A potent antibiotic-loaded bone-cement implant against staphylococcal bone infections.

New antibiotics should ideally exhibit activity against drug-resistant bacteria, delay the development of bacterial resistance to them and be suitable for local delivery at desired sites of infection. Here, we report the rational design, via molecular-docking simulations, of a library of 17 candidate antibiotics against bone infection by wild-type and mutated bacterial targets. We screened this library for activity against multidrug-resistant clinical isolates and identified an antibiotic that exhibits potent activity against resistant strains and the formation of biofilms, decreases the chances of bacterial resistance and is compatible with local delivery via a bone-cement matrix. The antibiotic-loaded bone cement exhibited greater efficacy than currently used antibiotic-loaded bone cements against staphylococcal bone infections in rats. Potent and locally delivered antibiotic-eluting polymers may help address antimicrobial resistance.

Application of a Mytilus edulis-derived promoting calcium absorption peptide in calcium phosphate cements for bone.

The IEELEEELEAER peptide (PIE) identified from the protein hydrolysate of Mytilus edulis is reported to enhance osteoblast growth and differentiation, which also possesses a superior bone formation ability both in vitro and in vivo. Moreover, PIE bound to calcium spontaneously at the stoichiometry of 1:1, and there were amino nitrogen and carboxyl oxygen atoms in 2 glutamic acid residues at the calcium-binding sites in the PIE. The PIE-calcium complex facilitated calcium uptake through the Caco-2 cell monolayers. Incorporation of PIE into calcium phosphate cements enhanced calcium ion uptake and proliferation of osteoblasts and inhibit bacteria. This study suggest that calcium phosphate cements supplemented with PIE can serve as a potentially efficient material for bone graft used during spinal surgery.

Absorption and transport of a Mytilus edulis-derived peptide with the function of preventing osteoporosis.

The YPRKDETGAERT peptide (PME-1) identified from the Mytilus edulis proteins has been shown to promote the proliferation and differentiation of osteoblasts and it has good bone-forming activity in vitro. Further, PME-1 has been shown to prevent osteoporosis in vivo. PME-1 can be absorbed through the gastrointestinal tract, and the passing rate in monolayer Caco-2 cells was 6.57%. PME-1 can also enter the blood circulation and the concentration of PME-1 in serum reached the maximum, 61.06 ± 26.32 ng mL-1, 20 min after feeding. The multifunctional in vivo imager was used to further determine the distribution of the 5-FITC-(Acp)-YPRKDETGAERT peptide (PME-1-FITC) 2 h after feeding the peptide, and the result confirmed the above results and showed that a part of PME-1-FITC can affect bone in vivo. Therefore, PME-1 not only was easily absorbed in the gastrointestinal tract, but also has the potential beneficial effect on preventing osteoporosis.

Pharmacokinetics and Transport of an Osteogenic Dodecapeptide.

A dodecapeptide with the amino acid sequence of IEELEEELEAER (PIE), identified from Mytilus edulis proteolysis hydrolysates, has shown good bone-forming activity in previous studies. The pharmacokinetics and transport of the PIE peptide in vivo or in vitro were investigated in this study. The results showed that the PIE peptide can be transported into monolayer Caco-2 cells, and the PIE peptide was identified in the serum after the mice reached the highest value of 173.60 ± 60.30 ng/mL, in which it was quantified by an optimized mass spectrometry method. In addition, the PIE peptide has a promoting effect on the bone morphogenetic protein pathway at the gene and protein levels. According to the distribution of PIE-FITC in ovariectomized mice after orally administrated PIE-FITC, it was confirmed that it can enter the gastrointestinal tract and serum, and reach the bones. Taken together, the PIE peptide can be absorbed well both in vitro and in vivo, and it could promote pre-osteoblast differentiation factors.

A bovine lactoferrin-derived peptide induced osteogenesis via regulation of osteoblast proliferation and differentiation.

