Kayo Terada

Collagen-like polypeptide poly(Pro-Hyp-Gly) conjugated with Gly-Arg-Gly-Asp-Ser and Pro-His-Ser-Arg-Asn peptides enhances cell adhesion, migration, and stratification†

Collagens are widely used in medical applications, including as a scaffold for tissue regeneration. However, animal-derived collagens have several drawbacks, such as low thermal stability, nonspecific cell adhesion, antigenicity, and contamination with pathogenic substances. To overcome these problems, we chemically synthesized the collagen-like polypeptide, poly(prolyl-hydroxyprolyl-glycyl) (poly(Pro-Hyp-Gly)), which forms a collagen-like triple-helical structure and shows biodegradability and biocompatibility. Here, we designed a novel scaffold where fibronectin-derived Gly Arg-Gly-Asp-Ser (GRGDS) and Pro-His-Ser-Arg-Asn (PHSRN) peptides were simultaneously conjugated with poly(Pro-Hyp-Gly). We assessed cell adhesion and migration activities using NIH3T3 cells in the scaffold and stratification ofimmortalized rabbit corneal epithelial cells. Cell adhesion was enhanced in scaffolds with GRGDS, increased with increasing amounts of conjugated GRGDS, and was significantly higher than bovine type I atelocollagen but lower than bovine fibronectin. Interestingly, simultaneous conjugation of GRGDS and PHSRN synergistically enhanced cell migration. Scaffolds containing almost equal amounts of GRGDS and PHSRN showed significantly higher cell migration than bovine type I atelocollagen. Addition of free GRGDS completely inhibited cell migration on the scaffold, whereas addition of free PHSRN partially inhibited cell migration. These results suggest that GRGDS plays a definitive role, and PHSRN plays an additional role, in cell migration. Conjugation of GRGDS resulted in the same level of stratification of rabbit corneal epithelial cells compared with bovine type I atelocollagen and bovine fibronectin. Because the simultaneous conjugation of GRGDS and PHSRN on poly(Pro-Hyp-Gly) enhances cell adhesion, migration, and stratification, it may be a useful scaffold for tissue regeneration.

Polycondensation of Diketopiperazine‐based Dicarboxylic Acids with Diamines and Dibromoxylenes

Aspartic and glutamic acid‐based diketopiperazines, cyclo(l‐asparaginyl‐l‐asparaginyl) (DKPD) and cyclo(l‐glutaminyl‐l‐glutaminyl) (DKPE) were synthesized. Polycondensation of DKPD and DKPE with α,α′‐dibromoxylenes was carried out using K2CO3 as a base in DMF to obtain polymers with weight‐average molecular weights (M ws) of 1100–3500. Furthermore, polycondensation of DKPE with various diamines was carried out using 4‐[4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl]‐4‐methylmorpholinium chloride as a condensation agent in DMF to obtain polymers with M ws of 1200–4100. The polymers were insoluble in common organic solvents except DMF.

Encapsulation of rat bone marrow stromal cells using a poly-ion complex gel of chitosan and succinylated poly(Pro-Hyp-Gly).

Encapsulation of stem cells into a three‐dimensional (3D) scaffold is necessary to achieve tissue regeneration. Prefabricated 3D scaffolds, such as fibres or porous sponges, have limitations regarding homogeneous cell distribution. Hydrogels that can encapsulate cells such as animal‐derived collagen gels need adjustment of the pH and/or temperature upon cell mixing. In this report, we fabricated a poly‐ion complex (PIC) hydrogel of chitosan and succinylated poly(Pro–Hyp–Gly) and assessed its effect on cell viability after encapsulation of rat bone marrow stromal cells. PIC hydrogels were obtained successfully with a concentration of each precursor as low as 3.0–3.8 mg/ml. The maximum gelation and swelling ratios were achieved with an equal molar ratio (1:1) of anionic and cationic groups. Using chitosan acetate as a cationic precursor produced a PIC hydrogel with both a significantly greater gelation ratio and a better swelling ratio than chitosan chloride. Ammonium succinylated poly(Pro–Hyp–Gly) as an anionic precursor gave similar gelation and swelling ratios to those of sodium succinylated poly(Pro–Hyp–Gly). Cell encapsulation was also achieved successfully by mixing rat bone marrow stromal cells with the PIC hydrogel simultaneously during its formation. The PIC hydrogel was maintained in the culture medium for 7 days at 37°C and the encapsulated cells survived and proliferated in it. Although it is necessary to improve its functionality, this PIC hydrogel has the potential to act as a 3D scaffold for cell encapsulation and tissue regeneration. Copyright

