Small-Diameter Synthetic Vascular Graft Immobilized with the REDV Peptide Reduces Early-Stage Fibrin Clot Deposition and Results in Graft Patency in Rats.

Early-stage hemocompatibility is indispensable for manufacturing tissue-engineered vascular grafts used in regenerative medicine. In this study, we report the in vivo blood response and patency of small-diameter synthetic vascular grafts modified with the Arg-Glu-Asp-Val (REDV) peptide. Vascular grafts were prepared by casting REDV-conjugated poly(depsipeptide-co-caprolactone) on a stainless-steel mandril (diameter: 1.8 mm). After implanting the grafts into the abdominal aorta of rats for 24 h, all three control grafts without the peptide and three out of the four REDV (control sequence) peptide-modified grafts showed occlusion. The luminal surfaces of these grafts were covered with thick thrombi. In contrast, all the grafts containing the REDV peptide were patent, and their luminal surfaces were covered with a thin layer of fibrin. These results indicated that the REDV peptide on the luminal surface effectively reduced early-stage fibrin clot deposition and formed the pseudo-endothelium layer in a peptide sequence-specific manner, resulting in graft patency.

Simple and efficient method for consecutive inactivation-cryopreservation of porcine skin grafts.

We previously reported that inactivation treatment by high hydrostatic pressurization (HHP) has potential utility as a novel skin regeneration therapy for various skin tumors. In this study, we evaluated whether glycerol-cryopreservation could be applied in order to preserve inactivated skin by HHP using a porcine model. Twenty full-thickness skin grafts (1.5 × 1.5 cm) were prepared from a minipig. The skin samples were inactivated by the HHP in normal saline or glycerol/fructose solution, followed by cryopreservation for 5 weeks at - 80 °C in each same solution. Another 10 grafts immediately after inactivation were prepared as non-cryopreserved controls. Nine grafts in each group were randomly implanted on the fascia of a host pig and removed at 1, 4 and 11 weeks after grafting. All grafts showed engraftment macroscopically. Hematoxylin eosin staining showed the cellular components in all areas of the dermis at 4 and 11 weeks after grafting, and immunohistochemical staining for CD31 showed the presence of capillaries in the grafts in all groups. The surface and cross-sectional areas of grafts in the normal saline solution cryopreserved group decreased between 1 and 11 weeks, whereas these areas in the glycerol cryopreserved group did not decrease significantly. Glycerol cryopreservation may therefore be a simple and efficient method for preserving porcine skin inactivated by HHP.

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.

Label-Free Separation of Induced Pluripotent Stem Cells with Anti-SSEA-1 Antibody Immobilized Microfluidic Channel.

When induced pluripotent stem cells (iPSCs) are routinely cultured, the obtained cells are a heterogeneous mixture, including feeder cells and partially differentiated cells. Therefore, a purification process is required to use them in a clinical stage. We described a label-free separation of iPSCs using a microfluidic channel. Antibodies against stage-specific embryonic antigen 1 (SSEA-1) was covalently immobilized on the channel coated with a phospholipid polymer. After injection of the heterogeneous cell suspension containing iPSCs, the velocity of cell movement under a liquid flow condition was measured. The mean velocity of the cell movement was 2.1 mm/sec in the unmodified channel, while that in the channel with the immobilized-antibody was 0.4 mm/sec. The eluted cells were fractionated by eluting time. As a result, the SSEA-1 positive iPSCs were mainly contained in later fractions, and the proportion of iPSCs was increased from 43% to 82% as a comparison with the initial cell suspension. These results indicated that iPSCs were selectively separated by the microfluidic channel. This channel is a promising device for label-free separation of iPSCs based on their pluripotent state.

A Novel Strategy for Etiologic Factor Removal: Drug-Navigated Clearance System (DNCS).

