Composite hydrogels for nucleus pulposus tissue engineering

Tissue engineering offers a paradigm shift in the treatment of back pain. Engineered intervertebral discs could replace degenerated tissue and overcome the limitations of current treatments, which substantially alter the biomechanical properties of the spine. The centre of the disc, the nucleus pulposus, is an amorphous gel with a large bound water content and it can resist substantial compressive loads. Due to similarities in their compositions, hydrogels have frequently been considered as substitutes for the nucleus pulposus. However, there has been limited work characterising the time-dependent mechanical behaviour of hydrogel scaffolds for nucleus pulposus tissue engineering. Poroelastic behaviour, which plays a key role in nutrient transport, is of particular importance. Here, we investigate the time-dependent mechanical properties of gelatin and agar hydrogels and of gelatin-agar composites. The time-dependent properties of these hydrogels are explored using viscoelastic and poroelastic frameworks. Several gel formulations demonstrate comparable equilibrium elastic behaviour to the nucleus pulposus under unconfined compression, but permeability values that are much greater than those of the native tissue. A range of time-dependent responses are observed in the composite gels examined, presenting the opportunity for targeted design of custom hydrogels with combinations of mechanical properties optimized for tissue engineering applications. © 2011 Elsevier Ltd.

Composite electrospun gelatin fiber-alginate gel scaffolds for mechanically robust tissue engineered cornea.

A severe shortage of good quality donor cornea is now an international crisis in public health. Alternatives for donor tissue need to be urgently developed to meet the increasing demand for corneal transplantation. Hydrogels have been widely used as scaffolds for corneal tissue regeneration due to their large water content, similar to that of native tissue. However, these hydrogel scaffolds lack the fibrous structure that functions as a load-bearing component in the native tissue, resulting in poor mechanical performance. This work shows that mechanical properties of compliant hydrogels can be substantially enhanced with electrospun nanofiber reinforcement. Electrospun gelatin nanofibers were infiltrated with alginate hydrogels, yielding transparent fiber-reinforced hydrogels. Without prior crosslinking, electrospun gelatin nanofibers improved the tensile elastic modulus of the hydrogels from 78±19 kPa to 450±100 kPa. Stiffer hydrogels, with elastic modulus of 820±210 kPa, were obtained by crosslinking the gelatin fibers with carbodiimide hydrochloride in ethanol before the infiltration process, but at the expense of transparency. The developed fiber-reinforced hydrogels show great promise as mechanically robust scaffolds for corneal tissue engineering applications.

Composite electrospun gelatin fiber-alginate gel scaffolds for mechanically robust tissue engineered cornea

A severe shortage of good quality donor cornea is now an international crisis in public health. Alternatives for donor tissue need to be urgently developed to meet the increasing demand for corneal transplantation. Hydrogels have been widely used as scaffolds for corneal tissue regeneration due to their large water content, similar to that of native tissue. However, these hydrogel scaffolds lack the fibrous structure that functions as a load-bearing component in the native tissue, resulting in poor mechanical performance. This work shows that mechanical properties of compliant hydrogels can be substantially enhanced with electrospun nanofiber reinforcement. Electrospun gelatin nanofibers were infiltrated with alginate hydrogels, yielding transparent fiber-reinforced hydrogels. Without prior crosslinking, electrospun gelatin nanofibers improved the tensile elastic modulus of the hydrogels from 78±19. kPa to 450±100. kPa. Stiffer hydrogels, with elastic modulus of 820±210. kPa, were obtained by crosslinking the gelatin fibers with carbodiimide hydrochloride in ethanol before the infiltration process, but at the expense of transparency. The developed fiber-reinforced hydrogels show great promise as mechanically robust scaffolds for corneal tissue engineering applications. © 2013 Elsevier Ltd.

Electrospun fiber - Hydrogel composites for nucleus pulposus tissue engineering

New materials are needed to replace degenerated intervertebral disc tissue and to provide longer-term solutions for chronic back-pain. Replacement tissue potentially could be engineered by seeding cells into a scaffold that mimics the architecture of natural tissue. Many natural tissues, including the nucleus pulposus (the central region of the intervertebral disc) consist of collagen nanofibers embedded in a gel-like matrix. Recently it was shown that electrospun micro- or nano-fiber structures of considerable thickness can be produced by collecting fibers in an ethanol bath. Here, randomly aligned polycaprolactone electrospun fiber structures up to 50 mm thick are backfilled with alginate hydrogels to form novel composite materials that mimic the fiber-reinforced structure of the nucleus pulposus. The composites are characterized using both indentation and tensile testing. The composites are mechanically robust, exhibiting substantial strain-to-failure. The method presented here provides a way to create large biomimetic scaffolds that more closely mimic the composite structure of natural tissue. © 2012 Materials Research Society.

Gelatin nanofiber-reinforced alginate gel scaffolds for corneal tissue engineering.

