Circulating endothelial progenitor cells in small-diameter artificial blood vessel.

In the tissue engineering research field, the presence of circulating endothelial progenitor cells (EPCs) in the peripheral blood of adults represents a promising cell source to grow autologous endothelium on blood-contacting devices. Materials functionalized with EPC-specific molecules are an intriguing strategy to induce the homing and differentiation of the trapped EPCs into endothelial cells to generate a non-thrombogenic surface. Although the EPCs have been identified in adult peripheral blood about 30 years ago, in the subsequent literatures, the term "EPCs" has encompassed different cell populations with a mixed ability to contribute to the formation of blood vessels. This confusion is due to limited functional characterization of "EPCs", an improper nomenclature, and the poorly standardized protocols. This review will screen the literature about "EPCs" to propose a specific nomenclature, phenotypic characterization, and, eventually, a protocol to perform reliable experiments.

Design of in situ porcine closed-circuit system for assessing blood-contacting biomaterials.

The overall pre-clinical process of determining the blood compatibility of any medical device involves several stages. Although the primary purpose is to protect the patients, laboratory testing has been over-utilized for many years with a huge number of unnecessary animal tests being done routinely. Recently, the elimination of needless testing has become important in controlling the cost of healthcare and in addressing many issues related to the ethics of animal research. With this in mind, we designed a new in situ porcine closed-circuit system to study the complex interplay between platelets, coagulation proteins, and other cellular elements in pigs. We proved that this system can be implemented in blood compatibility testing and minimize the number of animals used in the experiments.

PreFlap: From Photoacoustic Tomography Images to Vascular Mapping Sheets for Improved Preoperative Flap Evaluation.

OBJECTIVE: Advancements in technology have improved image acquisition and processing in the field of medical imaging, giving medical doctors the tools to implement effective medical care. In plastic surgery, despite advances in anatomical knowledge and technology, problems in preoperative planning for flap surgery remain. METHODS: In this study, we propose a new protocol to analyze three-dimensional (3D) Photoacoustic tomography images and generate two-dimensional (2D) mapping sheets that can help surgeons identify perforators and the perfusion territory during preoperative planning. The core of this protocol is PreFlap, a new algorithm that converts 3D photoacoustic tomography images into 2D vascular mapping images. CONCLUSION: Experimental results demonstrate that PreFlap can improve preoperative flap evaluation, thus can greatly saving surgeons' time and improving surgical outcomes.

Noninvasive visualization of the midline-crossing arterial variation in the deep inferior epigastric artery perforator flap using photoacoustic tomography for application in patients with abdominal scars.

BACKGROUND: The importance of the subcutaneous arterial network crossing the midline in transverse abdominal flaps has been reported. Photoacoustic tomography can be used to noninvasively visualize subcutaneous vascular networks. We applied this novel technology preoperatively in patients undergoing breast reconstruction to detect individual variations in the midline-crossing arteries. METHODS: Six patients scheduled to undergo breast reconstruction using free deep inferior epigastric artery perforator flaps were examined. Each scan of the 12 × 8-cm region took approximately 8 min. The accuracy of the tentative artery evaluation defined by photoacoustic tomography was compared with the arterial phase detected by intraoperative indocyanine green angiography. The number of perforator vessels used for the flap, surgical time for flap elevation, and perfusion area ratio were compared with those of the control group. RESULTS: The average match rate between tentative artery prediction by photoacoustic tomography and arterial-phase assessment by intraoperative angiography in five patients was 79.8%. Each midline-crossing artery showed individual variations. The photoacoustic tomography group (PAT-1 to 5) showed 1.8 perforators per flap, 163 min for flap elevation, and 93% perfusion area, with no significant differences from the control group (N = 5). A 63-year-old woman (PAT-6) with abdominal scars, including a midline abdominal incision, showed a preserved midline-crossing artery. The planned single perforator deep inferior epigastric perforator flap was successfully applied to the contralateral perfusion area. CONCLUSIONS: Photoacoustic tomography noninvasively visualizes the subcutaneous midline-crossing arterial networks. Understanding individual vascular variations can support preoperative planning and surgical indication of abdominal flaps, especially in patients with postsurgical scars.

