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.

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.

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.

Long term observation of de novo adipogenesis using novel bioabsorbable implants with larger size in a porcine model.

Introduction: The regeneration of adipose tissue in patients after breast cancer surgery would be desirable without the use of growth factors or cells to avoid potential recurrence and metastasis. We reported that prolate spheroidal-shaped poly-L-lactic acid (PLLA) mesh implants of approximately 18-mm polar diameter and 7.5-mm greatest equatorial diameter containing collagen sponge (CS) would be replaced by regenerated adipose tissue after implantation, thereby suggesting an innovative method for breast reconstruction. Our study aimed to evaluate the adipose tissue regeneration ability of implant aggregates in a porcine model. Methods: We prepared implant aggregates consisting of thirty PLLA mesh implants containing CS packed in a woven poly (glycolic acid) bag. The implant aggregates were inserted under the mammary glands in the porcine abdomen for a year. Single and double groups were classified by inserting either one or two implant aggregates on each side of the abdomen, respectively. Results: In both groups, the volume of the implant aggregates decreased over time, and the formation of adipose tissue peaked between 6 and 9 months. Histologically, the formation of adipose tissue was confirmed in the area that was in contact with native adipose tissue. Conclusions: Our implant aggregates could induce the autologous adipose tissue after long term implantation in vivo, without the use of any growth factor or cell treatment, presenting a potential novel method of breast reconstruction.

A high-hydrostatic pressure device for nevus tissue inactivation and dermal regeneration for reconstructing skin defects after giant congenital melanocytic nevus excision: a clinical trial.

Background: A novel treatment has been developed to reconstruct large skin defects caused by the excision of giant congenital melanocytic nevi. It involves the reimplantation of high-hydrostatic pressurized nevus tissue as a cell-inactivated autologous scaffold for dermal regeneration, followed by the implantation of cultured epithelial autografts on the regenerated dermis. Because this treatment has shown promise in a first-in-human clinical trial which used a prototype pressure machine, a novel pressure device was specifically designed for clinical use. Methods: In a prospective investigator-initiated clinical trial involving three patients, we evaluated the safety and efficacy of the skin regeneration treatment using a pressure device. All three patients underwent surgical excision of the nevus tissue, primary reimplantation of the inactivated nevus tissue, and secondary implantation of cultured epithelial autografts. Results: Engraftment of inactivated nevus tissue and cultured epithelial autografts was successful in all three cases, with over 90% epithelialization at 8 weeks post-surgery. No serious adverse events or device malfunction were observed during the trial. Conclusion: The novel pressure device safely and effectively enabled dermal regeneration using the nevus tissue as an autologous scaffold. This innovative approach offers several advantages, including reduced invasiveness due to minimal sacrifice of normal skin for skin grafting and high curative potential resulting from full-thickness removal of the nevus tissue.

Immobilization of Arg-Gly-Asp peptides on silk fibroin via Gly-Ala-Gly-Ala-Gly-Ser sequences.

A simple method by which the functional peptide of Gly-Arg-Gly-Asp-Ser (GRGDS) is immobilized on the surface of silk fibroin (SF) films via Gly-Ala-Gly-Ala-Gly-Ser (GAGAGS) sequences is proposed. GAGAGS, a repeating amino acid sequence in the crystal region of Bombyx mori SF, performs a key role in interacting with and immobilizing SF molecules. Immobilization by this proposed method involves no chemical reaction, thereby preserving the original properties of the SF molecule. The density of GRGDS peptides existing on SF film was found to be higher in the GAGAGS-bound type than in the non-GAGAGS-bound type. Furthermore, results showed that the amount of immobilized (GAGAGS)GRGDS peptide increased as the ß-sheet crystallization was promoted in the SF film. Fibroblasts, which adhered to the surface of the SF film, showed more extensibility because of the (GAGAGS)GRGDS immobilization, which suggests that the cell adhesion activity of RGD is functioning effectively.

Donor and Recipient Adipose-Derived Mesenchymal Stem Cell Therapy for Rat Lung Transplantation.

