Biomaterials (v.31, #25)
Antibiotic-loaded biomaterials and the risks for the spread of antibiotic resistance following their prophylactic and therapeutic clinical use
by Davide Campoccia; Lucio Montanaro; Pietro Speziale; Carla Renata Arciola (pp. 6363-6377).
Antibiotic-loaded biomaterials are currently part of standard medical procedures for both local treatment and prevention of implant infections. The achievement of local delivery of significant quantities of active drugs directly at the site of infection, bypassing or reducing the risks of systemic effects, represents a strong point in favor of this approach. When the aim is to resolve an existing infection, controlled local release of antibiotics can be properly targeted based on the characteristics of the bacterial isolate obtained from the infection site. Under these circumstances the choice of the antibiotic is rational and this local administration route offers new unprecedented possibilities for an efficacious in situ treatment, avoiding the adverse effects of conventional systemic chemotherapies. Although the idea of self sterilizing implants is appealing, controversial is the use of antibiotic-loaded biomaterials in uninfected tissues to prevent implant infections. Systems designed for prolonged release of prophylactic inhibitory or subinhibitory amounts of antibiotics, in absence of strict harmonized guidelines, raise concerns for their still weakly proved efficacy but, even more, for their possible contribution to enhancing biofilm formation and selecting resistant mutants. This consideration holds especially true if the antibiotic-loaded represents the first-line treatment against multiresistant strains.
Keywords: Antibiotic-loaded biomaterials; Implant infections; Biofilm; Antibiotic resistance; Gene transfer
On the mechanical properties of hierarchically structured biological materials
by Sabine Bechtle; Siang Fung Ang; Gerold A. Schneider (pp. 6378-6385).
Many biological materials are hierarchically structured which means that they are designed from the nano- to the macro-scale in a sometimes self-similar way. There are lots of papers published including very detailed descriptions of these structures at all length scales – however, investigations of mechanical properties are most often focused on either nano-indentation or bulk mechanical testing characterizing properties at the smallest or largest size scale. Interestingly, there are hardly any investigations that systematically interconnect mechanical properties of different length scales. Nevertheless there are often conclusions drawn like the one that “biological materials exhibit their excellent mechanical properties due to their hierarchical structuring”. Thus, we think there is a gap and discrepancy between the detection and description of biological structures and the correlated determination and interpretation of their mechanical properties. Hence, in this paper we order hierarchically structured biological materials with high mineral content according to their hierarchical levels and attribute measured mechanical properties to them. This offers the possibility to gain insight into the mechanical properties on different hierarchical levels even though the entire biological materials were tested. On the other hand we use data of one material, namely enamel, where mechanical properties were measured on every length scale. This kind of data analysis allows to show how a theoretical model developed by Huajian Gao and co-workers can be used to get closer insights into experimental data of hierarchically structured materials.
Keywords: Hierarchical structures; Mechanical properties; Biomaterials; Biological materials; Enamel
The use of thermal printing to control the properties of calcium phosphate deposits
by Saeed Saber-Samandari; Kārlis A. Gross (pp. 6386-6393).
The objective of this work was to characterize the deposits of calcium phosphate produced by thermal printing in terms of structure, topography and mechanical properties. Hydroxyapatite was molten and directed to (a) a titanium target in relative motion and (b) stationary titanium substrates preheated to 100°C and 350°C. Scanning electron microscopy showed round-like deposits, but high resolution profilometry measured the profile. Micro-Raman spectroscopy and X-ray diffraction characterized the surface for structure, while nanoindentation revealed the hardness and elastic modulus. A symmetrical hemispherical deposit was formed on a surface in slow relative motion, but an off-centre shape formed at a higher relative speed. Deposits on preheated surfaces (100°C and 350°C) were identified as amorphous calcium phosphate. Nanoindentation revealed no significant difference in hardness between the amorphous deposits (4.0–4.4±0.3 GPa), but the elastic modulus increased from 65±4 GPa (annealed calcium phosphate reference) to 88±3 GPa (100°C surface) and then to 98±3 GPa (350°C substrate). The large change in elastic modulus is thought to arise from the dehydroxylation during thermal printing. Production of functional materials through crystallization is discussed to extend the range of possible microstructures. The characterization and testing approach is useful for hemispherical deposits produced by printing, coatings (laser ablation, thermal spraying, simulated body fluid) and melt extrusion elements in scaffolds.
Keywords: Printing; Mechanical properties; Nanoindentation; Amorphous calcium phosphate; Hydroxyapatite; Biomedical implants
CD47-dependent molecular mechanisms of blood outgrowth endothelial cell attachment on cholesterol-modified polyurethane
by Masako Ueda; Ivan S. Alferiev; Stacey B. Simons; Robert P. Hebbel; Robert J. Levy; Stanley J. Stachelek (pp. 6394-6399).
We previously showed that blood outgrowth endothelial cells (BOECs) had a high affinity for polyurethane (PU) covalently configured with cholesterol residues (PU-Chol). However, the molecular mechanisms responsible for this enhanced affinity were not determined. CD47, a multifunctional transmembrane glycoprotein involved in cellular attachment, can form a cholesterol-dependent complex with integrin αvβ3 and heterotrimeric G proteins. We tested herein the hypothesis that CD47, and the other components of the multi-molecular complex, enhance the attachment of BOECs to PU-Chol. Immunoprecipitation studies, of human and ovine BOECs, demonstrated that CD47 associates with integrin αv and integrin β3 as well as Gαi−2 protein. The three-fold increase in BOEC attachment to PU-Chol, compared to unmodified PU, was reversed with the addition of blocking antibodies specific for CD47 and integrin αv and integrin β3. Similar results were observed with the addition of methyl-beta-cyclodextrin (MβCD), a known disruptor of the CD47 complex as well as of the membrane cholesterol content, to seeded BOEC or PU-Chol films. Reducing CD47 expression, via lentivirus transduced shRNA, decreased BOEC binding to PU-Chol by 50% compared to control groups. These data are the first demonstration of a role for the CD47 cholesterol-dependent signaling complex in BOEC attachment onto synthetic surfaces.
Keywords: Cell adhesion; Endothelialisation; RGD peptide
Synergistic induction of bone formation by hOP-1, hTGF-β3 and inhibition by zoledronate in macroporous coral-derived hydroxyapatites
by Ugo Ripamonti; Roland M. Klar; Louise F. Renton; Carlo Ferretti (pp. 6400-6410).
