Effect of pH on dentin protease inactivation by carbodiimide.

A water-soluble crosslinking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), has been used as a pretreatment of acid-etched dentin to inactivate matrix-bound endogenous dentin proteases. The aim of this study was to evaluate the effect of pH on the inactivation capacity of EDC. Demineralized dentin beams (1 × 2 × 6 mm) were divided into six groups (n = 8 per group). Then, EDC (0.3 M) was solubilized in distilled water with pH of 2, 4, 6, 7, 9, or 11. Control EDC was solubilized in 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer and its pH was adjusted to 6. The dentin beams were pretreated for 1 min with EDC at each pH or with EDC in MES buffer at pH 6.0 and then incubated in 1 ml of simulated body fluid (pH 7.2) for 1, 3, 7, or 14 d. Untreated beams served as controls. At each study time-point, the dry mass of dentin beams was assessed and the incubation media were analyzed for carboxyterminal telopeptide of type-I collagen (ICTP) and C-terminal telopeptide of type I collagen (CTX) using specific ELISAs. Data were subjected to repeat-measures anova. The results of the study indicated that specimens pretreated with EDC in MES buffer showed the lowest collagen degradation in terms of mass loss and release of telopeptides, while specimens pretreated in alkaline media showed the highest collagen degradation. This study indicates that the pH of the EDC solution plays an important role in the stability of dentin protease inactivation.

ECDI-fixed allogeneic splenocytes combined with α1-antitrypsin prolong survival of rat renal allografts.

Pre- and post-transplant infusions of donor splenocytes treated with 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI-SPs) induce donor-specific tolerance and prolong rat renal allograft survival. However, proinflammatory cytokine production during peritransplantation negates the effects of ECDI-SPs. Therefore, we reasoned that blocking proinflammatory cytokines would promote long-term ECDI-SP-induced allograft survival. We therefore examined the effects of infusing ECDI-SPs alone or in combination with a short course of α1-Antitrypsin (AAT) on the long-term outcomes of a rat kidney allograft model. The data showed that ECDI-SPs+AAT promote renal allograft survival compared with ECDI-SPs alone. This effect was accompanied by expansion of Foxp3+ Tregs, enhanced alloantigen-specific Treg function, and modulation of expression levels of proinflammatory cytokines IL-1ß, IL-6, TNF-α, and the anti-inflammatory cytokine IL-10. In conclusion, our strategy of combining ECDI-SPs and AAT provides a promising approach for inducing specific transplant tolerance.

Tempering allorecognition to induce transplant tolerance with chemically modified apoptotic donor cells.

The development of organ transplantation as a therapy for end-stage organ failure is among the most significant achievements of 20th century medicine, but chronic rejection remains a barrier to achieving long-term success. Current therapeutic regimens consist of immunosuppressive drugs that are efficient at delaying rejection but are associated with significant risks such as opportunistic infections, toxicity, and malignancy. Thus, the induction of specific immune tolerance to transplant antigens is the coveted aim of researchers. The use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (ECDI)-treated, autoantigen-coupled syngeneic leukocytes has been developed as a specific immunotherapy in preclinical models of autoimmunity and is currently in a phase II clinical trial for the treatment of multiple sclerosis. In this review, we discuss the use of allogeneic ECDI-treated apoptotic donor leukocytes (allo-ECDI-SP) as a strategy for inducing antigen-specific tolerance in allogeneic transplantation. Allo-ECDI-SP therapy induces long-term systemic immune tolerance to transplant antigens by subverting alloimmune recognition and exploiting apoptotic cell uptake pathways to recapitulate innate mechanisms of peripheral tolerance. Lastly, we discuss potential indications and challenges for transitioning allo-ECDI-SP therapy into clinical practice.

Carbodiimide inactivation of MMPs and effect on dentin bonding.