Osteoporosis is a disease of aging, characterized by a decrease in bone quality and a reduction in bone strength. Promoting the activity of osteoblasts is a useful strategy for combating the progression of osteoporosis. As a novel bone growth factor, lactoferrin plays a role in the anabolic activity in bone by inducing the proliferation and differentiation of osteoblasts and inhibiting the formation of osteoclasts. However, potential peptides with osteogenic activity from lactoferrin have not been identified. In the present study, a peptide with osteogenic activity-LFP-C, fragment residues 624 to 632, derived from lactoferrin hydrolysates-was identified using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and screened using molecular docking analysis. The LFP-C peptide significantly increased the proliferation of mouse cell line MC3T3-E1 and had a promoting effect on alkaline phosphatase activity and calcium deposition. Moreover, LFP-C increased the proportion of osteoblasts in the G2 and M phases. The osteogenic mechanism of LFP-C was also studied by molecular docking. We found that LFP-C could bind to the key domain (Lys13-Thr15-Gln16-Leu17-Gly18-Asp22) of epidermal growth factor receptor, a vital receptor tyrosine kinase that leads to the activation of the mitogen-activated protein kinase pathway. The main interaction forces were interpolated charge, hydrophobicity, and hydrogen bonding. Results indicated that LFP-C may play an osteogenic role in a similar way to lactoferrin, by promoting the proliferation and differentiation of osteoblasts. The findings of this in vitro experiment also demonstrated that the molecular docking method could play a role in the screening process; this in silico approach allowed for faster and cheaper identification of a promising bioactive component.

Nutritional properties and osteogenic activity of enzymatic hydrolysates of proteins from the blue mussel (Mytilus edulis).

Seafood provides a range of health benefits due to its nutritional and bioactive components. However, the bioactive peptides derived from Mytilus edulis proteins were seldom reported, especially their beneficial effects related to bone growth in vitro and in vivo. In this study, the water soluble protein from Mytilus edulis was isolated and the osteogenic activity of Mytilus edulis protein was determined in vivo. The protein from Mytilus edulis was subjected to simulated digestion in vitro, and the hydrolysate of different stages for osteogenic activity by osteoblast proliferation. It was found that the hydrolysate, derived from proteins hydrolyzed by pepsin for 2 h and trypsin for 3 h, showed high osteogenic activity, which induced an increase of 35.56 ± 2.92% in mouse-MC3T3-E1-preosteoblast-cell growth and the alkaline-phosphatase activity was 2.94 ± 0.10 mU, which was an increase of 19.78% compared with that of the control. Moreover, the molecular weight distribution of the peptides and the composition of the free amino acids were determined in order to evaluate the nutritional properties. These findings showed that the water soluble protein from Mytilus edulis could be used in functional food as a bioactive ingredient, which would be beneficial for bone growth and health.

Bone formation activity of an osteogenic dodecapeptide from blue mussels (Mytilus edulis).

A novel osteogenic dodecapeptide peptide (PIE), IEELEEELEAER, was purified from the protein hydrolysate of blue mussels (Mytilus edulis). PIE was identified using a capillary electrophoresis electrospray ionization-quadrupole-time of flight mass spectrometer. PIE showed a good reduction in the bone loss in ovariectomized mice, and it also increased the bone mineral density of the ovariectomized mice. PIE has a high affinity with integrins (PDB: , ). There are 8 and 12 amino acid residues from PIE that interact with integrins and , respectively. PIE accelerates the transformation of G0/G1 phase cells into G2 M phase cells, which promotes the growth of osteoblasts. PIE (100 µg mL-1) can enhance alkaline phosphatase (ALP) activity by 26.48% compared with the control, and it also inhibits the growth of osteoclasts and tartrate resistant acid phosphatase (TRAP) activity. Therefore, PIE may contribute to preventing osteoporosis both in vitro and in vivo.

Identification and availability of peptides from lactoferrin in the gastrointestinal tract of mice.

Lactoferrin is a protein with multifunctional bioactivities. However, limited studies have reported the digestion behavior of lactoferrin in mice. In the present study, the metabolic fate of lactoferrin in the gastrointestinal tract (GIT) of mice was investigated. The mice were orally administered 4 mg of lactoferrin. The contents in the stomach, duodenum, jejunum and ileum were collected at 0.5 h, 1 h, 2 h and 3 h after administration. The peptides in the contents of the stomach, duodenum, jejunum and ileum were identified by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) coupled with capillary electrophoresis (CE). At 0.5 h after administration, 350, 47, 118 and 26 novel peptides were identified in the contents of the stomach, duodenum, jejunum and ileum, respectively. At 1 h after administration, 175, 23, 3 and 8 novel peptides were identified in the contents of the stomach, duodenum, jejunum and ileum, respectively. At 2 h and 3 h after administration, 96 and 2 novel peptides were identified in the contents of the stomach, respectively. Among these peptides, 10 peptides were generated from gastrointestinal digestions and could play physiological roles when they are absorbed through the intestine in their original form. This work provided some theoretical basis for clarifying the bioactive mechanisms of lactoferrin.