Evaluation of adipogenesis over time using a novel bioabsorbable implant without the addition of exogenous cells or growth factors.

Background: Breast reconstruction is crucial for patients who have undergone mastectomy for breast cancer. Our bioabsorbable implants comprising an outer poly-l-lactic acid mesh and an inner component filled with collagen sponge promote and retain adipogenesis in vivo without the addition of exogenous cells or growth factors. In this study, we evaluated adipogenesis over time histologically and at the gene expression level using this implant in a rodent model. Methods: The implants were inserted in the inguinal and dorsal regions of the animals. At 1, 3, 6, and 12 months post-operation, the weight, volume, and histological assessment of all newly formed tissue were performed. We analyzed the formation of new adipose tissue using multiphoton microscopy and RNA sequencing. Results: Both in the inguinal and dorsal regions, adipose tissue began to form 1 month post-operation in the peripheral area. Angiogenesis into implants was observed until 3 months. At 6 months, microvessels matured and the amount of newly generated adipose tissue peaked and was uniformly distributed inside implants. The amount of newly generated adipose tissue decreased from 6 to 12 months but at 12 months, adipose tissue was equivalent to the native tissue histologically and in terms of gene expression. Conclusions: Our bioabsorbable implants could induce normal adipogenesis into the implants after subcutaneous implantation. Our implants can serve as a novel and safe material for breast reconstruction without requiring exogenous cells or growth factors.

Three-dimensional analysis of load-dependent changes in the orientation of dermal collagen fibers in human skin: A pilot study.

The availability of quantitative structural data on the orientation of collagen fibers is of crucial importance for understanding the behavior of connective tissues. These fibers can be visualized using a variety of imaging techniques, including second harmonic generation (SHG) microscopy. However, characterization of the collagen network requires the accurate extraction of parameters from imaging data. To this end, several automated processes have been developed to identify the preferred orientation of collagen fibers. Common methods include fast Fourier transforms and curvelet transforms, but these tools are mostly used to infer a single preferred orientation. The purpose of this pilot study was to develop an easy procedure for comprehensively comparing multiple human skin samples with the goal of analyzing load-dependent changes via SHG microscopy. We created a 3D model based upon 2D image stacks that provide fiber orientation data perpendicular and parallel to the plane of the epidermis. The SHG images were analyzed by CurveAlign to obtain angle histogram plots containing information about the multiple fiber orientations in each single image. Subsequently, contour plots of the angle histogram intensities were created to provide a useful visual plotting method to clearly show the anomalies in the angle histograms in all samples. Our results provided additional details on how the collagen network carries a load. In fact, analysis of SHG images indicated that increased stretch was accompanied by an increase in the alignment of fibers in the loading direction. Moreover, these images demonstrated that more than one type of preferred orientation is present. In particular, the 3D network of fibers appears to have two preferred orientations in the planes both perpendicular and parallel to the plane of the epidermis.

Preoperative visualization of midline-crossing subcutaneous arteries in transverse abdominal flaps using photoacoustic tomography.