The use of protein cross-linking agents during bonding procedures has been recently proposed to improve bond durability. This study aimed to use zymography and in situ zymography techniques to evaluate the ability of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linker to inhibit matrix metalloproteinase (MMP) activity. The hypotheses tested were that: (1) bonding procedures increase dentin gelatinolytic activity and (2) EDC pre-treatment prevents this enzymatic activity. The zymographic assay was performed on protein extracts obtained from dentin powder treated with Optibond FL or Scotchbond 1XT with or without 0.3M EDC pre-treatment. For in situ zymography, adhesive/dentin interfaces were created with the same adhesives applied to acid-etched dentin slabs pre-treated or not with EDC conditioner. Zymograms revealed increased expression of dentin endogenous MMP-2 and -9 after adhesive application, while the use of EDC as a primer inactivated dentin gelatinases. Results of in situ zymograpy showed that hybrid layers of tested adhesives exhibited intense collagenolytic activity, while almost no fluorescence signal was detected when specimens were pre-treated with EDC. The correlative analysis used in this study demonstrated that EDC could contribute to inactivate endogenous dentin MMPs within the hybrid layer created by etch-and-rinse adhesives.

Novel collagen scaffolds prepared by using unnatural D-amino acids assisted EDC/NHS crosslinking.

This work discusses the preparation and characterization of novel collagen scaffolds by using unnatural D-amino acids (Coll-D-AAs)-assisted 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxyl succinimide(NHS)-initiated crosslinking. The mechanical strength, hydrothermal and structural stability, resistance to biodegradation and the biocompatibility of Coll-D-AAs matrices were investigated. The results from Thermo mechanical analysis, Differential scanning calorimetric analysis and Thermo gravimetric analysis of the Coll-D-AAs matrices indicate a significant increase in the tensile strength (TS, 180±3), % elongation (% E, 80±9), elastic modulus (E, 170±4) denaturation temperature (T d, 108±4) and a significant decrease in decomposition rate (Tg, 64±6). Scanning electron microscopic and Atomic force microscopic analyses revealed a well-ordered with properly oriented and well-aligned structure of the Coll-D-AAs matrices. FT-IR results suggest that the incorporation of D-AAs favours the molecular stability of collagen matrix. The D-AAs stabilizing the collagen matrices against degradation by collagenase would have been brought about by protecting the active sites in collagen. The Coll-D-AAs matrices have good biocompatibility when compared with native collagen matrix. Molecular docking studies also indicate better understanding of bonding pattern of collagen with D-AAs. These Coll-D-AAs matrices have been produced in high mechanical strength, thermally and biologically stable, and highly biocompatible forms that can be further manipulated into the functional matrix suitable in designing scaffolds for tissue engineering and regenerative medical applications.

Characteristics of thermostable endoxylanase and ß-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1 and its applicability in generating xylooligosaccharides and xylose from agro-residues.

An extremely thermophilic bacterial isolate that produces a high titer of thermostable endoxylanase and ß-xylosidase extracellularly in an inducible manner was identified as Geobacillus thermodenitrificans TSAA1. The distinctive features of this strain are alkalitolerance and halotolerance. The endoxylanase is active over a broad range of pH (5.0-10.0) and temperatures (30-100 °C) with optima at pH 7.5 and 70 °C, while ß-xylosidase is optimally active at pH 7.0 and 60 °C. The T 1/2 values of the endoxylanase and ß-xylosidase are 30 min at 80 °C, and 180 min at 70 °C, respectively. The endoxylanase activity is stimulated by dithiothreitol, but inhibited strongly by EDAC and Woodward's reagent K. N-BS and DEPC strongly inhibited ß-xylosidase. MALDI-ToF (MS/MS) analysis of tryptic digest of ß-xylosidase revealed similarity with that of G. thermodenitrificans NG 80-2, and suggested that this belongs to the GH 52 glycosyl hydrolase super family. The action of endoxylanase on birch wood xylan and agro-residues such as wheat bran and wheat straw liberated xylooligosaccharides similar to endoxylanases of the family 10 glycoside hydrolases, while the enzyme preparation having both endoxylanase and ß-xylosidase liberated xylose as main hydrolysis product.