Thirty coral-derived calcium carbonate-based macroporous constructs with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) were implanted in the rectus abdominis of three adult non-human primate Papio ursinus to investigate the intrinsic induction of bone formation. Macroporous constructs with 125μg human recombinant osteogenic protein-1 (hOP-1) or 125μg human recombinant transforming growth factor-β3 (hTGF-β3) were also implanted. The potential synergistic interaction between morphogens was tested by implanting binary applications of hOP-1 and hTGF-β3 5:1 by weight, respectively. To evaluate the role of osteoclastic activity on the implanted macroporous surfaces, coral-derived constructs were pre-loaded with 0.24mg of bisphosphonate zoledronate (Zometa®). To correlate the morphology of tissue induction with osteogenic gene expression and activation, harvested specimens on day 90 were analyzed for changes in OP-1 andTGF-β 3 mRNA synthesis by quantitative real-time polymerase chain reaction (qRT-PCR). The induction of bone formation in 7% HA/CC solo correlated with OP-1 expression. Massive bone induction formed by binary applications of the recombinant morphogens. Single applications of hOP-1 and hTGF-β3 also resulted in substantial bone formation, not comparable however to synergistic binary applications. Zoledronate-treated macroporous constructs showed limited bone formation and in two specimens bone formation was altogether absent; qRT-PCR showed a prominent reduction of OP-1 gene expression whilstTGF-β 3 expression was far greater than OP-1. The lack of bone formation by zoledronate-treated specimens indicates that osteoclastic activity on the implanted coral-derived constructs is critical for the spontaneous induction of bone formation. Indirectly, zoledronate-treated samples showing lack of OP-1gene expression and absent or very limited bone formation by induction confirm that the spontaneous induction of bone formation by coral-derived macroporous constructs is initiated by secreted BMPs/OPs, in context the OP-1 isoform.
Keywords: Osteoclastic topographical modifications; Bisphosphonate zoledronate Zometa; Osteogenic proteins; Synergistic induction of bone formation; Biomimetism
The role of matrix metalloproteinases in regulating neuronal and nonneuronal cell invasion into PEGylated fibrinogen hydrogels
by Offra Sarig-Nadir; Dror Seliktar (pp. 6411-6416).
Injured peripheral nerve tissue could benefit from biomaterial nerve guidance conduits (NGCs) that are designed to promote neuronal regeneration. Nerve regeneration is a complex multi-step process that involves the remodeling of the ECM surrounding the regenerating neural tissue. Hydrogel biomaterials have been used as provisional matrices to regulate this regeneration process by providing the desired physical properties and controllable degradation characteristics. The purpose of this investigation was to understand the mechanism by which nerve cells penetrate into a hydrogel made from PEGylated fibrinogen. In this context, the dorsal root ganglion (DRG) assay was used as an in vitro model to study the cellular invasion behavior of both neural and nonneuronal cells. Our hypothesis stipulated that DRG cells employ matrix metalloproteinases (MMPs) in order to degrade the dense hydrogel matrix and penetrate the biomaterial. Three dimensional (3D) DRG-hydrogel constructs were cultured with MMP inhibitors (MMPi) and the effect of the inhibitors on DRG cell outgrowth was investigated. We also examined the effect of inhibitors on two dimensional (2D) DRG cell outgrowth on PEGylated fibrinogen hydrogels and on tissue culture polystyrene (TCP). Our results demonstrate that DRG cell outgrowth into and onto PEGylated fibrinogen hydrogels was inhibited by MMPi and that the outgrowth characteristics was dependent on the type of inhibitor and its concentration. MMP-3i and MMP-8i decreased both neuronal and nonneuronal outgrowth, where MMP-3i had a stronger inhibitory effect on nonneuronal cells. MMP-2/9i, on the other hand, affected the neuronal outgrowth much more than the others. We concluded that MMPs play a central role in the process of DRG cell penetration into PEGylated fibrinogen hydrogels and may also regulate the adhesion, migration and elongation of neuronal cells on the surface of these hydrogel biomaterials.
Keywords: Matrix metalloproteinase inhibitor; Dorsal root ganglion; Neuronal outgrowth; Peripheral nerve regeneration; Biodegradable hydrogel
The enhancement of bone allograft incorporation by the local delivery of the sphingosine 1-phosphate receptor targeted drug FTY720
by Caren E. Petrie Aronin; Soo J. Shin; Kimberly B. Naden; Peter D. Rios Jr.; Lauren S. Sefcik; Sarah R. Zawodny; Namory D. Bagayoko; Quanjun Cui; Yusuf Khan; Edward A. Botchwey (pp. 6417-6424).
Poor vascularization coupled with mechanical instability is the leading cause of post-operative complications and poor functional prognosis of massive bone allografts. To address this limitation, we designed a novel continuous polymer coating system to provide sustained localized delivery of pharmacological agent, FTY720, a selective agonist for sphingosine 1-phosphate receptors, within massive tibial defects. In vitro drug release studies validated 64% loading efficiency with complete release of compound following 14 days. Mechanical evaluation following six weeks of healing suggested significant enhancement of mechanical stability in FTY720 treatment groups compared with unloaded controls. Furthermore, superior osseous integration across the host–graft interface, significant enhancement in smooth muscle cell investment, and reduction in leukocyte recruitment was evident in FTY720 treated groups compared with untreated groups. Using this approach, we can capitalize on the existing mechanical and biomaterial properties of devitalized bone, add a controllable delivery system while maintaining overall porous structure, and deliver a small molecule compound to constitutively target vascular remodeling, osseous remodeling, and minimize fibrous encapsulation within the allograft–host bone interface. Such results support continued evaluation of drug-eluting allografts as a viable strategy to improve functional outcome and long-term success of massive cortical allograft implants.
Keywords: Bone tissue engineering; Drug delivery; Angiogenesis; Osseointegration
The differential regulation of cell motile activity through matrix stiffness and porosity in three dimensional collagen matrices
by Miguel Miron-Mendoza; Joachim Seemann; Frederick Grinnell (pp. 6425-6435).
In three dimensional collagen matrices, cell motile activity results in collagen translocation, cell spreading and cell migration. Cells can penetrate into the matrix as well as spread and migrate along its surface. In the current studies, we quantitatively characterize collagen translocation, cell spreading and cell migration in relationship to collagen matrix stiffness and porosity. Collagen matrices prepared with 1–4mg/ml collagen exhibited matrix stiffness (storage modulus measured by oscillating rheometry) increasing from 4 to 60Pa and matrix porosity (measured by scanning electron microscopy) decreasing from 4 to 1 μm2. Over this collagen concentration range, the consequences of cell motile activity changed markedly. As collagen concentration increased, cells no longer were able to cause translocation of collagen fibrils. Cell migration increased and cell spreading changed from dendritic to more flattened and polarized morphology depending on location of cells within or on the surface of the matrix. Collagen translocation appeared to depend primarily on matrix stiffness, whereas cell spreading and migration were less dependent on matrix stiffness and more dependent on collagen matrix porosity.