BACKGROUND: Photoacoustic tomography is a noninvasive vascular imaging modality that uses near-infrared pulsed laser light and ultrasound to visualize vessels. We previously demonstrated the utility of photoacoustic tomography for anterolateral thigh flap surgery involving body-attachable vascular mapping sheets. However, it was not possible to obtain clear separate images of arteries and veins. In this study, we tried to visualize subcutaneous arteries that cross the midline of the abdomen, since these arteries are known to be important for obtaining large perfusion areas in transverse abdominal flaps. METHODS: Four patients scheduled to undergo breast reconstruction with abdominal flaps were examined. Photoacoustic tomography was performed preoperatively. The tentative arteries and veins were traced according to the S-factor, an approximate hemoglobin oxygen saturation parameter calculated using 2 laser excitation wavelengths (756 and 797 nm). Intraoperatively, arterial-phase indocyanine green (ICG) angiography was performed after abdominal flap elevation. Images of vessels speculated to be arteries by preoperative photoacoustic tomography were merged with those of intraoperative ICG angiography and analyzed in an 8 × 4-cm2 area below the umbilical region. RESULTS: The S-factor was used to visualize the midline-crossing subcutaneous arteries in all 4 patients. A matching analysis compared preoperative tentative arteries according to photoacoustic tomography with ICG angiography results in the 8 × 4-cm2 area below the umbilical region and indicated a 71.3-82.1% match (average: 76.9% match). CONCLUSIONS: This study demonstrates that the S-factor, a noninvasive, label-free imaging modality, can be used to successfully visualize subcutaneous arteries. This information can aid in selecting perforators for abdominal flap surgery.

Anti-platelet adhesion and in situ capture of circulating endothelial progenitor cells on ePTFE surface modified with poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and hemocompatible peptide 1 (HCP-1).

We recently reported in vitro suppression of platelet adhesion on expanded polytetrafluoroethylene (ePTFE) by surface grafting of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC). However, this may be inadequate for long-term hemocompatibility of blood-contacting biomaterials, and it has led us to develop a strategy of circulating mononuclear cell-capture. ePTFE was treated with argon (Ar) plasma, and grafted with 2-methacryloyloxyethyl phosphorylcholine (MPC) and methacrylic acid (MAA), by glycidyl methacrylate (GMA)-anchored graft polymerization. Next, it was immobilized with integrin α4ß1-positive circulating blood cell-specific peptides, i.e., the traditional arginine-glutamic acid-aspartic acid-valine (REDV), and our original hemocompatible peptide-1 (HCP-1). Both the surfaces retained the anti-platelet property just like the PMPC-grafted surface, and revealed considerable affinity to human umbilical vein endothelial cells (HUVEC), which is a well-known in vitro integrin α4ß1-positive model. Better HUVEC spreading and proliferation was also confirmed, in terms of the cell extension property. Since coagulation and endothelialization on the materials compete in the body, they cannot be properly evaluated separately, in vitro. They were assessed by using an in situ porcine closed-circuit system for 18 h in the present study. Our findings suggest that poly(MPC-co-MAA) is a great ePTFE surface modifier, exhibiting good hemocompatibility in association with REDV/HCP-1 immobilization, which suppresses anti-platelet adhesion and enhances circulating cell capture simultaneously.

The Implication of Spatial Statistics in Human Mesenchymal Stem Cell Response to Nanotubular Architectures.

INTRODUCTION: In recent years there has been ample interest in nanoscale modifications of synthetic biomaterials to understand fundamental aspects of cell-surface interactions towards improved biological outcomes. In this study, we aimed at closing in on the effects of nanotubular TiO2 surfaces with variable nanotopography on the response on human mesenchymal stem cells (hMSCs). Although the influence of TiO2 nanotubes on the cellular response, and in particular on hMSC activity, has already been addressed in the past, previous studies overlooked critical morphological, structural and physical aspects that go beyond the simple nanotube diameter, such as spatial statistics. METHODS: To bridge this gap, we implemented an extensive characterization of nanotubular surfaces generated by anodization of titanium with a focus on spatial structural variables including eccentricity, nearest neighbour distance (NND) and Voronoi entropy, and associated them to the hMSC response. In addition, we assessed the biological potential of a two-tiered honeycomb nanoarchitecture, which allowed the detection of combinatory effects that this hierarchical structure has on stem cells with respect to conventional nanotubular designs. We have combined experimental techniques, ranging from Scanning Electron (SEM) and Atomic Force (AFM) microscopy to Raman spectroscopy, with computational simulations to characterize and model nanotubular surfaces. We evaluated the cell response at 6 hrs, 1 and 2 days by fluorescence microscopy, as well as bone mineral deposition by Raman spectroscopy, demonstrating substrate-induced differential biological cueing at both the short- and long-term. RESULTS: Our work demonstrates that the nanotube diameter is not sufficient to comprehensively characterize nanotubular surfaces and equally important parameters, such as eccentricity and wall thickness, ought to be included since they all contribute to the overall spatial disorder which, in turn, dictates the overall bioactive potential. We have also demonstrated that nanotubular surfaces affect the quality of bone mineral deposited by differentiated stem cells. Lastly, we closed in on the integrated effects exerted by the superimposition of two dissimilar nanotubular arrays in the honeycomb architecture. DISCUSSION: This work delineates a novel approach for the characterization of TiO2 nanotubes which supports the incorporation of critical spatial structural aspects that have been overlooked in previous research. This is a crucial aspect to interpret cellular behaviour on nanotubular substrates. Consequently, we anticipate that this strategy will contribute to the unification of studies focused on the use of such powerful nanostructured surfaces not only for biomedical applications but also in other technology fields, such as catalysis.

