Repair potential of self-assembling peptide hydrogel in a mouse model of anterior cruciate ligament reconstruction.

Purpose: Establishing zonal tendon-to-bone attachment could accelerate the anterior cruciate ligament reconstruction (ACLR) rehabilitation schedule and facilitate an earlier return to sports. KI24RGDS is a self-assembling peptide hydrogel scaffold (SAPS) with the RGDS amino acid sequence. This study aimed to elucidate the therapeutic potential of KI24RGDS in facilitating zonal tendon-to-bone attachment after ACLR. Methods: Sixty-four C57BL/6 mice were divided into the ACLR + SAPS and ACLR groups. ACLR was performed using the tail tendon. To assess the maturation of tendon-to-bone attachment, we quantified the area of mineralized fibrocartilage (MFC) in the tendon graft with demeclocycline. Immunofluorescence staining of α-smooth muscle actin (α-SMA) was performed to evaluate progenitor cell proliferation. The strength of tendon-to-bone attachment was evaluated using a pull-out test. Results: The MFC and maximum failure load in the ACLR + SAPS group were remarkably higher than in the ACLR group on Day 14. However, no significant difference was observed between the two groups on Day 28. The number of α-SMA-positive cells in the tendon graft was highest on Day 7 after ACLR in both the groups and was significantly higher in the ACLR + SAPS group than in the ACLR group. Conclusion: This study highlighted the latent healing potential of KI24RGDS in facilitating early-stage zonal attachment of tendon grafts and bone tunnels post-ACLR. These findings may expedite rehabilitation protocols and shorten the timeline for returning to sports. Level of Evidence: Not applicable.

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.

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.

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.

Nutrient digestibility, fecal output of fractionated proteins, and ruminal fermentation parameters of goats fed a diet supplemented with spent green tea and black tea leaf silage.

Spent green and black tea leaf silage (GTS and BTS, respectively) was offered as a protein supplement to goats to examine in vivo digestibility, nitrogen balance, urinary excretion of purine derivatives, and ruminal fermentation. Four castrated goats were fed a basal diet supplemented with alfalfa hay cube (AHC), GTS, or BTS in a 4 × 4 Latin square design. Digestibilities of various nutrients except for nitrogen (N) fraction were unaffected by the type of supplement. Digestibility of acid detergent insoluble N (ADIN) in BTS treatment was a negative value and significantly lower than those in other treatments. Urinary N output and retained N were not significantly affected by the diets. The fecal output of neutral detergent insoluble nitrogen (NDIN) and ADIN in the BTS treatment was significantly higher than those in other treatments. Urinary excretion of purine derivatives was not affected by the treatments. Ruminal NH3 -N concentration in AHC and GTS treatments were not significantly different, but that in the BTS treatment was significantly lower than others. These results indicated that GTS is substitutable for AHC as a protein supplement, whereas BTS was able to bind proteins tightly in the digestive tract, which lowered ruminal N degradability and increase fecal N output.

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.

Design of RGDS Peptide-Immobilized Self-Assembling ß-Strand Peptide from Barnacle Protein.

We designed three types of RGD-containing barnacle adhesive proteins using self-assembling peptides. In the present study, three types of RGD-containing peptides were synthesized by solid-phase peptide synthesis, and the secondary structures of these peptides were analyzed by CD and FT-IR spectroscopy. The mechanical properties of peptide hydrogels were characterized by a rheometer. We discuss the correlation between the peptide conformation, and cell attachment and cell spreading activity from the viewpoint of developing effective tissue engineering scaffolds. We created a peptide-coated cell culture substrate by coating peptides on a polystyrene plate. They significantly facilitated cell adhesion and spreading compared to a non-coated substrate. When the RGDS sequence was modified at N- or C-terminal of R-Y, it was found that the self-assembling ability was dependent on the strongly affects hydrogel formation and cell adhesion caused by its secondary structure.

Feasibility of a self-assembling peptide hydrogel scaffold for meniscal defect: An in vivo study in a rabbit model.