Here, we propose a novel therapeutic concept named drug-navigated clearance system (DNCS), in which a "navigator" decreases the concentration of a target etiologic factor in the blood by steering it to an unusual metabolic pathway. The navigator is composed of protein A (ProA) and dextran sulfate (DexS) and it successfully navigated antibodies (ABs), a model etiologic factor of dilated cardiomyopathy, to hepatocytes in vitro in the presence of low-density lipoprotein (LDL). ProA captured the Fc region of the target antibody while the DexS bound to LDL via the well-known electrostatic interaction. The hepatocytes simultaneously took up LDL via the LDL-receptor and internalized the AB/ProA-DexS complex that was bound to LDL. Therefore, this process demonstrates our attempt to navigate the etiologic factor to an alternate target pathway such as the LDL salvage.

Verification of the Inactivation of Melanocytic Nevus in vitro Using a Newly Developed Portable High Hydrostatic Pressure Device.

High hydrostatic pressure (HHP) technology is a physical method for inactivating tissue. We reported that nevus specimens were inactivated after HHP at 200 MPa and that the inactivated nevus could be used as autologous dermis for covering skin defects. In this study, we verified the inactivation of nevus specimens using a newly developed portable HHP device which will be used in a clinical trial. Nevus tissue specimens were obtained from 5 patients (mean age 7.2 years, range 1-19). We cultured fibroblasts and nevus cells from the tissue specimens and then evaluated their inactivation after HHP at 200 MPa by confirming the attachment of the suspensions and by the live/dead staining of the suspensions, through the dissociation of the cells on chamber slides and by the live/dead staining of the remaining cells. The cells were also quantitatively evaluated by WST-8 assay. We then confirmed the inactivation of the nevus specimens after HHP using explant culture. Our results indicated that fibroblasts and nevus cells were inactivated after HHP at 200 MPa, with the exception of a small percentage of green-colored cells, which reflected the remaining activity of the cellular esterases after HHP. No cells migrated from the nevus specimens after HHP at 200 MPa. We verified the inactivation of fibroblasts and nevus cells cultured from nevus specimens, and in the nevus samples themselves after pressurization at 200 MPa using this device. This device could be used in clinical trials for giant congenital melanocytic nevi and may thus become useful in various medical fields.

Micro-CT evaluation of high pressure-decellularized cardiovascular tissues transplanted in rat subcutaneous accelerated-calcification model.

We have succeeded in reducing the calcification of acellular aortas or valves in porcine allogeneic system by removing the DNA and phospholipids, but its further reduction is desirable. Here, the calcification of the acellular tissue was evaluated in rat subcutaneous transplantation model which is known as calcification model. Acellular samples prepared by high-hydrostatic pressure (HHP) protocols with different washing media were implanted and the calcification was monitored under micro-computed tomography for 1 and 3 months. The amount of the calcium deposition was quantitatively evaluated by atomic absorption spectroscopy. A cell culture medium showed very good cell removal ability but led to severe calcification at 1 month, and surprisingly the calcium deposition increased as the washing period increased. This calcification was suppressed by removing the DNA fraction with high DNase concentration. On the other hand, the calcification was greatly reduced when washed with saline even at low DNase concentration after 2 weeks washing. These results suggest that the ion species in the washing medium and the residual DNase cooperatively affect the tendency of in vivo calcification, which led us to the possibility of reduced calcification of acellular cardiac tissues.

Photo-induced universal modification of small-diameter decellularized blood vessels with a hemocompatible peptide improves in vivo patency.