High Hydrostatic Pressure Therapy Annihilates Squamous Cell Carcinoma in a Murine Model.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers. In the treatment of cSCC, it is necessary to remove it completely, and reconstructive surgery, such as a skin graft or a local or free flap, will be required, depending on the size, with donor-site morbidity posing a burden to the patient. The high hydrostatic pressure (HHP) technique has been developed as a physical method of decellularizing various tissues. We previously reported that HHP at 200 MPa for 10 min could inactivate all cells in the giant congenital melanocytic nevus, and we have already started a clinical trial using this technique. In the present study, we explored the critical pressurization condition for annihilating cSCC cells in vitro and confirmed that this condition could also annihilate cSCC in vivo. We prepared 5 pressurization conditions in this study (150, 160, 170, 180, and 190 MPa for 10 min) and confirmed that cSCC cells were killed by pressurization at ≥160 MPa for 10 min. In the in vivo study, the cSCC cells inactivated by HHP at 200 MPa for 10 min were unable to proliferate after injection into the intradermal space of mice, and transplanted cSCC tissues that had been inactivated by HHP showed a decreased weight at 5 weeks after implantation. These results suggested that HHP at 200 MPa for 10 min was able to annihilate SCC, so HHP technology may be a novel treatment of skin cancer.

A comparison of the wound healing process after the application of three dermal substitutes with or without basic fibroblast growth factor impregnation in diabetic mice.

Pelnac GplusⓇ, IntegraⓇ, and TerudermisⓇ are approved artificial dermis products in Japan. Previously, we proved that Pelnac GplusⓇ was able to sustain basic fibroblast growth factor (bFGF) and accelerated wound healing by releasing impregnated bFGF. In this study, we impregnated Pelnac GplusⓇ, IntegraⓇ, and TerudermisⓇ with bFGF and compared the binding activity and wound-healing process. We applied bFGF to each material and compared the bFGF concentrations in the surrounding area after 24-h incubation. For the in vivo study, dermal substitutes were impregnated with bFGF and implanted into full-thickness wounds of BKS.Cg-+Leprdb/+Leprdb/Jcl mice. Wounds were evaluated at days 7, 14, and 21 after implantation. The in vitro study showed that bFGF is strongly bound to IntegraⓇ, followed by Pelnac GplusⓇ and TerudermisⓇ. The in vivo study showed that fibroblasts and capillaries had infiltrated into the Pelnac GplusⓇ but not the IntegraⓇ or TerudermisⓇ. Furthermore, long epithelium and wide granulation tissue were formed in the Pelnac GplusⓇ with bFGF group. The TerudermisⓇ with bFGF group had more capillaries than other groups, but only at the base of the wound. The combination of Pelnac GplusⓇ with bFGF may be a novel approach for treating full-thickness skin defects or chronic skin ulcers.