Keywords: Extracellular matrix; Collagen translocation; Cell migration; Cell spreading; Mechanoregulation
The quantification of single cell adhesion on functionalized surfaces for cell sheet engineering
by G. Weder; O. Guillaume-Gentil; N. Matthey; F. Montagne; H. Heinzelmann; J. Vörös; M. Liley (pp. 6436-6443).
The use of force spectroscopy to measure and quantify the forces involved in the adhesion of 3T3 fibroblasts to different chemically functionalized surfaces has been investigated. Cells were grown on glass surfaces as well as on surfaces used for cell sheet engineering: surfaces coated with polyelectrolyte multilayers (poly-l-lysine and hyaluronic acid) and thermally-responsive poly(N-isopropylacrylamide) (PNIPAM) brushes. Individual adherent cells were detached from their culture substrate using an AFM cantilever coated with fibronectin. The maximum forces of detachment of each cell were measured and taken as characteristic of the cellular adhesion. Large differences in cellular adhesion were observed on polyelectrolyte coatings depending on the number of polyelectrolyte layers. On PNIPAM-grafted surfaces, changes of more than an order of magnitude were observed in cell adhesion above and below the lower critical solution temperature. Glass surfaces patterned with periodic PNIPAM microdomains were also investigated, and it was shown that cellular adhesion could be reduced while keeping cellular morphology unchanged.
Keywords: Poly(N-isopropylacrylamide); AFM; Force spectroscopy; Surface modification; Fibroblasts
Cardiac repair with injectable cell sheet fragments of human amniotic fluid stem cells in an immune-suppressed rat model
by Yi-Chun Yeh; Wen-Yu Lee; Chu-Leng Yu; Shiaw-Min Hwang; Min-Fan Chung; Li-Wen Hsu; Yen Chang; Wei-Wen Lin; Ming-Song Tsai; Hao-Ji Wei; Hsing-Wen Sung (pp. 6444-6453).
Direct intramyocardial injection of the desired cell types in a dissociated form is a common route of cell transplantation for repair of damaged myocardium. However, following injection of dissociated cells, a massive loss of transplanted cells has been reported. In this study, human amniotic fluid stem cells (hAFSCs) were used as the cell source for the fabrication of cell sheet fragments, using a thermo-responsive methylcellulose hydrogel system. The fabricated hAFSC sheet fragments preserved the endogenous extracellular matrices (ECM) and retained their cell phenotype. Test samples were xenogenically transplanted into the peri-ischemic area of an immune-suppressed rat model at 1 week after myocardial infarction (MI) induction. There were four treatment groups ( n≧10): sham; saline; dissociated hAFSCs; and hAFSC sheet fragments. The results obtained in the echocardiography revealed that the group treated with hAFSC sheet fragments had a superior heart function to those treated with saline or dissociated hAFSCs. Due to their inherent ECM, hAFSC sheet fragments had a better ability of cell retention and proliferation than dissociated hAFSCs upon transplantation to the host myocardium. Additionally, transplantation of hAFSC sheet fragments stimulated a significant increase in vascular density, consequently contributing towards improved wall thickness and a reduction in the infarct size, when compared with dissociated hAFSCs. Our histological findings and qPCR analyses suggest that the transplanted hAFSCs can be differentiated into cardiomyocyte-like cells and cells of endothelial lineages and modulate expression of multiple angiogenic cytokines and cardiac protective factor with the potential to promote neo-vascularization, which evidently contributed to the improvement of ventricular function.
Keywords: Thermo-responsive hydrogel; Myocardial infarction; Cell transplantation; Angiogenesis; Cardiomyogenesis
Epoxy-amine synthesised hydrogel scaffolds for soft-tissue engineering
by Zuratul A.A. Hamid; Anton Blencowe; Berkay Ozcelik; Jason A. Palmer; Geoffrey W. Stevens; Keren M. Abberton; Wayne A. Morrison; Anthony J. Penington; Greg G. Qiao (pp. 6454-6467).
Highly porous and biodegradable hydrogels based on poly(ethylene glycol) (PEG) and cystamine (Cys) were fabricated using epoxy-amine chemistry and investigated as scaffolds for soft-tissue engineering. Whereas the application of fused-salt templates provided a comprehensive interconnecting pore morphology, the incorporation of a specially designed poly(ε-caprolactone) (PCL) cross-linker provided enhanced mechanical function without adversely effecting the scaffolds positive biological interactions. The addition of only 1.2 wt% of the PCL cross-linker was sufficient to provide improvements in the ultimate stress of 30–40%. In vitro studies not only confirmed the non-cytotoxic nature of the scaffolds, but also their degradation products, which were isolated and characterised by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionisation time-of-flight mass spectroscopy (MALDI ToF MS). In vivo trials were conducted over a period of 8 weeks through implantation of the scaffolds into the dorsal region of rats. At both 2 and 8 week time points the explants revealed complete infiltration by the surrounding tissue and the development of a vascular network to support the newly generated tissue, without an excessive foreign-body response.
Keywords: Polyethylene oxide; Scaffold; In vitro; test; In vivo; test; Hydrogel; Cell viability
Effect of functionalized polycaprolactone on the behaviour of murine preosteoblasts
by Olivier Drevelle; Eric Bergeron; Helena Senta; Marc-Antoine Lauzon; Sophie Roux; Guillaume Grenier; Nathalie Faucheux (pp. 6468-6476).
The efficiency of biomaterials used in bone repair depends greatly on their ability to interact with bone cells. Hence, we have functionalized polycaprolactone (PCL) films by peptides derived from the bone sialoprotein containing RGD sequence (pRGD), to increase their ability to interact with murine MC3T3-E1 preosteoblasts, and favour cell response to recombinant human bone morphogenetic protein-2 (rhBMP-2). RGE peptides (pRGE) were used as negative controls. The PCL films were hydrolyzed with NaOH and then carboxylic acid groups were activated to allow chemisorption of the peptides. Alkaline treatment increased the hydrophilicity of PCL films without significantly change their roughness. Peptide immobilization on PCL was checked by X-ray photoelectron spectroscopy. Hydrolyzed PCL films (Hydro PCL), which adsorbed fibronectin and vitronectin from serum after 1 h incubation, prevented the spreading of MC3T3-E1 preosteoblasts, while films bearing pRGD or pRGE did not. In contrast, MC3T3-E1 preosteoblasts attached to pRGD and incubated for 1 h in serum-free medium spread better than cells on Hydro PCL or pRGE. Only cells on pRGD had organized cytoskeleton, phosphorylated focal adhesion kinase on Y397 and responded to rhBMP-2 by activating Smad pathway. Thus, pRGD PCL may be used to favour bone cell cytoskeletal organization and response to rhBMP-2.