A severe shortage of donor cornea is now an international crisis in public health. Substitutes for donor tissue need to be developed to meet the increasing demand for corneal transplantation. Current attempts in designing scaffolds for corneal tissue regeneration involve utilization of expensive materials. Yet, these corneal scaffolds still lack the highly-organized fibrous structure that functions as a load-bearing component in the native tissue. This work shows that transparent nanofiber-reinforced hydrogels could be developed from cheap, non-immunogenic and readily available natural polymers to mimic the cornea's microstructure. Electrospinning was employed to produce gelatin nanofibers, which were then infiltrated with alginate hydrogels. Introducing electrospun nanofibers into hydrogels improved their mechanical properties by nearly one order of magnitude, yielding mechanically robust composites. Such nanofiber-reinforced hydrogels could serve as alternatives to donor tissue for corneal transplantation.

Measuring the compressive viscoelastic mechanical properties of human cervical tissue using indentation.

The human cervix is an important mechanical barrier in pregnancy which must withstand the compressive and tensile forces generated from the growing fetus. Premature cervical shortening resulting from premature cervical remodeling and alterations of cervical material properties are known to increase a woman׳s risk of preterm birth (PTB). To understand the mechanical role of the cervix during pregnancy and to potentially develop indentation techniques for in vivo diagnostics to identify women who are at risk for premature cervical remodeling and thus preterm birth, we developed a spherical indentation technique to measure the time-dependent material properties of human cervical tissue taken from patients undergoing hysterectomy. In this study we present an inverse finite element analysis (IFEA) that optimizes material parameters of a viscoelastic material model to fit the stress-relaxation response of excised tissue slices to spherical indentation. Here we detail our IFEA methodology, report compressive viscoelastic material parameters for cervical tissue slices from nonpregnant (NP) and pregnant (PG) hysterectomy patients, and report slice-by-slice data for whole cervical tissue specimens. The material parameters reported here for human cervical tissue can be used to model the compressive time-dependent behavior of the tissue within a small strain regime of 25%.

Metallothionein-2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization

The metallothionein-2 (MT-2) gene was isolated from the mandarin fish, one of the most important industrial aquatic animals in China, by using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of MT-2 comprised 60 amino acids and showed approximately 62.3% identity to human metallothionein. Its promoter region was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The MT-2 gene consists of 3 exons and 2 introns, extending approximately 900 bp of genomic sequence. Phylogenetic analysis clearly demonstrated that MT-2 formed a clade with fish metallothionein. The promoter region contained 5 putative metal-regulatory elements (MREs) and 1 TATA box. Real-time quantitative RT-PCR analysis revealed that MT-2 transcripts were significantly increased in the brain and gills and were stable in the muscles, liver, and trunk kidney in Cd2+-stimulated fish. Western blotting analysis demonstrated that the protein of the MT-2 gene was expressed mainly in the gills, liver, heart, trunk kidney, muscle, and intestine; it was weakly detected in the brain and head kidney. Moreover, the MT-2 protein was immunohistochemically detected in the cytoplasm in the liver and trunk kidney. All the above results revealed that the mandarin fish MT-2 would be a useful biomarker for metal pollution. (C) 2008 Published by Elsevier Inc.

Effects of dietary manganese on growth and tissue manganese concentrations of juvenile gibel carp, Carassius auratus gibelio

A 68-day growth trial was conducted in a flow-through system to determine the effect of dietary manganese levels on growth and tissue manganese concentration of juvenile gibel carp (Carassius auratus gibelio). Seven purified diets containing 7.21, 8.46, 9.50, 10.50, 13.03, 19.72 and 22.17 mg manganese (as manganic sulfate) per kilogram diet were fed to triplicate groups of fish (initial weight 3.21 +/- 0.01 g). The results showed that dietary manganese levels did not significantly affect feed intake of the fish. Specific growth rate, feed efficiency, total hepatic superoxide dismutase activity, carcass and skeletal manganese concentration increased significantly with increased dietary manganese(P < 0.05) while condition factor decreased significantly(P < 0.05). It was concluded that dietary requirement of manganese was 13.77 mg Mn per kilogram diet. Carcass and skeletal manganese concentration could also be used to evaluate the manganese requirement. Total hepatic superoxide dismulase activity was not a sensitive indicator for dietary requirement.

Tissue distribution and depuration of the extracted hepatotoxic cyanotoxin microcystins in crucian carp (Carassius carassius) intraperitoneally injected at a sublethal dose

An acute toxicity experiment was conducted by intraperitoneal injection with a sublethal dose of extracted microcystins (MCs), 50 mu g MC-LR (where L = leucine and R = arginine) equivalent/kg body weight (BW), to examine tissue distribution and depuration of MCs in crucian carp (Carassius carassius). Liver to body weight ratio increased at 3, 12, 24, and 48 h postinjection compared with that at 0 h (p < 0.05). MC concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, 48, and 168 h postinjection using liquid chromatography coupled with mass spectrometry (LC-MS). The highest concentration of MCs (MC-RR + MC-LR) was found in blood, 2 -270 ng/g dry weight (DW), followed by heart (3 -100 ng/g DW) and kidney (13 -88 ng/g DW). MC levels were relatively low in liver, gonad, intestine, spleen, and brain. MC contents in gills, gallbladder, and muscle were below the limit of detection. Significant negative correlation was present between MC-RR concentration in blood and that in kidney, confirming that blood was important in the transportation of MC-RR to kidney for excretion. Rapid accumulation and slow degradation of MCs were observed in gonad, liver, intestine, spleen, and brain. Only 0.07% of injected MCs were detected in liver. The recovery of MCs in liver of crucian carp seemed to be dose dependent.