Decellularized vessels (DVs) have the potential to serve as available grafts for small-diameter vascular (<6 mm) reconstruction. However, the absence of functional endothelia makes them likely to trigger platelet aggregation and thrombosis. Luminal surface modification is an efficient approach to prevent thrombosis and promote endothelialization. Previously, we identified a hemocompatible peptide, HGGVRLY, that showed endothelial affinity and antiplatelet ability. By conjugating HGGVRLY with a phenylazide group, we generated a photoreactive peptide that can be modified onto multiple materials, including non-denatured extracellular matrices. To preserve the natural collagen of DVs as much as possible, we used a lower ultrahydrostatic pressure than that previously reported to prepare decellularized grafts. The photoreactive HGGVRLY peptide could be modified onto DV grafts via UV exposure for only 2 min. Modified DVs showed improved endothelial affinity and antiplatelet ability in vitro. When rat abdominal aortas were replaced with DVs, modified DVs with more natural collagen demonstrated the highest patent rate after 10 weeks. Moreover, the photoreactive peptide remained on the lumen surface of DVs over two months after implantation. Therefore, the photoreactive peptide could be efficiently and sustainably modified onto DVs with more natural collagens, resulting in improved hemocompatibility. STATEMENT OF SIGNIFICANCE: We employed a relatively lower ultrahydrostatic pressure to prepare decellularized vessels (DVs) with less denatured collagens to provide a more favorable environment for cell migration and proliferation. The hemocompatibility of DV luminal surface can be enhanced by peptide modification, but undenatured collagens are difficult to modify. We innovatively introduce a phenylazide group into the hemocompatible peptide HGGVRLY, which we previously identified to possess endothelial affinity and antiplatelet ability, to generate a photoreactive peptide. The photoreactive peptide can be efficiently and stably modified onto DVs with more natural collagens. DV grafts modified with photoreactive peptide exhibit enhanced in vivo patency. Furthermore, the sustainability of photoreactive peptide modification on DV grafts within bloodstream is evident after two months of transplantation.

Widely distributable and retainable in-situ gelling material for treating myocardial infarction.

Intramyocardial hydrogel injection is a promising therapy to prevent negative remodeling following myocardial infarction (MI). In this study, we report a mechanism for in-situ gel formation without external stimulation, resulting in an injectable and tissue-retainable hydrogel for MI treatment, and investigate its therapeutic outcomes. A liquid-like polymeric solution comprising poly(3-acrylamidophenylboronic acid-co-acrylamide) (BAAm), polyvinyl alcohol (PVA), and sorbitol (S) increases the viscous modulus by reducing the pre-added sorbitol concentration is developed. This solution achieves a sol-gel transition in-vitro in heart tissue by spontaneously diffusing the sorbitol. After intramyocardial injection, the BAAm/PVA/S with lower initial viscous modulus widely spreads in the myocardium and gelate compared to a viscoelastic alginate (ALG) hydrogel and is retained longer than the BAAm/S solution. Serial echocardiogram analyses prove that injecting the BAAm/PVA/S into the hearts of subacute MI rats significantly increases the fraction shortening and ejection shortening and attenuates the expansion of systolic LV diameter for up to 21 d after injection compared to the saline injection as a control, but the ALG injection does not. In addition, histological evaluation shows that only the BAAm/PVA/S decreases the infarct size and increases the wall thickness 21 d after injection. The BAAm/PVA/S intramyocardial injection is better at restraining systolic ventricular dilatation and cardiac failure in the rat MI model than in the control groups. Our findings highlight an effective injectable hydrogel therapy for MI by optimizing injectability-dependent distribution and retention of injected material. STATEMENT OF SIGNIFICANCE: In-situ gelling material is a promising strategy for intramyocardial hydrogel injection therapy for myocardial infarction (MI). Since the sol-gel transition of reported materials is driven by external stimulation such as temperature, pH, or ultraviolet, their application in vivo remains challenging. In this study, we first reported a synthetic in-situ gelling material (BAAm/PVA/S) whose gelation is stimulated by spontaneously reducing pre-added sorbitol after contacting the heart tissue. The BAAm/PVA/S solution spreads evenly, and is retained for at least 21 d in the heart tissue. Our study demonstrated that intramyocardial injection of the BAAm/PVA/S with more extensive distribution and longer retention had better effects on preventing LV dilation and improving cardiac function after MI than that of viscoelastic ALG and saline solution. We expect that these findings provide fundamental information for the optimum design of injectable biomaterials for treating MI.