The inner avascular zone of the meniscus has limited healing capacity as the area is poorly vascularized. Although peptide hydrogels have been reported to regenerate bone and cartilage, their effect on meniscus regeneration remains unknown. We tested whether the self-assembling peptide hydrogel scaffold KI24RGDS stays in the meniscal lesion and facilitates meniscal repair and regeneration in an induced rabbit meniscal defect model. Full-thickness (2.0 mm diameter) cylindrical defects were introduced into the inner avascular zones of the anterior portions of the medial menisci of rabbit knees (n = 40). Right knee defects were left empty (control group) while the left knee defects were transplanted with peptide hydrogel (KI24RGDS group). Macroscopic meniscus scores were significantly higher in the KI24RGDS group than in the control group at 2, 4, and 8 weeks after surgery. Histological examinations including quantitative and qualitative scores indicated that compared with the control group, the reparative tissue in the meniscus was significantly enhanced in the KI24RGDS group at 2, 4, 8, and 12 weeks after surgery. Immunohistochemical staining showed that the reparative tissue induced by KI24RGDS at 12 weeks postimplantation was positive for Type I and II collagen. KI24RGDS is highly biocompatible and biodegradable, with strong stiffness, and a three dimensional structure mimicking native extracellular matrix and RGDS sequences that enhance cell adhesion and proliferation. This in vivo study demonstrated that KI24RGDS remained in the meniscal lesion and facilitated the repair and regeneration in a rabbit meniscal defect model.

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.

Combination of Copper Ions and Nucleotide Generates Aggregates from Prion Protein Fragments in the N-Terminal Domain.

BACKGROUND: It has been previously found that PrP23-98, which contains four highly conserved octarepeats (residues 60-91) and one partial repeat (residues 92-96), polymerizes into amyloid-like and proteinase K-resistant spherical aggregates in the presence of NADPH plus copper ions. OBJECTIVE: We aimed to determine the requirements for the formation of these aggregates. METHODS: In this study, we performed an aggregation experiment using N-acetylated and Camidated PrP fragments of the N-terminal domain, Octa1, Octa2, Octa3, Octa4, PrP84-114, and PrP76-114, in the presence of NADPH with copper ions, and focused on the effect of the number of copper-binding sites on aggregation. RESULTS: Among these PrP fragments, Octa4, containing four copper-binding sites, was particularly effective in forming aggregates. We also tested the effect of other pyridine nucleotides and adenine nucleotides on the aggregation of Octa4. ATP was equally effective, but NADH, NADP, ADP, and AMP had no effect. CONCLUSION: The phosphate group on the adenine-linked ribose moiety of adenine nucleotides and pyridine nucleotides is presumed to be essential for the observed effect on aggregation. Efficient aggregation requires the presence of the four octarepeats. These insights may be helpful in the eventual development of therapeutic agents against prion-related disorders.

Arg-Glu-Asp-Val Peptide Immobilized on an Acellular Graft Surface Inhibits Platelet Adhesion and Fibrin Clot Deposition in a Peptide Density-Dependent Manner.

Acellular blood vessels possess high potential to be used as tissue-engineered vascular scaffolds. Previously, a high patency was achieved for an Arg-Glu-Asp-Val (REDV) peptide-immobilized small-diameter acellular graft in a minipig model. Results revealed the potential of the peptide to capture a circulating cell and also to suppress fibrin clot deposition. Here, the effect of REDV peptide density on the blood response under ex vivo blood perfusion conditions was investigated. When endothelial cells or platelets were seeded under static conditions, the number of adherent endothelial cells increased with the increase in peptide density. Platelets scarcely adhered on the surface where the peptide density was above 18.9 × 10-4 molecules per nm3. Fibrin clot deposition and circulating cell capture were evaluated in a minipig extracorporeal circulatory system. The fibrin clot did not form on the peptide-immobilized surface, in the range of peptide modification density that was evaluated, whereas the unmodified surface was covered with microthrombi. REDV-specific blood circulating cells were captured on the peptide-immobilized surface with a density above 18.9 × 10-4 molecules per nm3. These results illustrated, under ex vivo blood perfusion conditions, that the REDV-immobilized acellular surface was able to capture cells and also suppress platelet adhesion and fibrin clot deposition in a peptide density-dependent manner.

Nutritive evaluation of spent green and black tea leaf silages by in vitro gas production characteristics, ruminal degradability and post-ruminal digestibility assessed with inhibitory activity of their tannins.

Spent tea leaf contains high levels of crude protein, suggesting that it may be used as an alternative source for ruminant feeding. We assessed the nutritive characteristics of spent green tea leaf silage (GTS) and black tea leaf silages (BTS) in comparison with soybean meal (SBM) and alfalfa hay cube (AHC) using in vitro assay. The effects of tannin on the nutritive characteristics were also evaluated by adding polyethylene glycol (PEG) as a tannin-binding agent. The amount of gas production was greater for SBM, followed by AHC, GTS, and BTS. A significant improvement in gas production upon addition of PEG was observed only for BTS. Ruminal protein degradability and post-ruminal digestibility was higher for SBM, followed by AHC, GTS, and BTS. The presence of PEG significantly increased ruminal degradability and post-ruminal protein digestibility for GTS and BTS, but not for AHC. The increment of protein digestibility by PEG was much greater for BTS than for GTS, indicating that GTS tannins suppress protein digestibility slightly, whereas BTS tannins do so strongly. According to these results, GTS but not BTS has a potential as an alternative to AHC as a ruminant feedstuff.