Isolation and Characterization of Peptides from Mytilus edulis with Osteogenic Activity in Mouse MC3T3-E1 Preosteoblast Cells.

Seafood provides a range of health benefits because of its high protein levels. In this study, a novel peptide, YPRKDETGAERT, was identified from NHA-2 of Mytilus edulis by capillary-electrophoresis electrospray ionization-quadrupole-time of flight (CESI-Q-TOF). Peptide YPRKDETGAERT showed the highest affinity among all the peptides, with -CDOCKER energy values of 204.482 kcal/mol on one integrin (PDB: 3VI4 ) and 210.16 kcal/mol on another integrin (PDB: 1L5G ). The secondary mass spectrogram at the m/ z of peptide YPRKDETGAERT was 1422.53 Da, which was determined by CESI-Q-TOF. Peptide YPRKDETGAERT induced an increase of 28.27 ± 3.66% in mouse-MC3T3-E1-preosteoblast-cell growth. The alkaline-phosphatase activity of peptide YPRKDETGAERT was 2.79 ± 0.07 mU, which was an increase of 21.25% compared with that of the control. These results provide theoretical and practical insights for the preparation and application of osteogenic peptides in the functional-foods industry.

Identification and mechanism evaluation of a novel osteogenesis promoting peptide from Tubulin Alpha-1C chain in Crassostrea gigas.

Marine shellfish provides a series of biofunctionality account of its high-protein level. In this study, the osteogenic effect of a novel peptide, YRGDVVPK, from Crassostrea gigas protein hydrolysates on preosteoblast MC3T3-E1 proliferation was examined. Synthetic peptide with 100 nM significantly promoted the proliferation of MC3T3-E1 cells for a treatment of 72 h assayed by MTT method, and which was confirmed by the increase of alkaline phosphatase (ALP) activity. The peptide, YRGDVVPK, was docked with integrin α5ß1 (PDB ID: 3VI4), which is a surface receptor of MC3T3-E1. The interaction of the peptide with integrin α5ß1 (PDB ID: 3VI4) was analyzed by the molecular modeling algorithm of CDOCKER, which showed a more stable combination than the original ligand. The results suggested the novel peptide could promote the preosteoblast MC3T3-E1 proliferation probably by activating the signaling pathway of MAPK, which is induced through binding with peptide YRGDVVPK.

Protein cross-linking and the Maillard reaction decrease the solubility of milk protein concentrates.

Milk protein concentrate (MPC) is a widely used material in the food industry. However, despite its widespread use, the mechanism underlying the decreased solubility of MPC that occurs during storage has not yet been clarified. In this study, the solubility changes, protein cross-linking, and Maillard reaction and the relationships between them were investigated in modified MPC powders (MMPC) containing different concentrations of protein and/or lactose stored at 50°C for 15-45 days. The results demonstrated that both the protein and lactose contents affected solubility. The proteins interacted through hydrogen bonding, disulfide bonding, hydrophobic interactions, and nondisulphide covalent bonding, which led to cross-linking. The Maillard reaction promoted protein cross-linking and was in turn influenced by protein cross-linking. The Maillard reaction was slower when the degree of protein cross-linking was greater. These results improve our understanding of the mechanism leading to poor solubility of MPC powders during storage.

Characterization of natural hydroxyapatite originated from fish bone and its biocompatibility with osteoblasts.

Hydroxyapatite (HAP) was very attractive for using as bone implant material for a long period due to the close similarity with natural bone in composition and osteoconductive properties. In this study, three kinds of natural HAP (nHAP) derived from rainbow trout (Onchorynchus mkiss), cod (Gadus) and salmon (Oncorhynchus keta) bones were prepared using thermal calcination method for the first time. Resultant nHAPs were characterized by fourier transform infrared spectroscopy (FT-IR), x-ray diffraction analysis (XRD), field-emission scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analysis. Biocompatibility of calcined nHAP was evaluated through MTT cell viability assay and alkaline phosphatase activity experiment using mouse preosteoblast MC3T3-E1. Results of cell experiment indicated that the nHAP originated from rainbow trout and salmon bones showed better biological compatibility compared with the nHAP originated from cod bone and chemical synthetic HAP (cHAP). This is most likely attributed to the different element composition in nHAP, i.e., the nHAP derived from rainbow trout and salmon bones showed the presence of CO32- and Mg2+. Therefore, the nHAP originated from rainbow trout and salmon bones have a great potential for application as implant material substitute in bone tissue engineering and the natural waste fish bone product can be used for hydroxyapatite synthesis as a part of bio-waste management.