Improving hemocompatibility of decellularized vascular tissue by structural modification of collagen fiber.

Decellularized vascular tissue has high potential as a tissue-engineered vascular graft because of its similarity to native vessels in terms of mechanical strength. However, exposed collagen on the tissue induces blood coagulation, and low hemocompatibility is a major obstacle to its vascular application. Here we report that freeze-drying and ethanol treatment effectively modify collagen fiber structure and drastically reduce blood coagulation on the graft surface without exogenous chemical modification. Decellularized carotid artery of ostrich was treated with freeze-drying and ethanol solution at concentrations ranging between 5 and 99.5 %. Collagen fiber distance in the graft was narrowed by freeze-drying, and the non-helical region increased by ethanol treatment. Although in vitro blood coagulation pattern was similar on the grafts, platelet adhesion on the grafts was largely suppressed by freeze-drying and ethanol treatments. Ex vivo blood circulation tests also indicated that the adsorption of platelets and Von Willebrand Factor was largely reduced to approximately 80 % by ethanol treatment. These results indicate that structural modification of collagen fibers in decellularized tissue reduces blood coagulation on the surface by inhibiting platelet adhesion.

Biodistribution of vaccines comprised of hydrophobically-modified poly(γ-glutamic acid) nanoparticles and antigen proteins using fluorescence imaging.

Fluorophores-modified nanoparticles comprised of poly(γ-glutamic acid)-phenylalanine (γ-PGA-Phe-633) and ovalbumin (OVA-750) termed NPs-633/OVA-750 were prepared to assess their biodistribution using an in vivo fluorescence imager. Dynamic light scattering measurements indicated that NPs-633/OVA-750 were about 200nm in diameter. The release of encapsulated OVA from NPs-633 in PBS was negligible (∼10%) for a week. When subcutaneously injected, the localization period of OVA-750-encapsulated into NPs-633 at the site of injection (SOI) was much longer than that of free OVA-750, but was shorter as compared to a mixture with aluminum hydroxide. The NPs-633 disappeared at the SOI and major organs within 1month after administration. Moreover, intravenously and intraperitoneally administered NPs-633 were mainly observed at the liver, and there was more rapid clearance from all organs as compared with non-biodegradable NPs. These fast clearance and degradation characteristics of γ-PGA-Phe NPs will be important not only for avoiding undesired adverse effects, but also for inducing a strong vaccine effect.

Prevention of anastomotic stenosis for decellularized vascular grafts using rapamycin-loaded boronic acid-based hydrogels mimicking the perivascular tissue function.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) induces graft anastomotic stenosis, resulting in graft failure. Herein, we developed a drug-loaded tissue-adhesive hydrogel as artificial perivascular tissue to suppress VSMCs proliferation. Rapamycin (RPM), an anti-stenosis drug, is selected as the drug model. The hydrogel was composed of poly (3-acrylamidophenylboronic acid-co-acrylamide) (BAAm) and polyvinyl alcohol. Since phenylboronic acid reportedly binds to sialic acid of glycoproteins which is distributed on the tissues, the hydrogel is expected to be adherent to the vascular adventitia. Two hydrogels containing 25 or 50 mg/mL of BAAm (BAVA25 and BAVA50, respectively) were prepared. A decellularized vascular graft with a diameter of <2.5 mm was selected as a graft model. Lap-shear test indicates that both hydrogels adhered to the graft adventitia. In vitro release test indicated that 83 and 73 % of RPM in BAVA25 and BAVA50 hydrogels was released after 24 h, respectively. When VSMCs were cultured with RPM-loaded BAVA hydrogels, their proliferation was suppressed at an earlier stage in RPM-loaded BAVA25 hydrogels compared to RPM-loaded BAVA50 hydrogels. An in vivo preliminary test reveals that the graft coated with RPM-loaded BAVA25 hydrogel shows better graft patency for at least 180 d than the graft coated with RPM-loaded BAVA50 hydrogel or without hydrogel. Our results suggest that RPM-loaded BAVA25 hydrogel with tissue adhesive characteristics has potential to improve decellularized vascular graft patency.

