Yasuo Sakai

Promotion by Collagen Tripeptide of Type I Collagen Gene Expression in Human Osteoblastic Cells and Fracture Healing of Rat Femur

Peptides produced by the enzymatic degradation of collagens are reported to have various activities of biological and medical interest. The mechanisms underlying their actions are, however, poorly understood. We have produced, by collagenase digestion of type I collagen, a highly purified, non-antigenic, and low allergenic tripeptide fraction (collagen tripeptide, Ctp). We report here the effects of Ctp on the in vivo bone fracture healing and in vitro calcification of osteoblastic cells. An oral administration of Ctp to rats with a femur fracture accelerated the fracture healing. Ctp apparently stimulated the calcification of human osteoblastic cells in culture. This osteotrophic effect was accompanied by a significant increase in type I collagen protein production and its mRNA levels. DNA microarray and quantitative RT-PCR analyses demonstrated that Ctp upregulated the bone-specific transcription factor, Osterix, suggesting that the induction of type I collagen gene expression by Ctp was mediated by upregulation of this factor.

Absorption and plasma kinetics of collagen tripeptide after peroral or intraperitoneal administration in rats

Collagen tripeptide (CTP) is a collagen-derived compound containing a high concentration of tripeptides with a Gly-X-Y sequence. In this study, the concentrations and metabolites of CTP were monitored in rat plasma after its administration. We performed a quantitative analysis using high-performance liquid chromatography tandem mass spectrometry according to the isotopic dilution method with stable isotopes. We confirmed that the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp were transported into the plasma. Dipeptides, which are generated by degradation of the N- or C-terminus of the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp, were also present in plasma. The plasma kinetics for peroral and intraperitoneal administration was similar. In addition, tripeptides and dipeptides were detected in no-administration rat blood. The pharmacokinetics were monitored in rats perorally administered with Gly-[3H]Pro-Hyp. Furthermore, CTP was incorporated into tissues including skin, bone, and joint tissue. Thus, administering collagen as tripeptides enables efficient absorption of tripeptides and dipeptides. Graphical abstract Plasma kinetics of peptides in the two routes of administration, and autoradiographs after peroral administration of Gly-[3H]Pro-Hyp.

Interaction of (+)-Catechin with a Lipid Bilayer Studied by the Spin Probe Method

The interaction of (+)-catechin with a lipid bilayer was examined by the spin probe method. The spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was dissolved in an aqueous dipalmitoylphosphatidylcholine (DPPC) dispersion containing (+)-catechin. The temperature dependence of the TEMPO parameter was measured. The increase of this parameter due to pretransition was eliminated by the addition of (+)-catechin, suggesting that it was adsorbed to the lipid membrane surface in the gel state, which hindered the change of the membrane from a flat to wavy structure. In the temperature region of the main transition, the TEMPO parameter increased rapidly, then gradually with increasing temperature, which could be explained by the eutectic phase diagram. The rotational correlation time of a spin probe 16-doxylstearic acid and the order parameter of 5-doxylstearic acid in the aqueous dispersion system of egg yolk phosphatidylcholine revealed that the motion of the alkyl chain in the liquid crystal state was hindered in the center of the membrane as well as near the surface by the adsorption of (+)-catechin.

Purification and Properties of a New Type of Protease Produced by Microbacterium liquefaciens

A bacterium, identified as Microbacterium liquefaciens MIM-CG-9535-I, was isolated from a soil sample taken from the industrial site of a gelatin manufacturer. A new type of protease, which restrictively decomposes gelatin at one or two positions, was purified from the bacterial culture. The molecular mass of the purified enzyme was 21 kDa by SDS–polyacrylamide gel electrophoresis. The purified enzyme specifically degraded the α-chain of gelatin with a molecular weight of 100 kDa into two peptides of 60 kDa and 40 kDa. Native collagen was not a substrate for the enzyme.

Absorption and Urinary Excretion of Peptides after Collagen Tripeptide Ingestion in Humans

Collagen tripeptide (CTP) is a collagen hydrolysate containing a high concentration of tripeptides with a Gly-X-Y sequence, such as Gly-Pro-Hyp. To test the effects of this preparation, we compared the absorption of peptides in humans after ingestion of a tripeptide fraction of CTP (CTP-100), a CTP preparation containing ca. 50% Gly-X-Y tripeptides (CTP-50), and a collagen peptide that did not contain tripeptides (CP). The postprandial levels of Gly-Pro-Hyp and Pro-Hyp in the plasma increased in those subjects who ingested CTP-100 and CTP-50, and were higher with greater Gly-Pro-Hyp ingestion. This demonstrated that collagen hydrolysates were efficiently absorbed when the collagen was ingested in the tripeptide form. Gly-Pro-Hyp and Pro-Hyp were also found in the urine after ingestion of CTP-100 or CTP-50. Similar to the results for the plasma concentration, the urinary excretion of Gly-Pro-Hyp and Pro-Hyp was also dependent on the amount of Gly-Pro-Hyp ingested. This indicates that ingested Gly-Pro-Hyp and generated Pro-Hyp were relatively stable in the body and were transported to the urine in the peptide form. The concentration of Hyp-Gly in the plasma was low after the ingestion of CP and CTP-100 but higher after the ingestion of CTP-50. Overall, our results suggest that tripeptides derived from collagen are absorbed efficiently by the body.

