A decellularized porcine pulmonary valved conduit embedded with gelatin.

To prepare a tissue-engineered pulmonary valved conduit (PVC) with good tensile strength and biocompatibility. Sixty adult porcine PVCs were used to determine the optimal decellularization time. Five juvenile porcine decellularized PVCs and five juvenile porcine crosslinked PVCs were subsequently prepared according to the optimized decellularization and crosslinking methods. All PVCs were implanted into juvenile sheep for 8 months and then were harvested for staining. With a low concentration of detergent (0.25% Triton X-100+0.25% sodium deoxycholate), the decellularization effect on porcine PVCs was complete by 24 hours, and there was minimal damage to the matrix. Gelatin embedding and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) crosslinking improved the biomechanical properties of decellularized PVCs and reduced their immunogenicity. After implantation, the diameter and thickness of the PVCs in the decellularized and crosslinked groups increased significantly. In both groups, the conduits were unobstructed, with soft and smooth inner walls and without thrombosis, ulceration or neoplasia. The valves slightly degenerated with mild to moderate regurgitation. CD31-positive endothelial cells were visible on the inner surface of the conduits and valves. Scattered smooth muscle actin-positive cells were found in the middle layer of the conduit. The percentage of CD4- and CD68-positive cells and the calcium content were highest in decellularized porcine PVCs and lowest in ovine PVCs. The percentage of the matrix that was laminin-positive in decellularized and crosslinked porcine PVCs was lower than it was in ovine PVCs. Gelatin-embedded and EDC-crosslinked porcine PVCs can be "hosted" in sheep, with good biocompatibility, growth potential, and reduced calcification.

Simple and nonradioactive detection of microRNAs using digoxigenin (DIG)-labeled probes with high sensitivity.

The discovery of microRNAs (miRNAs), which are ∼21-23 nucleotides that can regulate targeted mRNA by transcript cleavage or protein translation suppression, has changed the landscape of biomedical field greatly. At present, Northern blot analysis based on radioisotopes is still the most popular method on the detection of miRNAs for its high sensitivity. However, radioisotopes have been known for certain disadvantages, such as instability, expense, and safety; thus, developing a nonradioactive and highly sensitive method is needed. Here, we report a simple, nonradioactive, and sensitive method for miRNAs detection based on 5'-phos-3'-DIG-labeled probes prepared through splinted ligation and EDC cross-linking (DSLE). The method was more sensitive than traditional Northern blots with a DIG-labeled DNA probe and can detect as low as 2 fmol of miRNAs. The whole procedure can be completed within 6-8 h. DSLE method is very convenient, cost-effective, time-saving, and highly sensitive.

Northern Blot Detection of Tiny RNAs.

Successful detection of very small RNAs (tiny RNAs, ~8-15 nt in length) by northern blotting depends on tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'-32P-end-label. Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate size (~50-400 nt).

Characterization of covalent crosslinking strategies for synthesizing DNA-based bioconjugates.

An adapted strategy from the conventional 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) crosslinking method was developed to form a covalently coupled phosphoramidated single stranded DNA (ssDNA). Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) results demonstrated that the phosphoramidated ssDNA conjugate is stable for several days, and that phosphoramidation occurred exclusively at the 5' phosphate of ssDNA. A reversed phase high-performance liquid chromatography (RP-HPLC) method with UV detection was developed to determine the yield of conjugates. The methods coefficients of variation (%CV) were less than 6%, and biases ranged from - 5.1 - 1.2%. The conjugate yield via the conventional EDC method was 68.3 ± 2.2%, while that of the adapted EDC/Imidazole method was 79.0 ± 2.4% (n = 10). This study demonstrates a convenient one pot strategy for crosslinking biological molecules.

Targeted protein delivery: carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds.

Tissue engineering response may be tailored via controlled, sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional (3D) ice-templated collagen scaffolds. However, the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored. Here, we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide) microparticles. We probe the effects of subsequent N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride crosslinking on protein release, using microparticles with different internal protein distributions. Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug. The scaffolds display a homogeneous microparticle distribution, and a reduction in pore size and percolation diameter with increased microparticle addition, although these values did not fall below those reported as necessary for cell invasion. The protein distribution within the microparticles, near the surface or more deeply located within the microparticles, was important in determining the release profile and effect of crosslinking, as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold. Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release. Protein located within the bulk of the microparticles, was protected from the crosslinking reaction and no delay in the overall release profile was seen.

Fundamental insight into the effect of carbodiimide crosslinking on cellular recognition of collagen-based scaffolds.