Superfine Magnetic Resonance Imaging of the Cerebrovasculature Using Self-Assembled Branched Polyethylene Glycol-Gd Contrast Agent.

Magnetic resonance angiography is an attractive method for the visualization of the cerebrovasculature, but small-sized vessels are hard to visualize with the current clinically approved agents. In this study, a polymeric contrast agent for the superfine imaging of the cerebrovasculature is presented. Eight-arm polyethylene glycol with a molecular weight of ≈17 000 Da conjugated with a Gd chelate and fluorescein (F-8-arm PEG-Gd) is used. The relaxivity rate is 9.3 × 10-3 m-1 s-1 , which is threefold higher than that of free Gd chelate. Light scattering analysis reveals that F-8-arm PEG-Gd is formed by self-assembly. When the F-8-arm PEG-Gd is intravenously injected, cerebrovasculature as small as 100 µm in diameter is clearly visualized. However, signals are not enhanced when Gd chelate and Gd chelate-conjugated 8-arm PEG are injected. Furthermore, small vasculature around infarct region in rat stroke model can be visualized. These results suggest that F-8-arm PEG-Gd enhances the MR imaging of cerebrovasculature.

Direct surface modification of metallic biomaterials via tyrosine oxidation aiming to accelerate the re-endothelialization of vascular stents.

Rapid in-situ re-endothelialization of coronary stents is one of the most effective approaches to inhibit late thrombosis and restenosis. Strut surfaces allowing excellent adhesion and migration of endothelial cells and endothelial progenitor cells may accelerate in-situ re-endothelialization. Here, a well-known endothelial cell adhesive peptide, Arg-Glu-Asp-Val (REDV), was directly immobilized onto metallic surfaces by means of single-step tyrosine oxidation with copper chloride (II) and hydrogen peroxide, which we recently reported as a new biomaterial modification technique. REDV immobilization on a 316L stainless steel plate improved endothelial cell adhesion and effectively suppressed platelet adhesion in vitro. In addition, a Co-Cr stent immobilized with Ac-Tyr-Gly-Gly-Gly-Arg-Glu-Asp-Val (Y-REDV) was implanted into a rabbit abdominal aorta. On 7 days postimplantation, 80% of the strut surface of the Y-REDV-immobilized stent was covered by a thin neointimal layer and was similar in appearance to native endothelium. Restenosis and late thrombosis were not observed in the Y-REDV-immobilized stent for 42 days. These findings suggest that direct immobilization of Y-REDV peptide onto metallic biomaterials by tyrosine oxidation is effective for promoting in-situ re-endothelialization in vascular stents. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 491-499, 2018.

Effect of Chaperones on Prion Protein PrP23-98 Aggregation In Vitro.

Recent studies have indicated that PrP23-98, an N-terminal portion of PrP, polymerizes into amyloid-like and proteinase K (PK)-resistant aggregates in the presence of NADPH with copper ions [19], and then that CRT suppressed aggregation of PrP23-98 and also promoted solubilization of the aggregates [18]. As it is interesting to find out whether other chaperones can inhibit aggregation of PrP23-98 in vitro similar to CRT, this study was conducted to determine whether BiP, Grp94, PDI Grp58 and heat shock cognate protein70 (Hsc70) can inhibit aggregation of PrP23-98 in vitro. The present results indicated that Grp94 suppressed aggregation of PrP23-98, but that Grp94 could not mediate solubilization occurred in the aggregates in contrast to CRT. Other chaperons induced aggregation of PrP23-98 in the absence of NADPH.

Functional peptide KP24 enhances submandibular gland tissue growth in vitro.

INTRODUCTION: Salivary gland hypofunction, also known as xerostomia, occurs as a result of radiotherapy for head and neck cancer, autoimmune diseases, or aging. Xerostomia leads to oral health problems and thus affects the quality of life. Biological salivary gland tissue generated in vitro would provide an alternative mode of treatment for this disease. METHODS: To develop a novel method for modulating salivary gland tissue growth in vitro, we prepared a KP24 peptide-immobilized hydrogel sheet, wherein the peptide comprised repeating proline and lysine sequences, and evaluated the effect of this peptide on salivary gland tissue growth. RESULTS: We found that the KP24 peptide has the potential to enhance glandular tissue growth in vitro. This enhancement is associated with neurite outgrowth and increasing neural innervation. CONCLUSION: KP24 peptide modified material would be a promising material for the modulation of salivary gland tissue growth in vitro.

Peptide-modified Substrate for Modulating Gland Tissue Growth and Morphology In Vitro.