Keywords: Cell signalling; RGD peptides; Vinculin; Actin
Functionalization of matrices by cyclically stretched osteoblasts through matrix targeting of VEGF
by Céline Faure; Laurence Vico; Philippe Tracqui; Norbert Laroche; Arnaud Vanden-Bossche; Marie-Thérèse Linossier; Aline Rattner; Alain Guignandon (pp. 6477-6484).
Vascular endothelial growth factor A (VEGF) plays a central role in load-induced bone gain. We previously showed that increasing cyclic stretch frequency from 0.05 to 5 Hz induce parallel increased in entrapment of VEGF (mVEGF) into osteoblast secreted extracellular matrix. We ask in this study if mVEGF could be protective against apoptotic signals and biologically active in vitro on endothelial cell migration as well as in vivo on angiogenesis. We established that mechanically–induced VEGF entrapment using stretched silicone membrane was saturable after 3 exposures at high frequency stretches (5Hz). We found that mVEGF stimulates microvascular cells migration and enhanced angiogenesis more importantly than VEGF 165 controls suggesting the absence of potent anti-angiogenic factors in our functionalized matrices. Indeed we found that the anti-angiogenic factors, tissue inhibitor of metalloproteinase (TIMP2) and pigment epithelium-derived factor (PEDF) were specifically downregulated for 5 Hz stretch and that the release of these potent factors was increased for low frequency of stretch (0.05 Hz). This study qualifies high frequency cyclic stretch as an interesting approach for surfaces activation of deformable biomaterials.
Keywords: Cyclic strain; Osteoblasts; Endothelial cells; VEGF
The role of pharmacologically active microcarriers releasing TGF-β3 in cartilage formation in vivo by mesenchymal stem cells
by Carine Bouffi; Olivier Thomas; Claire Bony; Alexandra Giteau; Marie-Claire Venier-Julienne; Christian Jorgensen; Claudia Montero-Menei; Danièle Noël (pp. 6485-6493).
Cartilage engineering using mesenchymal stem cells (MSC) will require the use of a scaffold which will act as a support for cell adhesion keeping the cells in the cartilage defect. Optimally, a tissue engineered construct should allow sustained delivery of bioactive factors capable of inducing MSC differentiation into chondrocytes and should be easily injected inside the cartilage lesions to avoid surgical operations. We therefore developed pharmacologically active microcarriers (PAM) made of poly-lactic-co-glycolic acid (PLGA) produced using an oil-in-water (o/w) emulsion method. The microspheres were coated with a biomimetic surface of fibronectin (FN) and engineered to release TGF-β3 as a chondrogenic differentiation factor. When human MSCs were incubated in vitro with TGF-β3 releasing FN-coated PAMs in chondrogenic medium, they firmly adhered onto the surface of PAMs rapidly forming cell aggregates. After 3 weeks, strong up-regulation of cartilage-specific markers was observed both at the mRNA and protein level whereas osteogenic or adipogenic genes could not be detected. Importantly, implantation of MSC/TGF-β3 releasing PAM complexes in SCID mice resulted in the formation of histologically resembling cartilage which stained positive for chondrocyte markers, collagen II and aggrecan. The present study demonstrated that functionalized PLGA-based microparticles can provide an appropriate environment for chondrogenic differentiation of MSCs and should contribute to injectable biomedical device development improving in vivo cartilage engineering.
Keywords: Microspheres; Scaffolds; Mesenchymal stromal cells; Cell differentiation; Chondrogenesis
Three dimensionally flocculated proangiogenic microgels for neovascularization
by Ross J. DeVolder; Hyun-Joon Kong (pp. 6494-6501).
Microparticles encapsulating regenerative medicines have been used in tissue engineering because of their several advantages, including non-invasive drug delivery and controllable drug release rates. However, microparticles implanted in tissue defects are readily displaced by external mechanical forces, decreasing their regenerative efficacy. We hypothesized that a drug-encapsulated colloidal gel formed through colloidal attraction between microparticles would resist displacement at an implant site, and subsequently improve therapeutic efficacy. This hypothesis was examined using a colloidal gel formed from the mixing of negatively charged microgels composed of poly(ethylene glycol) (PEG) and poly(sodium acrylate), and positively charged microgels composed of PEG and poly(vinyl benzyl trimethyl ammonium chloride). The structural strength of the colloidal gel could be tuned with the zeta potential and volumetric ratios of the oppositely charged microgels. Furthermore, the implantation of the colloidal gel, encapsulating vascular endothelial growth factor, significantly increased the vascular density while limiting host inflammation, as compared with the implantation of unary microgel suspensions. This study demonstrates an enhancement in the efficacy of microparticle drug delivery systems by tuning rheological properties of suspensions, which should be useful for the design of a wide array of particulate systems for both tissue engineering and drug delivery.
Keywords: Microsphere; Drug delivery; Viscoelasticity; Angiogenesis; In vivo test; Hydrogel
An injectable calcium phosphate-alginate hydrogel-umbilical cord mesenchymal stem cell paste for bone tissue engineering
by Liang Zhao; Michael D. Weir; Hockin H.K. Xu (pp. 6502-6510).
The need for bone repair has increased as the population ages. Stem cell-scaffold approaches hold immense promise for bone tissue engineering. However, currently, preformed scaffolds for cell delivery have drawbacks including the difficulty to seed cells deep into the scaffold, and inability for injection in minimally-invasive surgeries. Current injectable polymeric carriers and hydrogels are too weak for load-bearing orthopedic applications. The objective of this study was to develop an injectable and mechanically-strong stem cell construct for bone tissue engineering. Calcium phosphate cement (CPC) paste was combined with hydrogel microbeads encapsulating human umbilical cord mesenchymal stem cells (hUCMSCs). The hUCMSC-encapsulating composite paste was fully injectable under small injection forces. Cell viability after injection matched that in hydrogel without CPC and without injection. Mechanical properties of the construct matched the reported values of cancellous bone, and were much higher than previous injectable polymeric and hydrogel carriers. hUCMSCs in the injectable constructs osteodifferentiated, yielding high alkaline phosphatase, osteocalcin, collagen type I, and osterix gene expressions at 7 d, which were 50–70 fold higher than those at 1 d. Mineralization by the hUCMSCs at 14 d was 100-fold that at 1 d. In conclusion, a fully injectable, mechanically-strong, stem cell–CPC scaffold construct was developed. The encapsulated hUCMSCs remained viable, osteodifferentiated, and synthesized bone minerals. The new injectable stem cell construct with load-bearing capability may enhance bone regeneration in minimally-invasive and other orthopedic surgeries.
Keywords: Injectable scaffold; Umbilical cord stem cells; Calcium phosphate cement; Minimally-invasive; Osteogenic differentiation; Load-bearing
The control of stem cell morphology and differentiation by hydrogel surface wrinkles
by Murat Guvendiren; Jason A. Burdick (pp. 6511-6518).