Site specific properties of carious dentin matrices biomodified with collagen cross-linkers.

PURPOSE: To assess in non-cavitated carious teeth the mechanical properties of dentin matrix by measuring its reduced modulus of elasticity and the effect of dentin biomodification strategies on three dentin matrix zones: caries-affected, apparently normal dentin below caries-affected zone and sound dentin far from carious site. METHODS: Nano-indentations were performed on dentin matrices of carious molars before and after surface modification using known cross-linking agents (glutaraldehyde, proanthocyanidins from grape seed extract and carbodiimide). RESULTS: Statistically significant differences were observed between dentin zones of demineralized dentin prior to surface biomodification (P < 0.05). Following surface modification, there were no statistically significant differences between dentin zones (P < 0.05). An average increase of 30-fold, 2-fold and 2.2-fold of the reduced modulus of elasticity was observed following treatments of the three dentin zones with proanthocyanidin, carbodiimide and glutaraldehyde, respectively.

Effects of large-area irradiated laser phototherapy on peripheral nerve regeneration across a large gap in a biomaterial conduit.

This paper proposes a novel biodegradable nerve conduit comprising 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linked gelatin, annexed with ß-tricalcium phosphate (TCP) ceramic particles (EDC-Gelatin-TCP, EGT). In this study, the EGT-implant site in rats was irradiated using a large-area 660 nm AlGaInP diode laser (50 mW) to investigate the feasibility of laser stimulation in the regeneration of a 15-mm transected sciatic nerve. The animals were divided into three groups: a sham-irradiated group (EGT/sham); an experimental group undergoing low-level laser (LLL) therapy (EGT/laser); a control group undergoing autologous nerve grafts (autografts). Twelve weeks after implantation, walking track analysis showed a significantly higher sciatic functional index (p < 0.05) and improved toe spreading development in the EGT/laser and autograft groups than in the EGT/sham group. In electrophysiological measurement, both the mean peak amplitude and the area under the compound muscle action potential curves in the EGT/laser and autograft groups showed significantly improved functional recovery than the EGT/sham group (p < 0.05). Compared with the EGT/sham group, the EGT/laser and autograft groups displayed a reduction in muscular atrophy. Histomorphometric assessments revealed that the EGT/laser group had undergone more rapid nerve regeneration than the EGT/sham group. The laser-treated group also presented greater neural tissue area as well as larger axon diameter and thicker myelin sheath than the tube group without the laser treatment, indicating improved nerve regeneration. Thus, these assessments demonstrate that LLL therapy can accelerate the repair of a transected peripheral nerve in rats after being bridged with EGT conduit.

Design and analysis of braid-twist collagen scaffolds.

Collagen type I fiber-based scaffolds for anterior cruciate ligament (ACL) replacement were evaluated for their mechanical properties and their ability to promote cellular proliferation. Prior to scaffold formation, two crosslinking methods were investigated on individual reconstituted collagen type I fibers, ultraviolet radiation, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Crosslinking with EDC for 4 hr yielded mechanical properties similar to the human ACL; therefore, scaffold crosslinking was done with EDC for 4 hr. A braid-twist scaffold design was used, and scaffolds were left uncrosslinked, crosslinked after the addition of gelatin, or crosslinked without gelatin. The ultimate tensile strength, Young's modulus, and viscoelastic properties of the scaffolds were then evaluated. In order to assess cellular response on the scaffolds, primary rat ligament fibroblast cells were seeded upon the scaffolds. Cell activity was evaluated at days 7, 14, and 21 using a Cell Titer 96(®) AQueous One Solution Cell Proliferation Assay (MTS Assay). The mechanical testing results showed that among the three scaffold groups, the crosslinked scaffolds without gelatin displayed an ultimate tensile strength, Young's modulus, and viscoelastic properties that were closest to the human ACL. Improvements are still desired to enhance the mechanical compliance and ductility of these scaffolds. Cell activity was observed on all cell-seeded scaffolds by day 7, but by day 21 only the crosslinked scaffolds without gelatin displayed increased cellular activity compared with the negative controls. Although improvement is still needed, the results suggest that these scaffolds have the potential to contribute toward an ACL replacement strategy.

