated proliferation, migration, and differentiation. Urinary bladder ECM (a non-CNS ECM scaffold) was also cytocompatible and stimulated PC12 proliferation but inhibited migration rather than acting as a chemoattractant over the same concentration range while inducing greater rates of PC12 differentiation compared to CNS ECM. These results suggest that CNS ECM may provide tissue-specific advantages in CNS regenerative medicine applications and that ECM scaffolds in general may aid functional recovery after CNS injury.
Keywords: Extracellular matrix; Central nervous system; Scaffolds; Decellularization; Regenerative medicine; Tissue engineering;

The impact of adhesion peptides within hydrogels on the phenotype and signaling of normal and cancerous mammary epithelial cells by Michael S. Weiss; Beatriz Peñalver Bernabé; Ariella Shikanov; Dennis A. Bluver; Michael D. Mui; Seungjin Shin; Linda J. Broadbelt; Lonnie D. Shea (3548-3559).
The microenviroment contributes to directing mammary epithelial cell (MEC) development and the progression of breast cancer. Three-dimensional culture models have been used to support formation of structures that display varying degrees of disorganization that parallel the degree of cancer. Synthetic hydrogels were employed to investigate the mechanisms by which specific adhesion signals in the microenvironment directed development. Polyethylene glycol-based hydrogels supported 3D growth of MECs and directed formation of a range of phenotypes that were functions of genotype, and identity and concentration of adhesion peptides RGD and YIGSR. Non-cancerous and cancerous MECs responded differentially to the same adhesion cues and produced variable structural organizations. An analysis of dynamic signaling pathways revealed differential activities of transcription factors within the MAPK and JAK/STAT pathways in response to genotype and adhesion. These results directly implicate adhesion in cancer development and demonstrate that AP1, CREB, STAT1, and STAT3 all contribute to the genotype dependence of cellular response to adhesion peptides. The tools presented in this work could be applied to other systems and connect extracellular cues with intracellular signaling to molecularly dissect tissue development and further biomaterials development.
Keywords: Adhesion; Cell signaling; Hydrogel; Signal transducing mediator;

The Use of Silica Coated MnO Nanoparticles to Control MRI Relaxivity in Response to Specific Physiological Changes by Yi-Cheng Lee; Der-Yow Chen; Stephen J. Dodd; Nadia Bouraoud; Alan P. Koretsky; Kannan M. Krishnan (3560-3567).
MnO nanoparticles have been tested to engineer a delayed increase in MRI T1 relaxivity caused by cellular uptake via endocytosis into acidic compartments. Various coatings on core–shell structured MnO nanoparticles were tested for those that had the lowest T1 relaxivity at pH 7.4, a pH where MnO does not dissolve into Mn2+ ions. The rate of dissolution and release of Mn2+ of the different coated MnO particles as well as changes in T1 relaxivity were measured at pH 5, a pH routinely obtained in the endosomal-lysosomal pathway. Of a number of coatings, silica coated MnO (MnO@SiO2) had the lowest relaxivity at pH 7.4 (0.29 mm −1 sec−1). About one third of the MnO dissolved within 20 min and the T1 relaxivity increased to that of free Mn2+ (6.10 mm −1 sec−1) after three days at pH 5. MRI of MnO@SiO2 particles injected into the rat brain showed time-dependent signal changes consistent with the in vitro rates. Thalamocortical tract-tracing could be observed due to the released Mn2+. Intravenous infusion of MnO@SiO2 particles showed little enhancement in any tissue except gallbladder. The gallbladder enhancement was interpreted to be due to endocytosis by liver cells and excretion of Mn2+ ions into the gallbladder. The MnO@SiO2 core–shell nanoparticles show the best potential for delaying the release of MRI contrast until endocytosis into low pH compartments activate MRI contrast. The delayed enhancement may have benefits for targeting MRI contrast to specific cells and surface receptors that are known to be recycled by endocytosis.
Keywords: Manganese enhanced MRI; MnO nanoparticle; Contrast agent; pH-responsive;