In this study, we investigated human mesenchymal stem cell (hMSC) interactions with uniform hydrogels and hydrogels with lamellar or hexagonal surface wrinkles to elucidate our ability to control hMSC morphology and differentiation. Wrinkled hydrogels were prepared from photocurable poly(2-hydroxyethyl methacrylate) (PHEMA) precursor solutions containing ethylene glycol dimethacrylate as a crosslinker, using depth-wise gradients in crosslinking and subsequent buckling with swelling to generate wrinkles. A replica molding process was used to fabricate a series of gels with uniform mechanics, but altered surface wrinkle size and shape. We found that hMSCs attached to lamellar wrinkles spread by taking the shape of the pattern, exhibit high aspect ratios, and differentiate into an osteogenic lineage. In contrast, cells that attached inside the hexagonal patterns remain rounded with low spreading and differentiate into an adipogenic lineage. This work aids in the development of material-based cell culture and scaffold systems to direct stem cell differentiation.
Keywords: Hydrogel; Surface wrinkles; Stem cells; Morphology; Differentiation
Lipid-based nanoparticles with high binding affinity for amyloid-β1–42 peptide
by Marco Gobbi; Francesca Re; Mara Canovi; Marten Beeg; Maria Gregori; Silvia Sesana; Sandro Sonnino; Doriano Brogioli; Claudia Musicanti; Paolo Gasco; Mario Salmona; Massimo E. Masserini (pp. 6519-6529).
The neurotoxic beta-amyloid peptide (Aβ), formed in anomalous amounts in Alzheimer’s disease (AD), is released as monomer and then undergoes aggregation forming oligomers, fibrils and plaques in diseased brains. Aβ aggregates are considered as possible targets for therapy and/or diagnosis of AD. Since nanoparticles (NPs) are promising vehicles for imaging probes and therapeutic agents, we realized and characterized two types of NPs (liposomes and solid lipid nanoparticles, 145 and 76 nm average size, respectively) functionalized to target Aβ1–42 with high affinity. Preliminary immunostaining studies identified anionic phospholipids [phosphatidic acid (PA) and cardiolipin (CL)] as suitable Aβ1–42 ligands. PA/CL-functionalized, but not plain, NPs interacted with Aβ1–42 aggregates as indicated by ultracentrifugation experiments, in which binding reaction occurred in solution, and by Surface Plasmon Resonance (SPR) experiments, in which NPs flowed onto immobilized Aβ1–42. All these experiments were carried out in buffered saline. SPR studies indicated that, when exposed on NPs surface, PA/CL display very high affinity for Aβ1–42 fibrils (22–60 nm), likely because of the occurrence of multivalent interactions which markedly decrease the dissociation of PA/CL NPs from Aβ. Noteworthy, PA/CL NPs did not bind to bovine serum albumin. The PA/CL NPs described in this work are endowed with the highest affinity for Aβ so far reported. These characteristics make our NPs a very promising vector for the targeted delivery of potential new diagnostic and therapeutic molecules to be tested in appropriate animal models.
Keywords: Nanoparticles; Drug delivery; Affinity; Lipid; Liposome; Aβ-peptide
Heparin immobilized gold nanoparticles for targeted detection and apoptotic death of metastatic cancer cells
by Kyuri Lee; Hyukjin Lee; Ki Hyun Bae; Tae Gwan Park (pp. 6530-6536).
In the present study, heparin immobilized, multifunctional gold nanoparticles (AuNPs) were developed as a new class of theragnostic nanomaterials for metastatic cancer cell imaging and apoptosis. AuNPs were surface modified with fluorescent dye labeled heparin molecules to detect a metastatic stage of cancer cells that over-express heparin-degrading enzymes. The heparin immobilized AuNPs exhibited enhanced fluorescence signals by specific cleavage of heparin molecules from the surface of AuNPs by heparinase or heparanase secreted from metastatic cancer cells. In addition, heparin immobilized AuNPs that were additionally tethered with RGD peptides on the surface demonstrated highly specific apoptotic activities for selective cancer cells over-expressing RGD receptors on the membrane, revealing that internalized heparin within cells clearly triggered an apoptotic event. These results suggest that heparin immobilized AuNPs can be usefully exploited for optical imaging agents for metastatic tumors as well as therapeutic cancer treatment.
Keywords: Gold nanoparticles; Apoptosis; Molecular imaging; Targeted delivery; Heparin
Block-copolymer-stabilized iodinated emulsions for use as CT contrast agents
by Anke de Vries; Erica Custers; Johan Lub; Sandra van den Bosch; Klaas Nicolay; Holger Grüll (pp. 6537-6544).
The objective of this study was to develop radiopaque iodinated emulsions for use as CT blood pool contrast agents. Three hydrophobic iodinated oils were synthesized based on the 2,3,5-triiodobenzoate moiety and formulated into emulsions using either phospholipids or amphiphilic polymers, i.e. Pluronic F68 and poly(butadiene)- b-poly(ethylene glycol) (PBD-PEO), as emulsifiers. The size, stability and cell viability was investigated for all stabilized emulsions. Three emulsions stabilized with either lipids or PBD-PEO were subsequently tested in vivo as a CT blood pool contrast agent in mice. While the lipid-stabilized emulsions turned out unstable in vivo, polymer-stabilized emulsions performed well in vivo. In blood, a contrast enhancement of 220 Hounsfield Units (HU) was measured directly after intravenous administration of 520 mg I/kg. The blood circulation half-life of a PBD-PEO stabilized emulsion was approximately 3 h and no noticeable in vivo toxicity was observed. These results show the potential of above emulsions for use as blood pool agents in contrast enhanced CT imaging.
Keywords: Computed tomography (CT); Contrast agent; Emulsion; Nanoparticles; Iodine; X-ray imaging
The down regulation of target genes by photo activated DNA nanoscissors
by Tsung-Lin Tsai; Dar-Bin Shieh; Chen-Sheng Yeh; Yonhua Tzeng; Khant Htet; Kao-Shu Chuang; Jih Ru Hwu; Wu-Chou Su (pp. 6545-6554).
An artificial, targeted, light-activated nanoscissor (ATLANS) was developed for precision photonic cleavage of DNA at selectable target sequences. The ATLANS is comprised of nanoparticle core and a monolayer of hydrazone-modified triplex-forming oligonucleotides (TFOs), which recognize and capture the targeted DNA duplex. Upon photo-illumination ( λ = 460 nm), the attached hydrazone scissor specifically cleaves the targeted DNA at a pre-designed nucleotide pair. Electrophoretic mobility shift and co-precipitation assays revealed sequence-specific binding with the short-fragment and long-form plasmid DNA of both TFO and TFO-nanoparticle probes. Upon photo-illumination, ATLANS introduced a precise double-stranded break 12bp downstream the TFO binding sequence and down-regulated the target gene in HeLa cell system. Gold nanoparticles multiplexed the cutting efficiency and potential for simultaneous manipulation of multiple targets, as well as protected DNA from non-specific photo-damage. This photon-mediated DNA manipulation technology will facilitate high spatial and temporal precision in simultaneous silencing at the genome level, and advanced simultaneous manipulation of multiple targeted genes.