Hydrostatic pressure can induce apoptosis of the skin.

We previously showed that high hydrostatic pressure (HHP) treatment at 200 MPa for 10 min induced complete cell death in skin and skin tumors via necrosis. We used this technique to treat a giant congenital melanocytic nevus and reused the inactivated nevus tissue as a dermis autograft. However, skin inactivated by HHP promoted inflammation in a preclinical study using a porcine model. Therefore, in the present study, we explored the pressurization conditions that induce apoptosis of the skin, as apoptotic cells are not believed to promote inflammation, so the engraftment of inactivated skin should be improved. Using a human dermal fibroblast cell line in suspension culture, we found that HHP at 50 MPa for ≥ 36 h completely induced fibroblast cell death via apoptosis based on the morphological changes in transmission electron microscopy, reactive oxygen species elevation, caspase activation and phosphatidylserine membrane translocation. Furthermore, immunohistochemistry with terminal deoxynucleotidyl transferase dUTP nick-end labeling and cleaved caspase-3 showed most cells in the skin inactivated by pressurization to be apoptotic. Consequently, in vivo grafting of apoptosis-induced inactivated skin resulted in successful engraftment and greater dermal cellular density and macrophage infiltration than our existing method. Our finding supports an alternative approach to hydrostatic pressure application.

Exploration of the Pressurization Condition for Killing Human Skin Cells and Skin Tumor Cells by High Hydrostatic Pressure.

High hydrostatic pressure (HHP) is a physical method for inactivating cells or tissues without using chemicals such as detergents. We previously reported that HHP at 200 MPa for 10 min was able to inactivate all cells in skin and giant congenital melanocytic nevus (GCMN) without damaging the extracellular matrix. We also reported that HHP at 150 MPa for 10 min was not sufficient to inactivate them completely, while HHP at 200 MPa for 10 min was able to inactivate them completely. We intend to apply HHP to treat malignant skin tumor as the next step; however, the conditions necessary to kill each kind of cell have not been explored. In this work, we have performed a detailed experimental study on the critical pressure and pressurization time using five kinds of human skin cells and skin tumor cells, including keratinocytes (HEKas), dermal fibroblasts (HDFas), adipose tissue-derived stem cells (ASCs), epidermal melanocytes (HEMa-LPs), and malignant melanoma cells (MMs), using pressures between 150 and 200 MPa. We pressurized cells at 150, 160, 170, 180, or 190 MPa for 1 s, 2 min, and 10 min and evaluated the cellular activity using live/dead staining and proliferation assays. The proliferation assay revealed that HEKas were inactivated at a pressure higher than 150 MPa and a time period longer than 2 min, HDFas and MMs were inactivated at a pressure higher than 160 MPa and for 10 min, and ASCs and HEMa-LPs were inactivated at a pressure higher than 150 MPa and for 10 min. However, some HEMa-LPs were observed alive after HHP at 170 MPa for 10 min, so we concluded that HHP at a pressure higher than 180 MPa for 10 min was able to inactivate five kinds of cells completely.

Stress perturbation method for the assessment of cathodoluminescence probe response functions.

A method to determine the in-plane cathodoluminescence (CL) probe response function (PRF) (i.e., the function characterizing the in-plane luminescence intensity distribution within the electron probe volume) is proposed, which is based on "perturbing" the spectral position of a selected luminescence band using a highly graded stress field. The method is applied to the stress field developed ahead of the tip of an equilibrium crack in three different cases of CL bands, which arise from different structural phenomena: (i) the F(+) (oxygen excess) defect band in a nominally stoichiometric sapphire (alpha-Al(2)O(3)) single crystal; (ii) the chromophoric R-line in ruby lattice (alpha-Al(2-x)Cr(x)O(3)); and (iii) the near band-gap line in n-type GaN semiconductor crystal. A computer-aided data restoration procedure was applied to rationalize data retrieved from crack-tip line scans performed at different acceleration voltages. For the excitonic band-gap in GaN and for F(+) emission in sapphire the CL probe in the electron focal plane was found to be comparable, but not necessarily coincident, in size to the electron probe. On the other hand, the occurrence of self-absorption in the case of R-line photons in ruby resulted in a significantly broadened CL probe with respect to the average scattering length of electrons.