Pharmacokinetics of core-polymerized, boron-conjugated micelles designed for boron neutron capture therapy for cancer by Shogo Sumitani; Motoi Oishi; Tatsuya Yaguchi; Hiroki Murotani; Yukichi Horiguchi; Minoru Suzuki; Koji Ono; Hironobu Yanagie; Yukio Nagasaki (3568-3577).
Core-polymerized and boron-conjugated micelles (PM micelles) were prepared by free radical copolymerization of a PEG-b-PLA block copolymer bearing an acetal group and a methacryloyl group (acetal-PEG-b-PLA-MA), with 1-(4-vinylbenzyl)-closo-carborane (VB-carborane), and the utility of these micelles as a tumor-targeted boron delivery system was investigated for boron neutron capture therapy (BNCT). Non-polymerized micelles (NPM micelles) that incorporated VB-carborane physically showed significant leakage of VB-carborane (ca. 50%) after 12 h incubation with 10% fetal bovine serum (FBS) at 37 °C. On the other hand, no leakage from the PM micelles was observed even after 48 h of incubation. To clarify the pharmacokinetics of the micelles, 125I (radioisotope)-labeled PM and NPM micelles were administered to colon-26 tumor-bearing BALB/c mice. The 125I-labeled PM micelles showed prolonged blood circulation (area under the concentration curve (AUC): 943.4) than the 125I-labeled NPM micelles (AUC: 495.1), whereas tumor accumulation was similar for both types of micelles (AUCPM micelle: 249.6, AUCNPM micelle: 201.1). In contrast, the tumor accumulation of boron species in the PM micelles (AUC: 268.6) was 7-fold higher than the NPM micelles (AUC: 37.1), determined by ICP-AES. Thermal neutron irradiation yielded tumor growth suppression in the tumor-bearing mice treated with the PM micelles without reduction in body weight. On the basis of these data, the PM micelles represent a promising approach to the creation of boron carrier for BNCT.
Keywords: Boron neutron capture therapy; Poly(ethylene glycol)-block-poly(lactic acid) copolymer; Polymeric micelles; Drug delivery systems; Biodistribution; Nanoparticle-assisted BNCT;

Treatment of calcium channel blocker-induced cardiovascular toxicity with drug scavenging liposomes by Vincent Forster; Paola Luciani; Jean-Christophe Leroux (3578-3585).
Calcium channel blocker (CCB) overdose is potentially lethal. Verapamil and diltiazem are particularly prone to acute toxicity due to their dual effect on cardiac and vascular tissues. Unfortunately, conventional decontamination measures are ineffective in accelerating blood clearance and, to date, few efforts have been made to develop antidotes. To address the issue, injectable long-circulating liposomes bearing a transmembrane pH-gradient are proposed as efficient detoxifying agents of CCB poisoning. By scavenging the drug in situ, these circulating nanocarriers can restrict its distribution in tissues and hinder its pharmacological effect. In vitro, we showed that liposomes stability in serum and their ability to sequester CCBs could be finely-tuned by modulating their internal pH, surface charge, and lipid bilayer structure. Subsequently, we verified their efficacy in reversing the cardiovascular effects of verapamil in rats implanted with telemetric pressure/biopotential transmitters. In animals orally intoxicated to verapamil, an intravenous injection of the liposomal antidote rapidly attenuated the reduction in blood pressure. Areas under diastolic, systolic, and mean pressures curves were significantly reduced by up to 60% and the time to hemodynamic recovery was shortened from 19 to only 11 h. These findings confirm the protective effect of pH-gradient liposomes against cardiovascular failure after CBB intoxication, and endorse their potential as efficient, versatile antidotes.
Keywords: Blood pressure; Calcium channel blocker; Liposome; Intoxication; Drug uptake;