Keywords: Gold nanoparticles; Triplex-forming oligonucleotides (TFOs); Hydrazone; DNA cleavge; Gene manipulation
The biocompatibility of amino functionalized CdSe/ZnS quantum-dot-Doped SiO2 nanoparticles with primary neural cells and their gene carrying performance
by Giuseppe Bardi; Maria Ada Malvindi; Lisa Gherardini; Mario Costa; Pier Paolo Pompa; Roberto Cingolani; Tommaso Pizzorusso (pp. 6555-6566).
Nanoparticles have an enormous potential for the development of applications in biomedicine such as gene or drug delivery. We developed and characterized NH2 functionalized CdSe/ZnS quantum dot (QD)-doped SiO2 nanoparticles (NPs) with both imaging and gene carrier capabilities. We show that QD-doped SiO2 NPs are internalized by primary cortical neural cells without inducing cell death in vitro and in vivo. Moreover, the ability to bind, transport and release DNA into the cell allows GFP-plasmid transfection of NIH-3T3 and human neuroblastoma SH-SY5Y cell lines. QD-doped SiO2 NPs properties make them a valuable tool for future nanomedicine application.
Keywords: Silica nanoparticles; Neural cells; Biocompatibility; Gene transfer
Targeted imaging of tumor-associated M2 macrophages using a macromolecular contrast agent PG-Gd-NIR813
by Marites P. Melancon; Wei Lu; Qian Huang; Prakash Thapa; Dapeng Zhou; Chaan Ng; Chun Li (pp. 6567-6573).
Tumor-associated macrophages (TAMs) are diverse population containing multiple subtypes. M2 macrophages promote tumor growth and metastasis, in part by secreting a wide range of proangiogenic factors and growth factors. Selective depletion of M2 macrophages has been evaluated as a novel approach to anti-cancer therapy. In this study, a dual magneto-optical imaging probe, PG-Gd-NIR813 was synthesized and evaluated for non-invasive assessment of TAMs after intravenous injection. PG-Gd-NIR813 injected in nude rats bearing C6 tumors showed high uptake of the polymeric contrast agent in the tumor at 1 and 48 h after injection both in vivo and ex vivo optical imaging. T1-weighted MR imaging results showed accumulation of PG-Gd-NIR813 into the tumor necrotic area, which was confirmed by TUNEL staining of resected tumors. The uptake of PG-Gd-NIR813 within tumor necrosis decreased after animals were treated by the macrophage-depleting agent. Immunohistochemical staining demonstrated that PG-Gd-NIR813 colocalized with CD68 (marker for macrophages) and CD169 (marker for activated macrophages), but not with CD163 (residential macrophages). Using combined near-infrared fluorescence imaging and magnetic resonance imaging (MRI), we demonstrated that the accumulation of PG-Gd-NIR813 in tumors was mediated through M2 TAMs. Therefore, poly(l-glutamic acid) based reagents could be potentially used to image response to antitumor therapies targeted at M2 TAMs. Furthermore, poly(l-glutamic acid) is a promising carrier for candidate immunotherapeutics targeting M2 TAMs.
Biodistribution of PEG-modified gold nanoparticles following intratracheal instillation and intravenous injection
by Jens Lipka; Manuela Semmler-Behnke; Ralph A. Sperling; Alexander Wenk; Shinji Takenaka; Carsten Schleh; Thomas Kissel; Wolfgang J. Parak; Wolfgang G. Kreyling (pp. 6574-6581).
Besides toxicity tests, biokinetic studies are a fundamental part of investigations to evaluate a safe and sustainable use of nanoparticles. Today, gold nanoparticles (Au NPs) are known to be a versatile tool in different areas such as science, engineering or medicine. In this study, we investigated the biokinetics after intravenous and intratracheal applications of poly(ethylene glycol) (PEG) modified Au NPs compared to plain Au NPs. Radioactive-labeled Au NPs of 5 nm inorganic core diameter were applied to rats and the NP content in tissues, organs and excretion were quantified after 1-hour and 24-hours. After intravenous injection, a prolonged blood circulation time was determined for Au NPs with 10 kDa PEG chains. Non-PEGylated Au NPs and 750 Da PEG Au NPs accumulated mostly in liver and spleen. After intratracheal application the majority of all three types of applied NPs stayed in the lungs: the total translocation towards the circulation did not differ considerably after PEGylation of the Au NPs. However, a prolonged retention time in the circulation was detected for the small fraction of translocated 10 kDa PEG Au NPs, too.
Keywords: Radio-labeled gold nanoparticle; Surface modification; Polyethyleneglycol; Biokinetics
Imaging and cell targeting characteristics of magnetic nanoparticles modified by a functionalizable zwitterionic polymer with adhesive 3,4-dihydroxyphenyl-l-alanine linkages
by Lei Zhang; Hong Xue; Changlu Gao; Louisa Carr; Jinnan Wang; Baocheng Chu; Shaoyi Jiang (pp. 6582-6588).
Multifunctional magnetic nanoparticles (MNPs) modified by a zwitterionic polymer (pCBMA–DOPA2) containing one poly(carboxybetaine methacrylate) (pCBMA) chain and two 3,4-dihydroxyphenyl-l-alanine (DOPA) residue groups were developed. Results showed that MNPs modified by pCBMA were not only stable in complex media, but also provided abundant functional groups for ligand immobilization. The pCBMA–DOPA2 MNPs had a hydrodynamic particle size of about 130nm, a strong saturation magnetization of 110.2emu/g Fe and a high transverse relaxivity of 428mm−1s−1. Long-term stability in phosphate-buffered saline (PBS) and 10% NaCl solution was achieved for over six months. Compared to MNPs coated with dextran, pCBMA–DOPA2 MNPs presented better stability in 100% human blood serum at 37°C. Macrophage cell uptake studies revealed that the uptake ratio of pCBMA–DOPA2 MNPs was much lower than that of dextran MNPs. Furthermore, quantitative analysis results showed that after pCBMA–DOPA2 MNPs were conjugated with a targeting RGD peptide, uptake by human umbilical vein endothelial cell (HUVEC) was notably increased, which was further visualized by magnetic resonance imaging (MRI).