In vivo MR imaging for tumor-associated initial neovascularization by supramolecular contrast agents.

Microvascular imaging is required to understand tumor angiogenesis development; however, an appropriate whole-body imaging method has not yet been established. Here, we successfully developed a supramolecular magnetic resonance (MR) contrast agent for long-term whole-tissue observation in a single individual. Fluorescein- and Gd-chelate-conjugated polyethylene glycols (PEGs) were synthesized, and their structures were optimized. Spectroscopic and pharmacokinetic analyses suggested that the fluorescein-conjugated linear and 8-arm PEGs with a molecular weight of approximately 10 kDa were suitable to form a supramolecular structure to visualize the microvessel structure and blood circulation. Microvascular formation was evaluated in a glioma cell transplantation model, and neovascularization around the glioma tissue at 5 days was observed, with the contrast agent leaking out into the cancer tissue. In contrast, after 12 days, microvessel structures were formed inside the glioma tissue, but the agents did not leak out. These imaging data for the first time proved that the microvessels formed inside cancer tissues at the early stage are very leaky, but that they form continuous microvessels after 12 days.

Vascular tissue reconstruction by monocyte subpopulations on small-diameter acellular grafts via integrin activation.

Although the clinical application of cell-free tissue-engineered vascular grafts (TEVGs) has been proposed, vascular tissue regeneration mechanisms have not been fully clarified. Here, we report that monocyte subpopulations reconstruct vascular-like tissues through integrin signaling. An Arg-Glu-Asp-Val peptide-modified acellular long-bypass graft was used as the TEVG, and tissue regeneration in the graft was evaluated using a cardiopulmonary pump system and porcine transplantation model. In 1 day, the luminal surface of the graft was covered with cells that expressed CD163, CD14, and CD16, which represented the monocyte subpopulation, and they exhibited proliferative and migratory abilities. RNA sequencing showed that captured cells had an immune-related phenotype similar to that of monocytes and strongly expressed cell adhesion-related genes. In vitro angiogenesis assay showed that tube formation of the captured cells occurred via integrin signal activation. After medium- and long-term graft transplantation, the captured cells infiltrated the tunica media layer and constructed vascular with a CD31/CD105-positive layer and an αSMA-positive structure after 3 months. This finding, including multiple early-time observations provides clear evidence that blood-circulating monocytes are directly involved in vascular remodeling.

REDV-modified decellularized microvascular grafts for arterial and venous reconstruction.

Recently, a decellularized microvascular graft (inner diameter: 0.6 mm) modified with the integrin α4ß1 ligand, REDV, was developed to provide an alternative to autologous-vein grafting in reconstructive microsurgery, showing good early-stage patency under arterial flow in rats. This consecutive study evaluated its potential utility not only as an arterial substitute, but also as a venous substitute, using a rat-tail replantation model. Graft remodeling depending on hemodynamic status was also investigated. ACI rat tail arteries were decellularized via ultra-high-hydrostatic pressure treatment and modified with REDV to induce antithrombogenic interfaces and promote endothelialization after implantation. Grafts were implanted into the tail artery and vein to re-establish blood circulation in amputated Lewis rat tails (n = 12). The primary endpoint was the survival of replants. Secondary endpoints were graft patency, remodeling, and regeneration for 6 months. In all but three cases with technical errors or postoperative self-mutilation, tails survived without any evidence of ischemia or congestion. Six-month Kaplan-Meier patency was 100% for tail-artery implanted grafts and 62% for tail-vein implanted grafts. At 6 months, the neo-tunica media (thickness: 95.0 µm in tail-artery implanted grafts, 9.3 µm in tail-vein implanted grafts) was regenerated inside the neo-intima. In conclusion, the microvascular grafts functioned well both as arterial and venous paths of replanted-rat tails, with different remodeling under arterial and venous conditions.