Lactoferrin preserves bone homeostasis by regulating the RANKL/RANK/OPG pathway of osteoimmunology.

Osteoporosis can be classified as an inflammatory disease and the crosstalk between the immune system and bone growth should be considered. The effects of bovine lactoferrin on bone by osteoimmunology were investigated in the present study. Ten week old female BALB/c mice were ovariectomized (OVX) and fed for 12 weeks with a control diet or lactoferrin (2, 20 and 100 mg kg-1 d-1). The results showed that following 12 weeks of treatment after surgery, OVX resulted in bone loss, but treatment with lactoferrin preserved bone homeostasis. Lactoferrin significantly improved bone mineral density, and increased the serum levels of alkaline phosphatase, while it decreased the serum levels of tartrate-resistant acid phosphatase. Furthermore, according to micro-computed tomography (micro-CT), the bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) were elevated. The results indicated that the structure model index (SMI) was reduced in the OVX + LF groups. Additionally, lactoferrin enhanced the Max-Load and Max-Stress values of the femur, and increased the content of Ca and P. However, lactoferrin suppressed the RANKL/OPG ratio in OVX mice. Moreover, interferon-γ, interleukin-5 and interleukin-10 were elevated significantly in the OVX + LF groups. Lactoferrin had a positive effect on the bone micro-environment and might be a pleiotropic protein for the prevention and treatment of estrogen-dependent bone loss via the osteoimmunology pathway.

Complementation of UPLC-Q-TOF-MS and CESI-Q-TOF-MS on identification and determination of peptides from bovine lactoferrin.

Digested peptides of bovine lactoferrin as the functional hydrolysates were identified by the Q-TOF tandem mass spectrometry (Q-TOF-MS) coupled with ultra performance liquid chromatograph (UPLC) and capillary electrophoresis (CE). The former (UPLC-Q-TOF-MS) identified 106 peptides while the latter (CE-Q-TOF-MS) characterized 102 peptides after comparison of peptides in terms of their molecular weight (MW), mass-to-charge ratio (m/z), and isoelectric point (pI). In addition, the hydrophilic value, net charge (q), and molecular radius (r) of the peptides were calculated, and a correlation analysis of the two methods was conducted between the retention time (RT) and r/q ratio of the peptides in order to elucidate the different separation principles of the unique peptides. It was shown that the peptides with larger hydrophilic value were beneficial to be separated by UPLC, while the peptides with larger r/q ratio were beneficial to be separated by CE. Combination of the above mentioned two complementary techniques have confidently improved the sequence coverage of lactoferrin and enhanced the identification of peptides, which makes it up to 65.8% in this study.

Beneficial effects of polysaccharides on the solubility of Mytilus edulis enzymatic hydrolysates.

The low stability of proteins and peptides in liquid systems restricts the development of enzymatic hydrolysate. In this study, a variety of protein hydrolysates from Mytilus edulis (PHM) were prepared by enzymatic hydrolysis. The effects of four kinds of polysaccharides, CMC-Na, xanthan gum, ß-cyclodextrin, and Z-trim, on the stability of PHM were investigated by comparing the protein precipitation rate, particle size, zeta potential, microstructure, and fluorescence spectra. Results showed that the aqueous mixture enriched with proteins and peptides was stabilized by the addition of 1 mg/mL xanthan gum at pH 8, and it significantly prevented protein deposition. Particle size and zeta potential measurements confirmed the beneficial effects of xanthan gum on the stability of PHM. This study provides some theoretical references for the research and development of proteins from M. edulis.

Lactoferrin promotes MC3T3-E1 osteoblast cells proliferation via MAPK signaling pathways.

Lactoferrin has attracted great attention as a potential functional factor to prevent osteoporosis due to its various bioactivities. However, the molecular mechanism underlining the osteogenic activity of lactoferrin is unclear. In this study, effect of lactoferrin on MC3T3-E1 osteoblast cells proliferation was determined using MTT assay, while MAPK signaling pathways related to proliferation of MC3T3-E1 osteoblast cells were investigated based on mRNA and protein expressions. The distribution of cells at different cell cycle stages was evaluated by flow cytometry. Our findings indicated that lactoferrin enhanced MC3T3-E1 osteoblast cells proliferation in a dose-dependent manner; namely, it increased the proportion of cells in S and G2/M phases. Furthermore, we also found that lactoferrin could stimulate ERK, JNK and p38 MAPK. The mRNA expression of MAPK were significantly enhanced after treatment of lactoferrin. Lactoferrin significantly promoted the activation-associated phosphorylation of ERK and p38 MAPK and prevented the activation of JNK. Additionally, lactoferrin could enhance c-Fos and c-Jun expression by 3 times and 26 times, respectively. These results indicated that lactoferrin induced MC3T3-E1 osteoblast cells proliferation through c-Fos and c-Jun by stimulating ERK, JNK and p38, elucidating the molecular basis of the osteogenic activity of lactoferrin on MC3T3-E1 osteoblast cells.