Anticoagulant activity of enzymatically synthesized amylose derivatives containing carboxy or sulfonate groups

Heparin is an extracellular matrix polysaccharide. It is widely employed as an anticoagulant and can be used to form an anticoagulant surface on various medical devices such as renal dialysis devices to prevent thrombosis. However, heparin may cause hemorrhage and thrombocytopenia. Moreover, commercially available heparin may be contaminated with viruses and allergens of animal origin, as it is derived mainly from porcine or bovine tissue. To avoid these problems, we prepared succinated and sulfonated enzymatically synthesized amylose (SucESA and SulfESA, respectively) and assessed their anticoagulant activity. SucESA and SulfESA inhibited factor Xa activity in normal human plasma to an equal extent. However, SucESA strongly inhibited thrombin activity, whereas SulfESA only inhibited it slightly. These results suggest that SucESA inhibits the activities of both factor Xa (or its upstream coagulation factors) and thrombin and that SulfESA inhibits only factor Xa activity (or that of its upstream coagulation factors). SucESA and SulfESA with a high degree of substitution strongly inhibited factor Xa and thrombin activity compared with those of the derivatives with a low degree of substitution, even when present in high concentrations. This suggests that the density of the anion group determines the degree of inhibition of factor Xa and thrombin activity. SucESA, which has a high molecular weight, inhibited thrombin activity to a greater degree than low molecular weight SucESA. Because SucESA and SulfESA inhibited both purified factor Xa and thrombin irrespective of the presence of antithrombin, it is suggested that SucESA and SulfESA inhibit via direct action with both enzymes.

Calcium deposition in photocrosslinked poly(Pro-Hyp-Gly) hydrogels encapsulated rat bone marrow stromal cells

Reproducing the features of the extracellular matrix is important for fabricating three‐dimensional (3D) scaffolds for tissue regeneration. A collagen‐like polypeptide, poly(Pro‐Hyp‐Gly), is a promising material for 3D scaffolds because of its excellent physical properties, biocompatibility, and biodegradability. In this paper, we present a novel photocrosslinked poly(Pro‐Hyp‐Gly) hydrogel as a 3D scaffold for simultaneous rat bone marrow stromal cell (rBMSC) encapsulation. The hydrogels were fabricated using visible‐light photocrosslinking at various concentrations of methacrylated poly(Pro‐Hyp‐Gly) (20–50 mg/ml) and irradiation times (3 or 5 min). The results show that the rBMSCs encapsulated in the hydrogels survived 7 days of incubation. Calcium deposition on the encapsulated rBMSCs was assessed with scanning electron microscope observation, Alizarin Red S, and von Kossa staining. The most strongly stained area was observed in the hydrogel formed with 30 mg/ml of methacrylated poly(Pro‐Hyp‐Gly) with 5‐min irradiation. These findings demonstrate that poly(Pro‐Hyp‐Gly) hydrogels support rBMSC viability and differentiation, as well as demonstrating the feasibility of using poly(Pro‐Hyp‐Gly) hydrogels as a cytocompatible, biodegradable 3D scaffold for tissue regeneration.

Preliminary Study on Gelation of Succinylated Poly(Pro-Hyp-Gly) and Chitosan by Polyion Complex Interaction for Cartilage Repair

The research focusing on injectable material for tissue engineering has encouraged the investigation of gel formation derived from succinylated poly(Pro-Hyp-Gly) and chitosan. This paper describes a preliminary study on gelation of both materials to obtain a new material for cartilage repair. Poly(Pro-Hyp-Gly) shows a collagen-like triple helical structure as a major component of extracellular matrix of cartilage. Furthermore, the cationic nature of chitosan is similar in chemical structure with various glycosaminoglicans in cartilage. Through polyion complex interaction between carboxy group of succinylated poly(Pro-Hyp-Gly) and amino group of chitosan, viscous solution containing gel particles was obtained.

SYNTHESIS OF NOVEL DIKETOPIPERAZINE DERIVATIVE AND OBSEVATION OF SELF-ASSEMBLED STRUCTURE

An N-monomethylated unsymmetrical diketopiperazine was synthesized from D-p-hydroxyphenylglycine and sarcosine, and condensed with trans-1,4-cyclohexanedicarboxylic acid to obtain the ester having diketopiperazine moieties at the both termini. Atomic force microscope measurement indicated that the ester formed a supramolecular structure aligned in a circular pattern based on hydrogen bonding between the amide groups of the diketopiperazine moieties.