Accelerated tissue regeneration in decellularized vascular grafts with a patterned pore structure.

Decellularized tissue is expected to be utilized as a regenerative scaffold. However, the migration of host cells into the central region of the decellularized tissues is minimal because the tissues are mainly formed with dense collagen and elastin fibers. This results in insufficient tissue regeneration. Herein, it is demonstrated that host cell migration can be accelerated by using decellularized tissue with a patterned pore structure. Patterned pores with inner diameters of 24.5 ± 0.4 µm were fabricated at 100, 250, and 500 µm intervals in the decellularized vascular grafts via laser ablation. The grafts were transplanted into rat subcutaneous tissue for 1, 2, and 4 weeks. All the microporous grafts underwent faster recellularization with macrophages and fibroblast cells than the non-porous control tissue. In the case of non-porous tissue, the cells infiltrated approximately 50% of the area four weeks after transplantation. However, almost the entire area was occupied by the cells after two weeks when the micropores were aligned at a distance of less than 250 µm. These results suggest that host cell infiltration depends on the micropore interval, and a distance shorter than 250 µm can accelerate cell migration into decellularized tissues.

Identification of circulating cells interacted with integrin α4ß1 ligand peptides REDV or HGGVRLY.

Recently, artificial blood vessels modified by integrin α4ß1 ligand, such as REDV, showed endothelialization improvement and antithrombotic properties have been reported. Early endothelialization was affected by the type of circulating cells captured by the peptide in the initial transplantation state, however, it is still not clarified. In this study, we identified in vitro circulating cells bound with the peptides arginine-glutamic acid-aspartic acid-valine (REDV) or histidine-glycine-glycine-valine-arginine-leucine-tyrosine (HGGVRLY). The effect of free C- or N-terminal of HGGVRLY on the type of peptide-binding cells was also studied. The rat circulating cells were isolated from blood and incubated with 5(6)-carboxyfluorescein (5/6-FAM, F) labeled F-REDV (C-terminal free), F-HGGVRLY (C-terminal free), or HGGVRLY-F (N-terminal free). Furthermore, peptide-binding cells were identified by co-staining with various antibodies labeled with PE, PerCP/Cy5.5, or APC. N-terminal free HGGVRLY-F was found to bind to more circulating cells than C-terminal free F-REDV and F-HGGVRLY. The ratio of integrin α4ß1 positive cell bound with F-REDV, F-HGGVRLY, or HGGVRLY-F reached over 90 %, demonstrating that HGGVRLY is also a ligand of integrin α4ß1. Among identified cell types, we found that F-REDV mainly bounds with EPC and BMSC, while F-HGGVRLY with BMSC. HGGVRLY-F bounds with EPC and BMSC, exhibiting a higher EPC binding ratio than F-REDV and F-HGGVRLY.

Endothelial cell adhesion and blood response to hemocompatible peptide 1 (HCP-1), REDV, and RGD peptide sequences with free N-terminal amino groups immobilized on a biomedical expanded polytetrafluorethylene surface.