Bioactive hydrolysates from casein: generation, identification, and in silico toxicity and allergenicity prediction of peptides.

BACKGROUND: Bioactive casein peptides have attracted considerable attention for their applications in industry. However, there is little clarity regarding mass spectrometric profiles for peptides in enzymatic hydrolysates of casein produced under varying conditions. In this study, the compositions of the peptides from casein hydrolysates were compared for different enzyme/substrate ratio (E/S) and hydrolysis times. The toxicity, allergenicity and bioactivity of the identified peptides were assessed in silico. RESULTS: A total of 70 unique peptides were identified, and there were 28, 21, 13 and 8 peptides from αs1 -casein, αs2 -casein, ß-casein and κ-casein respectively. The peptide number decreased with the increase in E/S and hydrolysis time. Moreover, peptides with relative molecular mass Mr ranging from 1000 to 1500 Da occupied the highest proportion of 31.43%, and almost all of the peptides showed Mr less than 5000 Da. In silico analysis showed that all of the peptides were non-toxic and non-allergenic, and several of them were assessed by PeptideRanker as having a relatively high likelihood of being bioactive peptides. CONCLUSIONS: Composition of the peptides in the casein hydrolysates varied with the enzymolysis conditions. This study's results may facilitate the production of target bioactive peptides by controlling E/S and hydrolysis time, which is beneficial for the application of casein peptides in the functional food industry. © 2017 Society of Chemical Industry.

Isolation and Characterization of Lactoferrin Peptides with Stimulatory Effect on Osteoblast Proliferation.

Lactoferrin is reported to be a potential food protein with osteogenic activity. However, the activity of lactoferrin peptides is questionable. In the present study, we isolated and characterized peptides from lactoferrin with stimulatory effect on osteoblast proliferation. Peptides from the lactoferrin pepsin hydrolysate were purified using cation-exchange and gel-filtration chromatography. Effects of different hydrolysates and peptides on the proliferation of osteoblast MC3T3-E1 cells were compared by MTT assay. Results showed that fraction P5-a from Superdex Peptide 10/300 GL gel chromatography showed better activity. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis and high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry confirmed that two peptides components of P5-a corresponded to fractions of 20-78 and 191-277 amino acids in Bos taurus lactoferrin molecule (GI: 221706349). These results will provide some theoretical and practical data for the preparation and application of osteogenic peptides in functional food industry.

Hydroxyapatite nanorod and microsphere functionalized with bioactive lactoferrin as a new biomaterial for enhancement bone regeneration.

Lactoferrin (LF) has been recently recognized as a promising new novel bone growth factor for the beneficial effects on bone cells and promotion of bone growth. Currently, it has been attracted wide attention in bone regeneration as functional food additives or a potential bioactive protein in bone tissue engineering. The present study investigated the possibility that hydroxyapatite (HAP) particles, a widely used bone substitute material for high biocompatibility and osteoconductivity, functionalized with lactoferrin as a composite material are applied to bone tissue engineering. Two kinds of hydroxyapatite samples with different sizes, including nanorods and microspheres particles, were functionalized with lactoferrin molecules, respectively. A detailed characterization of as-prepared HAP-LF complex is presented, combining thermal gravimetric analysis (TGA) and Fourier Transform Infrared Spectroscopy (FT-IR). Zeta potential and the analysis of electrostatic surface potential of lactoferrin were carried to reveal the mechanism of adsorption. The effects of HAP-LF complex on MC3T3-E1 osteoblast proliferation and morphology were systematically evaluated at different culture time. Interestingly, results showed that cell viability of HAP-LF group was significantly higher than HAP group indicating that the HAP-LF can improve the biocompatibility of HAP, which mainly originated from a combination of HAP-LF interaction. These results indicated that hydroxyapatite particles can work as a controlled releasing carrier of lactoferrin successfully, and lactoferrin showed better potentiality on using in the field of bone regeneration by coupling with hydroxyapatite. This study would provide a new biomaterial and might offer a new insight for enhancement of bone regeneration.