Molecular evolution of connective tissue growth factor in Cyprinidae (Teleostei : Cypriniformes)

Connective tissue growth factor (CTGF) plays an important role in regulation of cell growth, differentiation, apoptosis and individual development in animals. The study of sequences variation and molecular evolution of CTGF gene across various species of the cyprinid could be helpful for understanding of speciation and gene divergence in this kind of fish. In this study, 19 novel sequences of CTGF gene were obtained from the representative species of the family Cyprinidae using PCR amplification, cloning and sequencing. Phylogenetic relationships of Cyprinidae were reconstructed by neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian method. Oryzias latipes from the family Cyprinodontidae was assigned to be the outgroup taxon. Leuciscini and Barbini were clustered into the monophyletic lineages, respectively, with the high nodal supports. The estimation of the ratio of non-synonymous to synonymous substitution (dN/dS) for the various branches indicated that there stood the different evolution rates between the Leuciscini and the Barbini. With the ratio of dN/dS of the Leuciscini being lower than that of the Barbini, species within the Barbini were demonstrated to be subjected to the relatively less selection pressure and under the relaxable evolution background. A 6 by indel (insertion/deletion) was found at the 5' end of CTGF gene of Cyprinidae, and this 6 by deletion only appeared in the Leuciscini, which is a typical characteristic of the Leuciscini and provides evidence for the monophylogeny of the Leuciscini. For the amino acid sequences of CTGF protein, the most variations and indels were distributed in the signal region and IGFBP region of this protein, implying that these variations were correlated with the regulation of the CTGF gene expression and protein activity. (c) 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Bioelectronics meets nanomedicine for cardiovascular implants: PEDOT-based nanocoatings for tissue regeneration.

BACKGROUND: An exciting direction in nanomedicine would be to analyze how living cells respond to conducting polymers. Their application for tissue regeneration may advance the performance of drug eluting stents by addressing the delayed stent re-endothelialization and late stent thrombosis. METHODS: The suitability of poly (3, 4-ethylenedioxythiophene) (PEDOT) thin films for stents to promote cell adhesion and proliferation is tested in correlation with doping and physicochemical properties. PEDOT doped either with poly (styrenesulfonate) (PSS) or tosylate anion (TOS) was used for films' fabrication by spin coating and vapor phase polymerization respectively. PEGylation of PEDOT: TOS for reduced immunogenicity and biofunctionalization of PEDOT: PSS with RGD peptides for induced cell proliferation was further applied. Atomic Force Microscopy and Spectroscopic Ellipsometry were implemented for nanotopographical, structural, optical and conductivity measurements in parallel with wettability and protein adsorption studies. Direct and extract testing of cell viability and proliferation of L929 fibroblasts on PEDOT samples by MTT assay in line with SEM studies follow. RESULTS: All PEDOT thin films are cytocompatible and promote human serum albumin adsorption. PEDOT:TOS films were found superior regarding cell adhesion as compared to controls. Their nanotopography and hydrophilicity are significant factors that influence cytocompatibility. PEGylation of PEDOT:TOS increases their conductivity and hydrophilicity with similar results on cell viability with bare PEDOT:TOS. The biofunctionalized PEDOT:PSS thin films show enhanced cell proliferation. CONCLUSIONS: The application of PEDOT polymers has evolved as a new perspective to advance stents. GENERAL SIGNIFICANCE: In this work, nanomedicine involving nanotools and novel nanomaterials merges with bioelectronics to stimulate tissue regeneration for cardiovascular implants. This article is part of a Special Issue entitled Organic Bioelectronics - Novel Applications in Biomedicine.

Mechanics of biological networks: from the cell cytoskeleton to connective tissue.

From the cell cytoskeleton to connective tissues, fibrous networks are ubiquitous in metazoan life as the key promoters of mechanical strength, support and integrity. In recent decades, the application of physics to biological systems has made substantial strides in elucidating the striking mechanical phenomena observed in such networks, explaining strain stiffening, power law rheology and cytoskeletal fluidisation - all key to the biological function of individual cells and tissues. In this review we focus on the current progress in the field, with a primer into the basic physics of individual filaments and the networks they form. This is followed by a discussion of biological networks in the context of a broad spread of recent in vitro and in vivo experiments.