In vitro fabricated biological tissue would be a valuable tool to screen newly synthesized drugs or understand the tissue development process. Several studies have attempted to fabricate biological tissue in vitro. However, controlling the growth and morphology of the fabricated tissue remains a challenge. Therefore, new techniques are required to modulate tissue growth. RGD (arginine-glycine-aspartic acid), which is an integrin-binding domain of fibronectin, has been found to enhance cell adhesion and survival; it has been used to modify substrates for in vitro cell culture studies or used as tissue engineering scaffolds. In addition, this study shows novel functions of the RGD peptide, which enhances tissue growth and modulates tissue morphology in vitro. When an isolated submandibular gland (SMG) was cultured on an RGD-modified alginate hydrogel sheet, SMG growth including bud expansion and cleft formation was dramatically enhanced. Furthermore, we prepared small RGD-modified alginate beads and placed them on the growing SMG tissue. These RGD-modified beads successfully induced cleft formation at the bead position, guiding the desired SMG morphology. Thus, this RGD-modified material might be a promising tool to modulate tissue growth and morphology in vitro for biological tissue fabrication.

Tissue-engineered acellular small diameter long-bypass grafts with neointima-inducing activity.

Researchers have attempted to develop efficient antithrombogenic surfaces, and yet small-caliber artificial vascular grafts are still unavailable. Here, we demonstrate the excellent patency of tissue-engineered small-caliber long-bypass grafts measuring 20-30 cm in length and having a 2-mm inner diameter. The inner surface of an acellular ostrich carotid artery was modified with a novel heterobifunctional peptide composed of a collagen-binding region and the integrin α4ß1 ligand, REDV. Six grafts were transplanted in the femoral-femoral artery crossover bypass method. Animals were observed for 20 days and received no anticoagulant medication. No thrombogenesis was observed on the luminal surface and five cases were patent. In contrast, all unmodified grafts became occluded, and severe thrombosis was observed. The vascular grafts reported here are the first successful demonstrations of short-term patency at clinically applicable sizes.

Assessment of Anti-nutritive Activity of Tannins in Tea By-products Based on In vitro Rumen Fermentation.

Nutritive values of green and black tea by-products and anti-nutritive activity of their tannins were evaluated in an in vitro rumen fermentation using various molecular weights of polyethylene glycols (PEG), polyvinyl pyrrolidone (PVP) and polyvinyl polypyrrolidone as tannin-binding agents. Significant improvement in gas production by addition of PEG4000, 6000 and 20000 and PVP was observed only from black tea by-product, but not from green tea by-product. All tannin binding agents increased NH3-N concentration from both green and black tea by-products in the fermentation medium, and the PEG6000 and 20000 showed relatively higher improvement in the NH3-N concentration. The PEG6000 and 20000 also improved in vitro organic matter digestibility and metabolizable energy contents of both tea by-products. It was concluded that high molecular PEG would be suitable to assess the suppressive activity of tannins in tea by-products by in vitro fermentation. Higher responses to gas production and NH3-N concentration from black tea by-product than green tea by-product due to PEG indicate that tannins in black tea by-product could suppress rumen fermentation more strongly than that in green tea by-product.

Fermentation Characteristics, Tannin Contents and In vitro Ruminal Degradation of Green Tea and Black Tea By-products Ensiled at Different Temperatures.

Green and black tea by-products, obtained from ready-made tea industry, were ensiled at 10°C, 20°C, and 30°C. Green tea by-product silage (GTS) and black tea by-product silage (BTS) were opened at 5, 10, 45 days after ensiling. Fermentation characteristics and nutrient composition, including tannins, were monitored and the silages on day 45 were subjected to in vitro ruminal fermentation to assess anti-nutritive effects of tannins using polyethylene glycol (PEG) as a tannin-binding agent. Results showed that the GTS and BTS silages were stable and fermented slightly when ensiled at 10°C. The GTS stored at 20°C and 30°C showed rapid pH decline and high acetic acid concentration. The BTS was fermented gradually with moderate change of pH and acid concentration. Acetic acid was the main acid product of fermentation in both GTS and BTS. The contents of total extractable phenolics and total extractable tannins in both silages were unaffected by storage temperatures, but condensed tannins in GTS were less when stored at high temperature. The GTS showed no PEG response on in vitro gas production, and revealed only a small increase by PEG on NH3-N concentration. Storage temperature of GTS did not affect the extent of PEG response to both gas production and NH3-N concentration. On the other hand, addition of PEG on BTS markedly increased both the gas production and NH3-N concentration at any ensiled temperature. It can be concluded that tannins in both GTS and BTS suppressed rumen fermentation, and tannins in GTS did more weakly than that in BTS. Ensiling temperature for both tea by-products did not affect the tannin's activity in the rumen.