Blood compatibility generally requires two contradictory characteristics: reduced protein/platelet adhesion and excellent endothelium-related cell affinity. To understand the effect of cell adhesion peptides on blood compatibility, the peptides REDV, RGD, and hemocompatible peptide-1 (HCP-1) were immobilized on an expanded polytetrafluorethylene (ePTFE) surface and evaluated in vitro, in situ, and in vivo. Since the terminal amino groups of functional peptides often have an important effect, a cysteine residue was added to the C terminal and used for immobilization to keep the terminal amino groups free. Maleimide groups were added to carboxylic groups of highly hydrophilic and biologically inert (bioinert) polymer chains grafted onto ePTFE and coupled with cysteine residues. In vitro tests revealed that free N-terminal HCP-1 and RGD-immobilized surfaces improved the adhesion and spread of human umbilical vein endothelial cells (HUVECs), while, unexpectedly, a free N-terminal adjacent to REDV suppressed cell affinity. In situ evaluation with a porcine closed-circuit system for 2 h showed that no platelets adhered to the modified ePTFE sutures due to the bioinert graft chain containing phosphorylcholine groups. Simultaneously, leukocyte-related and endothelium-related cells were observed on RGD-immobilized ePTFE sutures because RGD was recognized by broad types of cells. These cells were not observed on the HCP-1- and REDV-immobilized ePTFE sutures, which may be due to insufficient exposure time. HCP-1-modified ePTFE graft implantation in a porcine femorofemoral (FF) bypass model for 10 days showed that the thrombus layer was clearly mitigated by HCP-1 immobilization. This study suggests that the HCP-1-immobilized ePTFE surface has potential for long-term application by mitigating thrombus and supporting endothelial cell adhesion.

Impact of REDV peptide density and its linker structure on the capture, movement, and adhesion of flowing endothelial progenitor cells in microfluidic devices.

Ligand-immobilization to stents and vascular grafts is expected to promote endothelialization by capturing flowing endothelial progenitor cells (EPCs). However, the optimized ligand density and linker structure have not been fully elucidated. Here, we report that flowing EPCs were selectively captured by the REDV peptide conjugated with a short linker. The microchannel surface was modified with the REDV peptide via Gly-Gly-Gly (G3), (Gly-Gly-Gly)3 (G9), and diethylene glycol (diEG) linkers, and the moving velocity and captured ratio were evaluated. On the unmodified microchannels, the moving velocity of the cells exhibited a unimodal distribution similar to the liquid flow. The velocity of the endothelial cells and EPCs on the peptide-immobilized surface indicated a bimodal distribution, and approximately 20 to 30% of cells moved slower than the liquid flow, suggesting that the cells were captured and rolled on the surface. When the immobilized ligand density was lower than 1 molecule/nm2, selective cell capture was observed only in REDV with G3 and diEG linkers, but not in G9 linkers. An in silico study revealed that the G9 linker tends to form a bent structure, and the REDV peptide is oriented to the substrate side. These results indicated that REDV captured the flowing EPC in a sequence-specific manner, and that the short linker was more adequate.

A Novel Treatment for Giant Congenital Melanocytic Nevi Combining Inactivated Autologous Nevus Tissue by High Hydrostatic Pressure and a Cultured Epidermal Autograft: First-in-Human, Open, Prospective Clinical Trial.

BACKGROUND: Giant congenital melanocytic nevi are large skin lesions associated with a risk of malignant transformation. The authors developed a novel treatment to reconstruct full-thickness skin defects by combining an inactivated nevus as the autologous dermis and a cultured epidermal autograft. The first-in-human trial of this treatment was performed. METHODS: Patients with melanocytic nevi that were not expected to be closed by primary closure were recruited. The full-thickness nevus of the target was removed and inactivated by high hydrostatic pressurization at 200 MPa for 10 minutes. The inactivated nevus was sutured to the original site, and a cultured epidermal autograft was grafted onto it 4 weeks later. Patients were followed for up to 52 weeks. RESULTS: Ten patients underwent reimplantation of the pressurized nevus, and one patient dropped out. The recurrence of nevus at 52 weeks was not detected by pathological diagnosis in any patients. The L* value at 52 weeks was significantly higher than that of the target nevus. One patient received skin grafting due to contracture of the reconstructed skin. The epithelized area of the reconstructed skin, as the percentage of the original target nevus, was 55.5 ± 19.4 percent at 12 weeks and 85.0 ± 32.4 percent at 52 weeks. CONCLUSIONS: The inactivated nevus caused inflammation and contracture for several months. However, no recurrence was observed, and combination therapy using an inactivated nevus with a cultured epidermal autograft may therefore be a novel treatment of giant congenital melanocytic nevi. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.