BACKGROUND: Mesenchymal stem cells (MSCs) are beginning to be proven as immunosuppressant in the field of organ transplantation. However, the effects of MSC origin (donor or recipient) on immunosuppression are not clear. Hence, we investigated the effects of recipient and donor adipose-derived MSCs (ADMSCs) on immunosuppression in a rat lung transplantation model. METHODS: Subjects were divided into no treatment, tacrolimus administration, recipient ADMSC administration, donor ADMSC administration, and mixed donor and recipient ADMSC administration groups. ADMSC-administered groups were also treated with tacrolimus. Histologic study, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay, and polymerase chain reaction were used for various analyses. RESULTS: Fluorescently labeled ADMSCs were predominant in the grafted donor lung, but not in the recipient lung, on day 5. On day 7, the pathologic rejection grades of the grafted donor lung were significantly lower in the ADMSC-administered groups (P < .05) and did not differ among these groups. Although serum hepatocyte growth factor and vascular endothelial growth factor levels did not differ among the groups, interleukin 10 level was slightly higher in the ADMSC-administered groups. The numbers of infiltrating regulatory T cells in the grafted lung were significantly higher in the ADMSC-administered groups (P < .05) but did not differ with cell origin. Transcriptional analysis suggested interleukin 6 suppression to be the main overlapping immunosuppressive mechanism, regardless of origin. Therefore, a donor or recipient origin may not influence the immunosuppressive efficacy of ADMSCs in our rat lung transplantation model. CONCLUSIONS: Collectively, the results indicate that allogenic ADMSCs, regardless of their origin, may exert similar immunosuppressive effects in clinical organ transplantation.

Stable and direct coating of fibronectin-derived Leu-Asp-Val peptide on ePTFE using one-pot tyrosine oxidation for endothelial cell adhesion.

Expanded polytetrafluoroethylene (ePTFE) is widely used in clinical applications, such as in the manufacture of blood-contacting implantable devices, owing to its flexibility, biostability, and non-adhesiveness. Modification with peptides is an effective strategy to further improve the ePTFE function. However, the chemical stability of PTFE makes it difficult to modify with peptides. In this study, we reported a simple method for the dense and stable coating of biofunctional peptides on the ePTFE surface through the anchor sequence, Tyr-Lys-Tyr-Lys-Tyr-Lys (YK3). A peptide (YK3-LDV) incorporating the YK3 anchor and a ligand sequence for α4ß1 integrin, Leu-Asp-Val (LDV), was successfully coated on ePTFE grafts through one-pot oxidation. The peptide layer constructed via YK3-LDV coating on ePTFE was stable and resistant to extensive washing by aqueous solutions of highly concentrated salts and surfactants. YK3-LDV coating promoted the in vitro adhesion of endothelial cells to ePTFE. Furthermore, YK3-LDV coating accelerated the in vivo formation of neointima-like tissue in a rat model with an ePTFE patch implanted into the carotid artery.

Preliminary report of de novo adipogenesis using novel bioabsorbable implants and image evaluation using a porcine model.

Our bioabsorbable poly-L-lactic acid (PLLA) mesh implants containing collagen sponge are replaced with adipose tissue after implantation, and this is an innovative method for breast reconstruction. In this preliminary study, we investigated the formation of adipose tissue and evaluated the process via multimodal images in a porcine model using an implant aggregate to generate the larger adipose tissue. The implant aggregate consists of PLLA mesh implants containing collagen sponge and a poly-glycolic acid woven bag covering them. We inserted the implant aggregates under the porcine mammary glands. Magnetic resonance imaging (MRI), ultrasonography (USG), and 3-dimensional (3D) surface imaging and histological evaluations were performed to evaluate the formation of adipose tissue over time. The volume of the implant aggregate and the formed adipose tissue inside the implant aggregate could be evaluated over time via MRI. The space within the implant aggregate was not confirmed on USG due to the acoustic shadow of the PLLA threads. The change in volume was not confirmed precisely using 3D surface imaging. Histologically, the newly formed adipose tissue was confirmed on the skin side of the implant aggregate. This implant aggregate has the ability to regenerate adipose tissue, and MRI is an appropriate method for the evaluation of the volume of the implant aggregation and the formation of adipose tissue.

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.

Enhanced ß2-microglobulin binding of a "navigator" molecule bearing a single-chain variable fragment antibody for artificial switching of metabolic processing pathways.

Kidney dysfunction increases the blood levels of ß2-microglobulin (ß2-m), triggering dialysis-related amyloidosis. Previously, we developed a navigator molecule, consisting of a fusion protein of the N-terminal domain of apolipoprotein E (ApoE NTD) and the α3 domain of the major histocompatibility complex class I (MHC α3), for switching the metabolic processing pathway of ß2-m from the kidneys to the liver. However, the ß2-m binding of ApoE NTD-MHC α3 was impaired in the blood. In the current study, we replaced the ß2-m binding part of the navigator protein (MHC α3) with an anti-ß2-m single-chain variable fragment (scFv) antibody. The resultant ApoE NTD-scFv exhibited better ß2-m binding than ApoE NTD-MHC α3 in buffer, and even in serum. Similar to ApoE NTD-MHC α3, in the mice model ApoE NTD-scFv bound to the liver cells' surfaces in vitro and accumulated mainly in the liver, when complexed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Both ApoE NTD-MHC α3 + DMPC and ApoE NTD-scFv + DMPC significantly switched the ß2-m accumulation in mice from the kidneys to the liver, but only the ApoE NTD-scFv + DMPC group showed a significantly higher ratio of ß2-m accumulation in the liver versus the kidneys, compared with the control group. These results suggest that the enhanced ß2-m binding activity of the navigator molecule increased the efficiency of switching the metabolic processing pathway of the etiologic factor.