Herceptin conjugates linked by EDC boost direct tumor cell death via programmed tumor cell necrosis.

Tumor-targeted antibody therapy is one of the safest biological therapeutics for cancer patients, but it is often ineffective at inducing direct tumor cell death and is ineffective against resistant tumor cells. Currently, the antitumor efficacy of antibody therapy is primarily achieved by inducing indirect tumor cell death, such as antibody-dependent cell cytotoxicity. Our study reveals that Herceptin conjugates, if generated via the crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), are capable of engendering human epidermal growth factor receptor 2 (Her2) positive tumor cells death. Using a high-performance liquid chromatography (HPLC) system, three peaks with estimated molecular weights of antibody monomer, dimer, and trimer were isolated. Both Herceptin trimer and dimer separated by HPLC induced significant levels of necrotic tumor cell death, although the trimer was more effective than the dimer. Notably, the Herceptin trimer also induced Herceptin-resistant tumor cell death. Surprisingly different from the known cell death mechanism that often results from antibody treatment, the Herceptin trimer elicited effective and direct tumor cell death via a novel mechanism: programmed cell necrosis. In Her2-positive cells, inhibition of necrosis pathways significantly reversed Herceptin trimer-induced cell death. In summary, the Herceptin trimer reported herein harbors great potential for overcoming tumor cell resistance to Herceptin treatment.

Impact of carbondiimide crosslinker used for magnetic carbon nanotube mediated GFP plasmid delivery.

1-Ethyl-3-(3-dimethylaminopropyl) carbondiimide hydrochloride (EDC) is commonly used as a crosslinker to help bind biomolecules, such as DNA plasmids, with nanostructures. However, EDC often remains, after a crosslink reaction, in the micro-aperture of the nanostructure, e.g., carbon nanotube. The remaining EDC shows positive green fluorescent signals and makes a nanostructure with a strong cytotoxicity which induces cell death. The toxicity of EDC was confirmed on a breast cancer cell line (MCF-7) and two leukemic cell lines (THP-1 and KG-1). The MCF-7 cells mainly underwent necrosis after treatment with EDC, which was verified by fluorescein isothiocyanate (FITC) annexin V staining, video microscopy and scanning electronic microscopy (SEM). If the EDC was not removed completely, the nanostructures with remaining EDC produced a green fluorescent background that could interfere with flow cytometry (FACS) measurement and result in false information about GFP plasmid delivery. Effective methods to remove residual EDC on macromolecules were also developed.

Application of carbodiimide derivatized synovial fluid to enhance extrasynovial tendon gliding ability.

PURPOSE: To investigate the effects of surface modification of extrasynovial tendon with a carbodiimide derivatized synovial fluid (SF) on the gliding ability of extrasynovial tendon for a possible tendon graft application. METHODS: We used 63 peroneus longus tendons from canine hind legs. We immediately assessed 3 tendons morphologically using a scanning electron microscope (SEM); these served as the normal tendon group. The other 60 tendons were randomly assigned to each of 6 experimental groups treated with (1) control (saline); (2) 1% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) plus 1% N-hydroxysuccinimide (NHS) (cd only); (3) 1% EDC/NHS plus 10% gelatin (cd-G); (4) SF plus 1% EDC/NHS plus 10% gelatin (cd-SF-G); (5) SF only; or (6) SF plus 1% EDC/NHS (cd-SF). We measured the gliding resistance for 1,000 cycles of simulated flexion-extension motion. We also observed the tendon surface smoothness by SEM. RESULTS: Compared with the first cycle in each group, the gliding resistance after 1,000 cycles of tendon motion was significantly increased in the control, cd only, cd-gelatin, SF only, and cd-SF groups (p<.05). In contrast, we found no significant difference in gliding resistance between the first cycle and 1,000 cycles for the cd-SF-G-treated group. In addition, the gliding resistance in the cd-SF, cd-G, and cd-SF-G groups was significantly lower than the control group after 1,000 cycles of tendon motion (p<.05) and the gliding resistance of the cd-SF-G group was significantly lower than both the cd-G and cd-SF groups (p<.05). On SEM, the surface treated with cd-SF-G was smooth after 1,000 cycles, whereas the other surfaces were rough. CONCLUSIONS: Surface modification of extrasynovial tendon with cd-SF-G improves tendon gliding ability. This treatment may be useful clinically in improving the outcomes of tendon autografts.