Evolution of Phage Display Approaches to Select Highly Specific Hemocompatible Peptides.

The ideal blood-contacting surface would support endothelial cell lining and suppress platelet adhesion, but, in synthetic biomaterials, these issues often conflict with each other. The reconciliation of this dichotomy may arise by modifying the biomaterial surfaces with "smart" peptides. Phage display is a powerful method for discovering unique peptides capable of binding to target molecules, but the selection of peptides binding to intact cells is an intricate process. In fact, the target molecules are often hindered by the extremely complex composition of cell membrane. In this work, the traditional phage display screening approach against endothelial progenitor cells (EPCs) was implemented with the introduction of (1) a negative selection step against platelets and (2) the target affinity scoring function phage binding index. The peptide candidates were used to modify an expanded polytetrafluoroethylene (ePTFE) surface to demonstrate that one of them not only has high affinity for EPCs but also simultaneously decreases thrombus formation.

Collagen/Gelatin Sponges (CGSs) Provide Both Protection and Release of bFGF: An In Vitro Study.

It has been reported that collagen/gelatin sponges (CGSs) are able to sustain the release of basic fibroblast growth factor (bFGF) for approximately 10 days via the formation of ion complexes between bFGF and gelatin. CGSs impregnated with bFGF have been proven to promote dermis-like tissue formation in various in vivo studies and clinical trials. However, the bioactivities of bFGF released from CGSs have not been explored in vitro. In this study, we explored the ability of CGS impregnated with bFGF, stored at 37°C for up to 14 days, to promote fibroblast proliferation and the sustained release of bFGF. We analyzed the cellular viability and proliferation in 2D and in 3D cell cultures, by a CCK-8 assay. Furthermore, in order to characterize the morphological alteration of fibroblasts, we studied 3D cultures by microscopy with a scanning electron microscope (SEM) and a confocal microscope. Our analyses revealed that the fibroblasts were elongated and flanked each other. They infiltrated and migrated inside the CGSs and were oriented along the CGS structure. Thus, these data prove that CGSs protect and sustain the efficient release of growth factor for more than 7 days.

The sustained release of basic fibroblast growth factor accelerates angiogenesis and the engraftment of the inactivated dermis by high hydrostatic pressure.

We developed a novel skin regeneration therapy combining nevus tissue inactivated by high hydrostatic pressure (HHP) in the reconstruction of the dermis with a cultured epidermal autograft (CEA). The issue with this treatment is the unstable survival of CEA on the inactivated dermis. In this study, we applied collagen/gelatin sponge (CGS), which can sustain the release of basic fibroblast growth factor (bFGF), to the inactivated skin in order to accelerate angiogenesis. Murine skin grafts from C57BL6J/Jcl mice (8 mm in diameter) were prepared, inactivated by HHP and cryopreserved. One month later, the grafts were transplanted subcutaneously onto the back of other mice and covered by CGS impregnated with saline or bFGF. Grafts were harvested after one, two and eight weeks, at which point the engraftment was evaluated through the histology and angiogenesis-related gene expressions were determined by real-time polymerase chain reaction. Histological sections showed that the dermal cellular density and newly formed capillaries in the bFGF group were significantly higher than in the control group. The relative expression of FGF-2, PDGF-A and VEGF-A genes in the bFGF group was significantly higher than in the control group at Week 1. This study suggested that the angiogenesis into grafts was accelerated, which might improve the engraftment of inactivated dermis in combination with the sustained release of bFGF by CGSs.

A surface graft polymerization process on chemically stable medical ePTFE for suppressing platelet adhesion and activation.