Keywords: Zwitterionic; Iron oxide nanoparticles; Biomimetic linkage; Ultra-low fouling; MRI (magnetic resonance imaging); Targeting
PEGlated magnetic polymeric liposome anchored with TAT for delivery of drugs across the blood-spinal cord barrier
by Hanjie Wang; Shuangnan Zhang; Zhenyu Liao; Chunyuan Wang; Yang Liu; Shiqing Feng; Xinguo Jiang; Jin Chang (pp. 6589-6596).
Due to the existence of the blood-spinal cord barrier (BSCB), many therapeutic macromolecular agents, such as drugs, protein and gene, cannot pass through this barrier to reach the site of injury, all of which restricts the treatment of spinal cord injuries (SCI). In this study, TAT-conjugated PEGlated Magnetic polymeric liposomes (TAT-PEG-MPLs) formed from PEGlated amphiphilic octadecyl quaternized carboxymethyl chitosan (PEG-OQCMC), cholesterol (Chol), superparamagnetic nanoparticles, and transactivating-transduction protein (TAT), were prepared successfully and evaluated the properties in vitro and in vivo. The result indicated that TAT-PEG-MPLs were spherical in solution, with significantly small mean diameter (83.2 nm) and excellent magnetism (magnetization saturation values of 43.5 emu/g). In vitro experiment, the uptake of PEG-MPLs with TAT by MCF-7 cells was greater than that of the PEG-MPLs without TAT. Most importantly, in vivo experiment, a low MRI signal was observed in the T2-weighted images; Histological analysis, Cryo-TEM and flame atomic absorption spectrophotometry revealed that TAT-PEG-MPLs nanoparticles significantly accumulated around the site of the SCI even inside the nerve cells. These nanoparticles may provide a promising carrier to locate to the lesion site, deliver therapeutic macromolecular agents across the BSCB and penetrate into the nerve cells for the treatment of SCI.
Keywords: Chitin/chitosan; Drug delivery; Liposome, MRI(magnetic resonance imaging)Nanopartilce; Surface modification
The in vitro stability and in vivo pharmacokinetics of curcumin prepared as an aqueous nanoparticulate formulation
by Chandana Mohanty; Sanjeeb K. Sahoo (pp. 6597-6611).
Curcumin, the natural anticancer drug and its optimum potential is limited due to lack of solubility in aqueous solvent, degradation at alkaline pH and poor tissue absorption. In order to enhance its potency and improve bioavailability, we have synthesized curcumin loaded nanoparticulate delivery system. Unlike free curcumin, it is readily dispersed in aqueous medium, showing narrow size distribution ˜192 nm ranges (as observed by microscope) with biocompatibility (confocal studies and TNF-α assay). Furthermore, it displayed enhanced stability in phosphate buffer saline by protecting encapsulated curcumin against hydrolysis and biotransformation. Most importantly, nanoparticulate curcumin was comparatively more effective than native curcumin against different cancer cell lines under in vitro condition with time due to enhanced cellular uptake resulting in reduction of cell viability by inducing apoptosis. Molecular basis of apoptosis studied by western blotting revealed blockade of nuclear factor kappa B (NFκB) and its regulated gene expression through inhibition of IκB kinase and Akt activation. In mice, nanoparticulate curcumin was more bioavailable and had a longer half-life than native curcumin as revealed from pharmacokinetics study. Thus, the results demonstrated nanoparticulate curcumin may be useful as a potential anticancer drug for treatment of various malignant tumors.
Keywords: Apoptosis; Bioavailability; Cancer therapy; Curcumin; Nanoparticle
A near-infrared fluorescent heptamethine indocyanine dye with preferential tumor accumulation for in vivo imaging
by Chao Zhang; Tao Liu; Yongping Su; Shenglin Luo; Ying Zhu; Xu Tan; Song Fan; Lilong Zhang; Yue Zhou; Tianmin Cheng; Chunmeng Shi (pp. 6612-6617).
Near-infrared (NIR) fluorescence imaging holds great promise for tumor imaging due to low tissue autofluorescence and deep tissue penetration. However, most tumor-targeting fluorescent probes require combination of targeting agents and fluorescent reporters. In this study, we described a NIR heptamethine cyanine dye, IR-780 iodide, with preferential accumulation in multiple tumor cells without the necessity of chemical conjugation. The IR-780 iodide was found to retain in tumors but not normal cells in multiple tumor xenografts in nude mice and chemically-induced lung tumors in C57BL/6 mice. The fluorescent signal of tumors could persist at least 20 days with a significant signal-to-backgroud ratio. As a lipophilic cation, a predominant accumulation of IR-780 iodide was shown in the mitochondria of tumor cells owing to the high magnitude of mitochondrial membrane potential in tumor cells than normal cells. We further showed that the transportation of IR-780 iodide into tumor cells was mediated by the organic anion transporter peptides (OATPs) because the dye accumulation was significantly inhibited by sulfobromophthalein (BSP), a competitive inhibitor of OATPs. Our study shows that IR-780 iodide that preferentially accumulates in tumor cells and is natively NIR fluorescent would be useful in tumor detection.
Keywords: In vivo test; Fluorescence; Tumor targeting; Heptamethine dye
Mineralisation of reconstituted collagen using polyvinylphosphonic acid/polyacrylic acid templating matrix protein analogues in the presence of calcium, phosphate and hydroxyl ions
by Young Kyung Kim; Li-sha Gu; Thomas E. Bryan; Jong R. Kim; Liang Chen; Yan Liu; James C. Yoon; Lorenzo Breschi; David H. Pashley; Franklin R. Tay (pp. 6618-6627).
The complex morphologies of mineralised collagen fibrils are regulated through interactions between the collagen matrix and non-collagenous extracellular proteins. In the present study, polyvinylphosphonic acid, a biomimetic analogue of matrix phosphoproteins, was synthesised and confirmed with FTIR and NMR. Biomimetic mineralisation of reconstituted collagen fibrils devoid of natural non-collagenous proteins was demonstrated with TEM using a Portland cement-containing resin composite and a phosphate-containing fluid in the presence of polyacrylic acid as sequestration, and polyvinylphosphonic acid as templating matrix protein analogues. In the presence of these dual biomimetic analogues in the mineralisation medium, intrafibrillar and extrafibrillar mineralisation via bottom-up nanoparticle assembly based on the non-classical crystallisation pathway could be identified. Conversely, only large mineral spheres with no preferred association with collagen fibrils were observed in the absence of biomimetic analogues in the medium. Mineral phases were evident within the collagen fibrils as early as 4 h after the initially-formed amorphous calcium phosphate nanoprecursors were transformed into apatite nanocrystals. Selected area electron diffraction patterns of highly mineralised collagen fibrils were nearly identical to those of natural bone, with apatite crystallites preferentially aligned along the collagen fibril axes.