Immobilization of a phosphonated analog of matrix phosphoproteins within cross-linked collagen as a templating mechanism for biomimetic mineralization.

Immobilization of phosphoproteins on a collagen matrix is important for the induction of intrafibrillar apatite mineralization. Unlike phosphate esters, polyphosphonic acid has no reactive sites for covalent binding to collagen amine groups. Binding of poly(vinyl phosphonic acid) (PVPA), a biomimetic templating analog of matrix phosphoproteins, to collagen was found to be electrostatic in nature. Thus, an alternative retention mechanism was designed for immobilization of PVPA on collagen by cross-linking the latter with carbodiimide (EDC). This mechanism is based on the principle of size exclusion entrapment of PVPA molecules within the internal water compartments of collagen. By cross-linking collagen with EDC, a zero length cross-linking agent, the sieving property of collagen is increased, enabling the PVPA to be immobilized within the collagen. The absence of covalent cross-linking between PVPA and collagen was confirmed by Fourier transform infrared spectroscopy. Based on these results, a concentration range for immobilized PVPA to template intrafibrillar apatite deposition was established and validated using a single layer reconstituted type I collagen mineralization model. In the presence of a polyacrylic acid-containing mineralization medium optimal intrafibrillar mineralization of the EDC-cross-linked collagen was achieved using 500 and 1000 µg ml⁻¹ PVPA. The mineralized fibrils exhibited a hierarchical order of intrafibrillar mineral infiltration, as manifested by the appearance of electron-dense periodicity within unstained fibrils. Understanding the basic processes in intrafibrillar mineralization of reconstituted collagen creates opportunities for the design of tissue engineering materials for hard tissue repair and regeneration.

Mycobacterial cell-wall skeleton as a universal vaccine vehicle for antigen conjugation.

Mycobacterial cell-wall skeleton (CWS) is an immunoactive and biodegradable particulate adjuvant and has been used for immunotherapy in patients with cancer. The CWS of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS) was studied as a universal vaccine vehicle for antigen conjugation, to develop potentially effective and safe vaccines. Here, we describe experiments in which protein antigens, such as keyhole limpet haemocyanin (KLH), ovalbumin (OVA) and bovine serum albumin (BSA) were highly efficiently coupled to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS)-activated carboxyl groups of BCG-CWS, and tested the immunogenicity of OVA-conjugated BCG-CWS vaccine. We found that a strong immune response was induced in mice immunised with OVA-conjugated BCG-CWS, which was similar to the enhancement of the immune responses in mice immunised with OVA and complete Freund's adjuvant. Covalent conjugation of OVA to BCG-CWS was essential for Th1-skewed immune responses, with prominent expression of IFN-γ. Furthermore, antigen-conjugated BCG-CWS vaccine is simple to manufacture, safe, and easy to use. Our results suggest that mycobacterial CWS as a universal vaccine vehicle for conjugation of a wide variety of antigens constitutes a breakthrough for development of the most promising vaccines for infections, allergic diseases, and cancer.

Chemical and thermal cross-linking of collagen and elastin hydrolysates.