An effective surface grafting method for chemically inert and elaborately porous medical expanded-polytetrafluoroethylene (ePTFE) was developed. Although surface graft polymerization onto basic polymeric biomaterials has been widely studied, successful modification of the ePTFE surface has been lacking due to its high chemical resistance. Herein, we succeeded in surface graft polymerization onto ePTFE through glycidyl methacrylate (GMA) as a bridge linkage following argon (Ar) plasma treatment. The epoxy group of GMA was expected to react with the peroxide groups produced on ePTFE by Ar plasma exposure, and its methacrylic groups can copolymerize with various monomers. In the present study, we selected 2-methacryloyloxyethyl phosphorylcholine (MPC) as a model monomer and the blood compatibility of modified ePTFE was evaluated. Two sequences of surface grafting were compared. In a two-step graft polymerization, GMA was first immobilized onto Ar plasma treated ePTFE, and then MPC was polymerized. In a one-step graft copolymerization, MPC and GMA were mixed and copolymerized simultaneously onto Ar plasma treated ePTFE, resulting in a poly(MPC-co-GMA) (PMG) graft surface. The roughness of the node-and-fibril structure of ePTFE was reduced by the uniform polymer layer, and the modified ePTFE had a good hydrophilic nature even after being stored in an aqueous environment for 30 days. The indispensable GMA in graft polymerization improved the surface grafting on ePTFE. The one-step and two-step graft polymerization methods could decrease the number of adhered platelets, and almost inhibit platelet activation. We concluded that graft polymerization with the GMA linker provides a novel strategy to modify the chemically inert ePTFE surfaces for functionalizing as new medical devices.

Novel peptides for small-caliber graft functionalization selected by a phage display of endothelial-positive/platelet-negative combined selection.

Coronary artery disease is the leading cause of death in the world. While transplantation of autologous vessels remains a viable option, often patients may have a limited availability of suitable veins/arteries. Nowadays, despite the efforts of research made towards developing synthetic vessels, the small-diameter conduits have not fully met clinical needs. Surface modifications to mimic the vascular wall or seeding of endothelial cells within the lumen of the graft prior implantation are common approaches. However, due to the difficulties of the seeding methods, the immobilization of short recognition sequences presenting cell binding motifs remains one of the most promising strategies. In this paper, about 600 endothelial cell specific peptides have been reviewed and compared with three sequences selected by an improved phage display biopanning protocol. Indeed, the standard protocol has been ameliorated by (i) a positive selection of Endothelial Progenitor Cell (EPC)-binding phage clones, (ii) a negative selection against platelet binding phage clones, and (iii) the introduction of the phage binding index (PBI), which is a target affinity scoring function. The selected peptides were used to modify the ePTFE surface, and our results indicated that the peptide modified surfaces may be useful in solving the problems of small calibre grafts.

Reliable assessment and quantification of the fluorescence-labeled antisense oligonucleotides in vivo.

The availability of fluorescent dyes and the advances in the optical systems for in vivo imaging have stimulated an increasing interest in developing new methodologies to study and quantify the biodistribution of labeled agents. However, despite these great achievements, we are facing significant challenges in determining if the observed fluorescence does correspond to the quantity of the dye in the tissues. In fact, although the far-red and near-infrared lights can propagate through several centimetres of tissue, they diffuse within a few millimetres as consequence of the elastic scattering of photons. In addition, when dye-labeled oligonucleotides form stable complex with cationic carriers, a large change in the fluorescence intensity of the dye is observed. Therefore, the measured fluorescence intensity is altered by the tissue heterogeneity and by the fluctuation of dye intensity. Hence, in this study a quantification strategy for fluorescence-labeled oligonucleotides was developed to solve these disadvantageous effects. Our results proved that upon efficient homogenization and dilution with chaotropic agents, such as guanidinium thiocyanate, it is possible to achieve a complete fluorescence intensity recovery. Furthermore, we demonstrated that this method has the advantage of good sensitivity and reproducibility, as well as easy handling of the tissue samples.