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.

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.

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.

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.

Artificial switching of the metabolic processing pathway of an etiologic factor, ß2-microglobulin, by a "navigator" molecule.

Metabolic pathways in the body are highly specific. Dysfunction of a metabolic pathway triggers the accumulation of its target substance. For example, kidney failure results in increased ß2-microglobulin blood levels, causing dialysis-related amyloidosis. Previously, we proposed a novel therapeutic concept, that is a removal of an etiologic factor of metabolic disease by artificial switching of its metabolic processing pathway, and tested this concept using in cultured cells. However, the feasibility of artificial metabolic switching in vivo remained unknown. Here, we show that a newly developed "navigator" molecule changes the metabolic processing pathway of ß2-microglobulin from the kidney to the liver in mouse. The artificial metabolic switching is achieved by the capture of the etiologic factor by the navigator, which then steers the etiologic factor to hepatic lysosomes via low-density lipoprotein receptors. These findings demonstrate that navigator-based artificial metabolic switching can be a therapeutic strategy for various diseases caused by metabolic disorders.

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.

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.

Cellular attachment behavior on biodegradable polymer surface immobilizing endothelial cell-specific peptide.

Small-caliber artificial blood vessels with inner diameters of smaller than 4 mm have not been put into practical use because of early thrombus formation and graft occlusion. To realize small-caliber artificial blood vessels with anti-thrombus property and long-term patency, one of the promising approaches is endothelialization of the lumen by tissue engineering approaches. Integrin α4ß1 on the endothelial cell membrane is known to act as a receptor for Arg-Glu-Asp-Val (REDV) tetra-peptide, and this peptide can be used as a specific ligand to introduce endothelial cell attachment onto the surfaces of polymer scaffold. In this study, biodegradable polymer surface immobilizing REDV peptide were prepared, and the specific attachment of endothelial cells on it was investigated as a preliminary study for tissue-engineered small-caliber blood vessels in a future application. We synthesized copolymer of ε-caprolactone and depsipeptide having reactive carboxylic acid side-chain groups (PGDCL), and REDV peptide was attached to the copolymer to give PGDCL-REDV. The attachment of human umbilical vein endothelial cells (HUVECs) were investigated for the blend polymer film prepared by mixing PGDCL and PGDCL-REDV. The obtained blend polymer films exhibited sequence- and cell-specific HUVECs attachment through REDV peptide recognition. This technique should be useful not only to obtain artificial blood vessels which induce endothelialization and but also to provide biodegradable scaffolds with specific ligands immobilized surfaces for tissue regeneration.

Modification of decellularized vascular xenografts with 8-arm polyethylene glycol suppresses macrophage infiltration but maintains graft degradability.

Because acellular vascular xenografts induce an immunological reaction through macrophage infiltration, they are conventionally crosslinked with glutaraldehyde (GA). However, the GA crosslinking reaction inhibits not only the host immune reaction around the graft but also the graft's enzymatic degradability, which is one of the key characteristics of acellular grafts that allow them to be replaced by host tissue. In this study, we used an 8-arm polyethylene glycol (PEG) to successfully suppress macrophage infiltration, without eliminating graft degradation. Decellularized ostrich carotid arteries were modified with GA or N-hydroxysuccinimide-activated 8-arm PEG (8-arm PEG-NHS), which has a molecular weight of 17 kDa. To evaluate the enzymatic degradation in vitro, the graft was immersed in a collagenase solution for 12 hr. The 8-arm PEG-modified graft was degraded to the same extent as the unmodified graft, but the GA-modified graft was not degraded. The graft was transplanted into rat subcutaneous tissue for up to 8 weeks. Although CD68-positive cells accumulated in the unmodified graft, they did not infiltrate into either modified graft. However, the GA-modified grafts calcified, but the 8-arm PEG-modified graft did not calcify after transplantation. These data suggested that 8-arm PEG-NHS is a promising modification agent for biodegradable vascular xenografts, to suppress acute macrophage infiltration only.