Effect of the up-front heat treatment of gelatin particles dispersed in calcium phosphate cements on the in vivo material resorption and concomitant bone formation

Calcium phosphate cements (CPCs), consisting of a mixture of calcium phosphate powders and setting liquid, have been widely used in orthopedic applications. One of the drawbacks of CPCs is their poor resorbability in the living body, which hinders substitution with natural bones. One of the strategies to facilitate the resorption of CPCs is the incorporation of bioresorbable or water-soluble pore-generating particles (porogens), such as gelatin, in the CPC matrices. In spite of numerous reports, however, little is known about the effect of the dissolution/resorption rate of the porogens on concomitant bone regeneration. In the present study, we prepared preset CPCs dispersed with 10 mass% of low-endotoxin gelatin particles 200–500 μm in diameter having different heat-treatment histories, therefore exhibiting different dissolution rate, and then the obtained CPC/gelatin composites were evaluated for in vivo resorption and concomitant in vivo bone formation behaviors. As the results, the dispersion of gelatin particles markedly promoted in vivo resorption of CPC, and enhanced concomitant bone formation, connective tissue formation, osteoblast proliferation, and vascularization. The dissolution/resorption rate was able to be controlled by changing the up-front heat-treatment temperature. In particular, when CPC/gelatin composites were implanted in distal metaphysis of rabbits, the optimum dissolution/resorption was attained by heat-treating gelatin particles at 383 K for 24 h before dispersing in CPC.Graphical abstractQuick resorption of calcium phosphate cement and concomitant bone formation by dispersing properly heat-treated with gelatin particles.

Anti-inflammatory effect of collagen tripeptide in atopic dermatitis

BACKGROUND Atopic dermatitis (AD) is a chronic, pruritic inflammatory skin disease in which type 2 allergic inflammation plays an important role. Collagen tripeptide (CTP) is a functional collagen fraction with a high content of Gly-X-Y tripeptides. OBJECTIVE To examine the effect of CTP on inflammation in AD. METHODS Levels of inflammatory cytokines and chemokines, such as thymus- and activation-regulated chemokine (TARC), macrophage-derived chemokine, and thymic stromal lymphopoietin (TSLP), were examined in human keratinocytes supplemented with or without CTP under AD-like inflammation. To evaluate the functional effect of CTP, a migration assay was performed using the supernatants of cultured keratinocytes treated with CTP. The signaling pathway for CTP inhibitory activity was also determined. Additionally, we conducted a clinical trial with seventeen AD patients who were assigned randomly to receive daily for 12 weeks either 3.9g of a CTP product or normal collagen peptides (CP). The eruption area, severity scoring of atopic dermatitis (SCORAD), skin hydration, transepidermal water loss (TEWL), and itching score were evaluated. The levels of TARC, serum IgE, lactate dehydrogenase, and eosinophil counts at week 12 were also compared with those at the start of administration. RESULTS In human keratinocytes, TARC and TSLP mRNA and protein levels were inhibited significantly by CTP treatment under AD-like inflammation. Supernatants obtained from CTP-treated keratinocytes inhibited cell migration. STAT1 phosphorylation was significantly decreased by CTP in a dose-dependent manner. In the clinical trial, 13 patients (7 for CTP, 6 for CP) completed the study. The eruption area, SCORAD, and TEWL at week 12 were reduced significantly compared with the initial values in the CTP but not CP group. A significant reduction in the serum TARC level was observed only in the CTP group at week 12. Other blood parameters were not improved in either group. CONCLUSION CTP may have therapeutic benefit for AD by inhibiting type 2-skewed allergic inflammation.

Enhanced bone formation in the vicinity of porous β-TCP scaffolds exhibiting slow release of collagen-derived tripeptides

It has been experimentally proven that orally ingested collagen-derived tripeptides (Ctp) are quickly absorbed in the body and effectively promote the regeneration of connective tissues including bone and skin. Ctp are capable to activate osteoblasts and fibroblasts, which eventually promotes tissue regeneration. Based on these findings, a hypothesis was formulated in this study that direct delivery of Ctp to bone defect would also facilitate tissue regeneration as well as oral administration. To test the hypothesis, we prepared a bone augmentation material with the ability to slowly release Ctp, and investigated its in vivo bone regeneration efficacy. The implant material was porous β-tricalcium phosphate (β-TCP) scaffold which was coated with a co-precipitated layer of bone-like hydroxyapatite and Ctp. The β-TCP was impregnated with approximately 0.8%(w/w) Ctp. Then, the Ctp-modified β-TCP was implanted into bone defects of Wistar rats to evaluate in vivo efficacy of Ctp directly delivered from the material to the bone defects. The control was pristine porous β-TCP. In vitro tests showed that Ctp were steadily released from the co-precipitated layer for approximately two weeks. The Ctp-modified scaffolds significantly promoted new bone formation in vivo in their vicinity as compared with pristine β-TCP scaffolds; 6 weeks after the implantation, Ctp-modified scaffolds promoted twice as much bone formation as the control implants. Consequently, we achieved the slow and steady release of Ctp, and found that direct delivery of Ctp from implant materials was effective for bone regeneration as well as oral administration.Graphical abstractA β-TCP scaffold capable of slowly releasing bone-enhancing substances significantly promoted bone formation.