Research on the development of collagen constructs is extremely important in the field of tissue engineering. Collagen scaffolds for numerous tissue engineering applications are frequently crosslinked with 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride (EDC) in the presence of N-hydroxy-succinimide (NHS). Despite producing scaffolds with good biocompatibility and low cellular toxicity the influence of EDC/NHS crosslinking on the cell interactive properties of collagen has been overlooked. Here we have extensively studied the interaction of model cell lines with collagen I-based materials after crosslinking with different ratios of EDC in relation to the number of carboxylic acid residues on collagen. Divalent cation-dependent cell adhesion, via integrins α1ß1, α2ß1, α10ß1 and α11ß1, were sensitive to EDC crosslinking. With increasing EDC concentration, this was replaced with cation-independent adhesion. These results were replicated using purified recombinant I domains derived from integrin α1 and α2 subunits. Integrin α2ß1-mediated cell spreading, apoptosis and proliferation were all heavily influenced by EDC crosslinking of collagen. Data from this rigorous study provides an exciting new insight that EDC/NHS crosslinking is utilising the same carboxylic side chain chemistry that is vital for native-like integrin-mediated cell interactions. Due to the ubiquitous usage of EDC/NHS crosslinked collagen for biomaterials fabrication this data is essential to have a full understanding in order to ensure optimized collagen-based material performance. STATEMENT OF SIGNIFICANCE: Carbodiimide stabilised collagen is employed extensively for the fabrication of biologically active materials. Despite this common usage, the effect of carbodiimide crosslinking on cell-collagen interactions is unclear. Here we have found that carbodiimide crosslinking of collagen inhibits native-like, whilst increasing non-native like, cellular interactions. We propose a mechanistic model in which carbodiimide modifies the carboxylic acid groups on collagen that are essential for cell binding. As such we feel that this research provides a crucial, long awaited, insight into the bioactivity of carbodiimide crosslinked collagen. Through the ubiquitous use of collagen as a cellular substrate we feel that this is fundamental to a wide range of research activity with high impact across a broad range of disciplines.

Effect of carbodiimide cross-linking of decellularized porcine pulmonary artery valvular leaflets.

Decellularization provides low immunogenicity and is only slightly subject to calcification in tissue engineering. However, the mechanical properties of the tissues are weakened after decellularization. We adopted cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to treat decellularized porcine pulmonary artery valvular leaflets to improve their mechanical properties. Twenty porcine pulmonary artery valvular leaflets were divided into three groups: the fresh control group A, group B treated with trypsin and Triton X-100 to remove cells, and group C cross-linked with EDC after decellularization. All samples were evaluated the physical and mechanical properties and were then subcutaneously embedded in rabbits. These valvular leaflets were removed after 1, 2, or 4 weeks and checked for pathological changes. The cells of the valvular leaflets were completely removed. The thickness of the valvular leaflets was thinner in group B than in group A (P<0.01). In the subcutaneous embedding of the group B samples, there was mild immunological response after 1-2 weeks, and parts of the scaffolds were degraded. After 4 weeks, fibroblasts had grown into the scaffolds. In group C, there was an increase in the tensile strength and thermal shrinkage temperature in group C compared with group B (P<0.01). In subcutaneous embedding of the group C samples, there was a mild immunological response after 1-2 weeks. The fibroblasts had grown into the samples. The EDC-based cross-linking procedure can enhance the tensile strength of decellularized pulmonary artery valvular leaflets and both decrease the valvular leaflets' rejection and promote tissue regeneration in vivo.

Decellularized porcine pulmonary arteries cross-linked by carbodiimide.

The physical properties of the tissues are weakened after decellularization, and the exposed collagen fibers are prone to thrombogenesis. Several studies have proven that the use of carbodiimide (EDC) as a cross-linking agent can improve the properties of decellularized xenogeneic scaffold materials. We adopted EDC for the treatment of porcine pulmonary arteries in an effort to improve the physical properties of these arteries following decellularization. Twenty porcine pulmonary arteries were randomly divided into 3 groups. The control group (group A) consisted of fresh porcine pulmonary arteries with no further processing; group B was treated with trypsin and the detergent Triton X-100 to remove cells; and group C was cross-linked with EDC after trypsin and Triton X-100 treatment, as in group B. The pulmonary arteries were assessed based on water content, thickness, tensile strength, and thermal shrinkage temperature, to evaluate the physical properties of all of the samples. The scaffolds were then subcutaneously embedded in rabbits. These constructs were removed after 4 weeks and checked. The cells and matrix components of the arterial walls were removed and the fibrous scaffolds were retained. In group B, the moisture content of the pulmonary arterial walls was increased; and the thickness of the walls and the tensile strength of the pulmonary arteries were decreased in comparison with group A. In subcutaneous embedding of the group B samples in rabbits, after 4 weeks, fibroblasts had grown into the scaffolds and regenerated the tissue. The water content was decreased in the pulmonary arterial walls, there was an increase in the tensile strength and the thermal shrinkage temperature in group C compared with group B. The EDC-based cross-linking procedure can enhance the tensile strength of decellularized pulmonary arteries and decrease scaffold rejection and degradation and promote tissue regeneration in vivo.