Diketopiperazine supramolecule derived from hydroxyphenylglycine

A diketopiperazine (DKP) having long alkyl chains was synthesized from D-p-hydroxyphenylglycine, and the formation of supramolecules was examined. The 1 H NMR and UV-vis spectroscopic measurements and molecular modeling have suggested that the DKP molecules are aligned based on hydrogen bonding between the amide groups, and there is no stacking between the phenyl groups.

Cytocompatible polyion complex gel of poly(Pro-Hyp-Gly) for simultaneous rat bone marrow stromal cell encapsulation

Abstract Polyion complex (PIC) gel of poly(Pro-Hyp-Gly) was successfully fabricated by simply mixing polyanion and polycation derivatives of poly(Pro-Hyp-Gly), a collagen-like polypeptide. The polyanion, succinylated poly(Pro-Hyp-Gly), and the polycation, arginylated poly(Pro-Hyp-Gly), contain carboxy (pKa = 5.2) and guanidinium (pKa = 12.4) groups, respectively. Mixing the polyanion and the polycation at physiological pH (pH = 7.4) resulted in PIC gel. The hydrogel formation was optimum at an equimolar ratio of carboxy to guanidinium groups, suggesting that ionic interaction is the main determinant for the hydrogel formation. The hydrogel was successfully used for simultaneous rat bone marrow stromal cell encapsulation. The encapsulated cells survived and proliferated within the hydrogel. In addition, the cells exhibited different morphology in the hydrogel compared with cells cultured on a tissue culture dish as a two-dimensional (2D) control. At day one, a round morphology and homogeneous single cell distribution were observed in the hydrogel. In contrast, the cells spread and formed a fibroblast-like morphology on the 2D control. After three days, the cells in the hydrogel maintained their morphology and some of them formed multicellular aggregates, which is similar to cell morphology in an in vivo microenvironment. These results suggest that the PIC gel of poly(Pro-Hyp-Gly) can serve as a cytocompatible three-dimensional scaffold for stem cell encapsulation, supporting their viability, proliferation, and in vivo-like behavior.

Collagen-Like Polypeptide Poly(Pro-Hyp-Gly) Conjugated with Fibronectin- Derived Peptides Enhances the Cell Adhesion, Migration and Stratification

Animal-derived collagens are used as a scaffold for tissue regeneration. However, the collagens have several drawbacks, such as nonspecific cell adhesion, low thermal stability, residual antigenicity, and contamination of pathogenic substances. To overcome these problems, we reported the chemical synthesis of the collagen-like polypeptide, poly(prolyl-hydroxyprolyl-glycyl) (poly(PHG)), which forms a collagen-like triple-helical structure and has biodegradability and biocompatibility. Here, we designed a new scaffold where fibronectin-derived RGD, Gly-Arg-Gly-Asp-Ser (GRGDS) or cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGDfK), and Pro-His-Ser- Arg-Asn (PHSRN) peptides were simultaneously conjugated on poly(PHG). We assessed NIH3T3 cell adhesion and migration and stratification of immortalized rabbit corneal epithelial (RCE) cells on the scaffold. Cell adhesion was enhanced with increasing in the amount of the conjugated RGD. Cell adhesion activity of conjugated RGD were significantly higher than bovine type I atelocollagen but lower than bovine fibronectin. Cell adhesion activity of cRGDfK-conjugated poly(PHG) was higher than that of GRGDS below the conjugation ration of 150 nmol/mg. This result suggests that cRGDfK is suitable for interaction with cell surface receptor, integrin, because RGD sequence is located in a loop region of fibronectin. Interestingly, simultaneous conjugation of GRGDS and PHSRN synergistically enhanced cell migration. Scaffolds containing almost equal amounts of GRGDS and PHSRN showed significantly higher cell migration activity than bovine type I atelocollagen. Addition of free GRGDS completely inhibited cell migration on the scaffold, whereas addition of free PHSRN partially inhibited cell migration. These results suggest that GRGDS plays a definitive role, and PHSRN plays an additional role, in cell migration. Conjugation of GRGDS resulted in the same level of stratification of RCE cells compared with bovine type I atelocollagen and bovine fibronectin. Because the simultaneous conjugation of GRGDS and PHSRN on poly(PHG) enhances cell adhesion, migration and stratification, it may be a useful scaffold for tissue regeneration.