Keywords: Extrafibrillar mineralisation; Intrafibrillar mineralisation; Matrix protein analogues; Reconstituted collagen fibrils; Tissue engineering materials
Mussel-inspired transformation of CaCO3 to bone minerals
by Sungjin Kim; Chan Beum Park (pp. 6628-6634).
We report a mussel-inspired route to create carbonated bone hydroxyapatite from CaCO3 vaterite microspheres. When catechol-containing dopamine, a biomimetic small molecule of mussel adhesive proteins, was incorporated during the mineralization of CaCO3, the oxidative polymerization of dopamine stabilized the formation of spherical vaterite, the most unstable phase among CaCO3 crystalline structures. Thus-formed vaterite microspheres were readily transformed to carbonated hydroxyapatite crystals when incubated in a simulated body fluid at human body temperature. We found that dopamine not only stabilized the vaterite phase but also influenced the level of conversion to carbonated hydroxyapatites. Considering that carbonated hydroxyapatites are highly bioresorbable, similar to natural bone and dentin, the synthesis of a mussel-inspired hybrid material showing good in vitro bone bioactivity should present a new prospect for future applications in the treatment of bone defects and bone degenerative diseases.
Keywords: Calcium carbonate; Vaterite; Bone minerals; Hydroxyapatite; Dopamine
The biomechanical characteristics of the bone-periodontal ligament-cementum complex
by Sunita P. Ho; Michael P. Kurylo; Tiffany K. Fong; Stephen S.J. Lee; Hanoch D. Wagner; Mark I. Ryder; Grayson W. Marshall (pp. 6635-6646).
The relative motion between the tooth and alveolar bone is facilitated by the soft-hard tissue interfaces which include periodontal ligament-bone (PDL-bone) and periodontal ligament-cementum (PDL-cementum). The soft-hard tissue interfaces are responsible for attachment and are critical to the overall biomechanical efficiency of the bone–tooth complex. In this study, the PDL-bone and PDL-cementum attachment sites in human molars were investigated to identify the structural orientation and integration of the PDL with bone and cementum. These attachment sites were characterized from a combined materials and mechanics perspective and were related to macro-scale function.High resolution complimentary imaging techniques including atomic force microscopy, scanning electron microscopy and micro-scale X-ray computed tomography (Micro XCT™) illustrated two distinct orientations of PDL; circumferential-PDL (cir-PDL) and radial-PDL (rad-PDL). Within the PDL-space, the primary orientation of the ligament was radial (rad-PDL) as is well known. Interestingly, circumferential orientation of PDL continuous with rad-PDL was observed adjacent to alveolar bone and cementum. The integration of the cir-PDL was identified by 1–2 μm diameter PDL-inserts or Sharpey’s fibers in alveolar bone and cementum. Chemically and biochemically the cir-PDL adjacent to bone and cementum was identified by relatively higher carbon and lower calcium including the localization of small leucine rich proteins responsible for maintaining soft-hard tissue cohesion, stiffness and hygroscopic nature of PDL-bone and PDL-cementum attachment sites. The combined structural and chemical properties provided graded stiffness characteristics of PDL-bone (Er range for PDL: 10–50MPa; bone: 0.2–9.6 GPa) and PDL-cementum (Er range for cementum: 1.1–8.3 GPa), which was related to the macro-scale function of the bone–tooth complex.
Keywords: Interfaces; Bone-tooth complex; Biomechanics; Fibrous joint; Cementum; Alveolar bone
Carbodiimide cross-linked amniotic membranes for cultivation of limbal epithelial cells
by David Hui-Kang Ma; Jui-Yang Lai; Hsiao-Yun Cheng; Chen-Chi Tsai; Lung-Kun Yeh (pp. 6647-6658).
In ophthalmic tissue engineering, amniotic membrane (AM) is one of the most prevalent natural matrices used for limbal epithelial cell (LEC) cultivation and transplantation. However, the application of AM as a scaffold is limited by its low biomechanical strength and rapid biodegradation. The present study reports the development of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)/ N-hydroxysuccinimide (NHS) cross-linked AM as an LEC carrier. The collagenous tissue materials were modified with varying cross-linker concentrations (0–0.25 mmol EDC/mg AM) and were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), ninhydrin assays, electron microscopy, light transmission measurements, mechanical and in vitro degradation tests, as well as diffusion permeability and cell culture studies. Our results showed that chemical cross-linking approaches saturation at concentrations of 0.05 mmol EDC/mg AM. The formation of cross-links (i.e., amide bonds) in the samples treated with 0.05 mmol EDC/mg AM may cause significant aggregation of tropocollagen molecules and collagen microfibrils without affecting cell morphology of biological tissues. With the optimum concentration of 0.05 mmol EDC/mg AM, chemical cross-linker could significantly enhance the mechanical and thermal stability, optical transparency, and resistance to collagenase digestion. Continuous permeation of albumin through the cross-linked AM would be helpful to cell growth over the matrix surface. In addition, the EDC cross-linked samples were able to support LEC proliferation and preserve epithelial progenitor cells in vitro and in vivo. It is concluded that the AM cross-linked with 0.05 mmol EDC/mg AM may be a potential biomaterial for regenerative medicine.
Keywords: Amniotic membrane; Carbodiimide cross-linking; Limbal epithelial cells; Cornea; Tissue engineering/regenerative medicine
Mechanical properties of DNA biofilms adsorbed on microcantilevers in label-free biodetections
by Neng-Hui Zhang; Jian-Zhong Chen; Jing-Jing Li; Zou-Qing Tan (pp. 6659-6666).
Biomolecule adsorption is a fundamental process in the design of biosensors. Mechanical/electrical/thermal properties of biofilms have great influences on biodetection signals. The double-stranded DNA (dsDNA) biofilm adhered on microcantilever is treated as a bending beam with a macroscopic elastic modulus in the viewpoint of continuum mechanics. Accounting for hydration force, electrostatic repulsion and conformational entropy, moment-angle diagrams of dsDNA biofilm in pure bending state are depicted with the help of the energy conservation law and a mesoscopic liquid crystal theory presented by Strey et al. An analytical model is provided to predict macroscopic elastic modulus of dsDNA biofilm as a function of nanoscopic properties of dsDNA, packing density, buffer salt concentration and etc. The parameters for microcantilever-DNA system are obtained by curve fitting with Stachowiak’s experimental data based on a modified Stoney’s formula. Elastic modulus grows exponentially with the enhancement of packaging density, but diminishes with the increase of buffer salt concentration, and its order is about 1 ∼ 10 MPa. Conformational entropy is one of predominant factors considered in near-surface system whether in high or low salt consternation.
Keywords: DNA; Adsorption; Biosensor; Biofilm; Mechanical properties; Surface energy