Chemical and thermal cross-linking of collagen soluble in acetic acid and elastin hydrolysates soluble in water have been studied. Solutions of collagen and elastin hydrolysates were treated using variable concentrations of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Moreover, diepoxypropylether (DEPE) has been used as cross-linking agent. Films made of collagen and elastin hydrolysates were also treated with temperature at 60°C and 100°C to get additional cross-links. The effect of cross-linking has been studied using FTIR spectroscopy, thermal analysis, AFM and SEM microscopy. Mechanical and surface properties of materials have been studied after cross-linking. It was found that thermal and mechanical properties of collagen and elastin materials have been altered after thermal treatment and after the reactions with EDC/NHS and/or DEPE. Surface properties of collagen materials after chemical cross-linking have been modified. Thermal and chemical cross-linking of collagen films lead to alteration of polarity of the surface.

Cationic nanoemulsions as non-viral vectors for plasmid DNA delivery.

Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for therapeutic gene delivery. Many cationic lipid carriers including liposomes, emulsions, and solid lipid nanoparticles are used to transfer plasmid DNA. Stable nanoemulsions were prepared and modified by conjugating fatty acids with cationic amino acids including lysine, arginine, and histidine with the help of carbodiimide. Concentrations of crosslinker and amino acids were optimized to obtain the maximal surface potential. The zeta potential and size distribution of the cationic nanoemulsions were measured using photon correlation spectroscopy. The morphology of nanoemulsion-DNA complexes was examined by transmission electron microscopy. The transfection efficiencies and cytotoxicity of three cationic nanoemulsions were evaluated using 3T3 fibroblast cells. The maximal zeta potentials of lysine-, arginine-, and histidine-modified nanoemulsions were 50, 43, and 7 mV, respectively. The transfection efficiencies of amino acid-modified nanoemulsions were in the order of lysine > arginine > histidine. Low cytotoxicities of these three amino acid-modified nanoemulsions were observed. A facile and inexpensive in situ modification for producing cationic nanoemulsions was developed. The results show the potential of amino acid-modified cationic nanoemulsions as non-viral vectors for gene delivery.

Long-term effect of carbodiimide on dentin matrix and resin-dentin bonds.

OBJECTIVES: To characterize the interaction of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide Hydrochloride (EDC) with dentin matrix and its effect on the resin-dentin bond. METHODS: Changes to the stiffness of demineralized dentin fragments treated with EDC/N-hydroxysuccinimide (NHS) in different solutions were evaluated at different time points. The resistance against enzymatic degradation was indirectly evaluated by ultimate tensile strength (UTS) test of demineralized dentin treated or not with EDC/NHS and subjected to collagenase digestion. Short- and long-term evaluations of the strength of resin-dentin interfaces treated with EDC/NHS for 1 h were performed using microtensile bond strength (microTBS) test. All data (MPa) were individually analyzed using ANOVA and Tukey HSD tests (alpha = 0.05). RESULTS: The different exposure times significantly increased the stiffness of dentin (p < 0.0001, control-5.15 and EDC/NHS-29.50), while no differences were observed among the different solutions of EDC/NHS (p = 0.063). Collagenase challenge did not affect the UTS values of EDC/NHS group (6.08) (p > 0.05), while complete degradation was observed for the control group (p = 0.0008, control-20.84 and EDC/NHS-43.15). EDC/NHS treatment did not significantly increase resin-dentin muTBS, but the values remained stable after 12 months water storage (p < 0.05). CONCLUSIONS: Biomimetic use of EDC/NHS to induce exogenous collagen cross-links resulted in increased mechanical properties and stability of dentin matrix and dentin-resin interfaces.

Tissue substitutes with improved angiogenic capabilities: an in vitro investigation with endothelial cells and endothelial progenitor cells.