Toxicogenomic analysis of a sustained release local anesthetic delivery system by Iris Shichor; Noam Shomron; Michael W. Lawlor; Seul A. Bae; Janet Zoldan; Robert Langer; Daniel S. Kohane (3586-3593).
Concerns over neurotoxicity have impeded the development of sustained release formulations providing prolonged duration local anesthesia (PDLA) from a single injection, for which there is an urgent clinical need. Here, we have used toxicogenomics to investigate whether nerve injury occurred during week-long continuous sciatic nerve blockade by microspheres containing bupivacaine, tetrodotoxin, and dexamethasone (TBD). Animals treated with amitriptyline solution (our positive control for local anesthetic-associated nerve injury) developed irreversible nerve blockade, had severely abnormal nerve histology, and the expression of hundreds of genes was altered in the dorsal root ganglia at 4 and 7 days after injection. In marked contrast, TBD-treated nerves reverted to normal function, were normal histologically and there were changes in the expression of a small number of genes. Toxicogenomic studies have great potential in delineating patterns of gene expression associated with specific patterns of tissue injury (e.g. amitriptyline neurotoxicity), and in identifying related changes in gene expression upon exposure to a drug, biomaterial, or drug delivery system.
Keywords: Toxicogenomics; Drug delivery; Biocompatibility; Prolonged duration local anesthesia; Neurotoxicity; Nerve injury;

Effect of molecular weight of amine end-modified poly(β-amino ester)s on gene delivery efficiency and toxicity by Ahmed A. Eltoukhy; Daniel J. Siegwart; Christopher A. Alabi; Jay S. Rajan; Robert Langer; Daniel G. Anderson (3594-3603).
Amine end-modified poly(β-amino ester)s (PBAEs) have generated interest as efficient, biodegradable polymeric carriers for plasmid DNA (pDNA). For cationic, non-degradable polymers, such as polyethylenimine (PEI), the polymer molecular weight (MW) and molecular weight distribution (MWD) significantly affect transfection activity and cytotoxicity. The effect of MW on DNA transfection activity for PBAEs has been less well studied. We applied two strategies to obtain amine end-modified PBAEs varying in MW. In one approach, we synthesized four amine end-modified PBAEs with each at 15 different monomer molar ratios, and observed that polymers of intermediate length mediated optimal DNA transfection in HeLa cells. Biophysical characterization of these feed ratio variants suggested that optimal performance was related to higher DNA complexation efficiency and smaller nanoparticle size, but not to nanoparticle charge. In a second approach, we used preparative size exclusion chromatography (SEC) to obtain well-defined, monodisperse polymer fractions. We observed that the transfection activities of size-fractionated PBAEs generally increased with MW, a trend that was weakly associated with an increase in DNA binding efficiency. Furthermore, this approach allowed for the isolation of polymer fractions with greater transfection potency than the starting material. For researchers working with gene delivery polymers synthesized by step-growth polymerization, our data highlight the potentially broad utility of preparative SEC to isolate monodisperse polymers with improved properties. Overall, these results help to elucidate the influence of polymer MWD on nucleic acid delivery and provide insight toward the rational design of next-generation materials for gene therapy.
Keywords: Polymer; Molecular weight; DNA; Nanoparticle; Gene therapy; Gene transfer;

Nano-carrier for gene delivery and bioimaging based on carbon dots with PEI-passivation enhanced fluorescence by Changjun Liu; Peng Zhang; Xinyun Zhai; Feng Tian; Wenchen Li; Jianhai Yang; Yuan Liu; Hongbo Wang; Wei Wang; Wenguang Liu (3604-3613).
Polyethylenimine (PEI) functionalized carbon dots (CD-PEI) were fabricated by one-step microwave assisted pyrolysis of glycerol and branched PEI25k mixture where the formation of carbon nanoparticles and the surface passivation were accomplished simultaneously. In this hybrid C-dot, PEI molecule played two key roles in the system − as a nitrogen-rich compound to passivate surface to enhance the fluorescence and as a polyelectrolyte to condense DNA. This CD-PEI was shown to be water soluble and emit stable bright multicolor fluorescence relying on excitation wavelength. The DNA condensation capability and cytotoxicity of CD-PEI could be regulated by pyrolysis time possibly due to the somewhat destruction of PEI during the formation of carbon dots. CD-PEI obtained at an appropriate pyrolysis time exhibited lower toxicity, higher or comparable gene expression of plasmid DNA in COS-7 cells and HepG2 cells relative to control PEI25k. Intriguingly, the CD-PEIs internalized into cells displayed tunable fluorescent emission under varying excitation wavelength, suggesting the potential application of CD-PEI in gene delivery and bioimaging.
Keywords: Carbon dots; Microwave; Gene delivery; Photoluminescence; Bioimaging;