The use of implantable biomaterials, such as artificial skin substitutes used for dermal defects, remains limited by the low angiogenic potential of these products. The rapid in vivo degradation of growth factors contributes to the limiting of angiogenesis in biomaterials. Here, we report on collagen sponges in which vascular endothelial growth factor (VEGF) was immobilized through physical binding to heparin, covalently incorporated in the matrix via cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide. The in vitro release of VEGF over time and endothelial cell proliferation were investigated in matrices modified at varying heparin to EDC ratios either nonloaded or loaded with VEGF. ELISA demonstrated a significantly slower in vitro release of VEGF over a period of 5 days from heparinized matrices as compared to their unmodified and cross-linked counterparts. The effects of these modifications on the proliferation of endothelial cells and endothelial progenitor cells were evaluated after 1, 3 and 5 days either according to the bromodeoxyuridine assay or total cell counting with a Neubauer chamber. The endothelial and endothelial progenitor cells cultured in contact with heparinized matrices loaded with VEGF revealed both the highest rate of DNA synthesis and the highest total cell count. Furthermore, these results show that the cross-linking of collagen matrices - both in the presence and absence of heparin - leads to increases of the proliferative activities. We can assume that these changes lead to matrices with increased angiogenic capabilities.

Carbodiimide conjugation of fibronectin on collagen basal lamina analogs enhances cellular binding domains and epithelialization.

To improve the regenerative potential of biomaterials used as bioengineered scaffolds, it is necessary to strategically incorporate biologically active molecules that promote in vivo cellular processes that lead to a fully functional tissue. This work evaluates the effects of strategically binding fibronectin (FN) to collagen basal lamina analogs to enhance keratinocyte functions necessary for complete skin regeneration. We found that FN that was passively adsorbed to collagen-glycosaminoglycan basal lamina analogs enhanced epithelial thickness and keratinocyte proliferation compared with nontreated basal lamina analogs at 3 days of air/liquid (A/L) interface culture. Additionally, we evaluated the availability of FN cellular binding site domains when FN was either passively adsorbed or [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride] conjugated to basal lamina analogs fabricated from collagen-glycosaminoglycan coprecipitate or self-assembled type I collagen. It was found that 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride conjugation of FN significantly enhanced FN binding site presentation as well as epithelial thickness. Overall, the results gained from this study will be used to improve the regenerative capacity of basal lamina analogs for bioengineered skin substitutes as well as the development of bioengineered scaffolds for other tissue engineering applications.

Antibody-labeled liposomes for CT imaging of atherosclerotic plaques: in vitro investigation of an anti-ICAM antibody-labeled liposome containing iohexol for molecular imaging of atherosclerotic plaques via computed tomography.

We evaluated the specific binding of anti-intercellular adhesion molecule 1 (ICAM-1) conjugated liposomes (immunoliposomes, or ILs) to activated human coronary artery endothelial cells (HCAEC) with the purpose of designing a computed tomographic imaging agent for early detection of atherosclerotic plaques. Covalent attachment of anti-ICAM-1 monoclonal antibodies to pre-formed liposomes stabilized with polyethylene glycol yielded ILs, with a coupling efficiency of the ICAM-1 to the liposomes of 10% to 24%. The anti-ICAM-1-labeled ILs had an average diameter of 136 nm as determined by dynamic light-scattering and cryogenic electron microscopy. The ILs' encapsulation of 5-[N-acetyl-(2,3-dihydroxypropyl)-amino)-N, N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-benzene-1,3-dicarboxamide (iohexol) was determined to be 18% to 19% by a dialysis technique coupled with ultraviolet detection of free iohexol. This encapsulation corresponded to 30 to 38 mg iodine per mL IL solution, and the ILs exhibited 91% to 98.5% iohexol retention at room temperature and under physiologic conditions. The specific binding of the ILs to cultured, activated HCAEC was measured using flow cytometry, enzyme-linked immunosorbent assays, and fluorescence microscopy. The immunosorbent assays demonstrated the specificity of binding of anti-ICAM-1 to ICAM-1 compared with control studies using nonspecific immunoglobulin G-labeled ILs. Flow cytometry and fluorescence microscopy experiments demonstrated the expression of ICAM-1 on the surface of activated HCAEC. Therefore, our iohexol-filled ILs demonstrated potential for implementation in computed tomographic angiography to noninvasively detect atherosclerotic plaques that are prone to rupture.