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Effect of caseinphosphopeptides on bone mass acquisition: in vitro biomolecular analysis on primary human osteoblasts
No full textL'importanza del calcio e i caseinofosfopeptidi: dalla nutrizione all'ingegneria tissutale
No full textBone is characterized by a physiologic turnover, in adult too: 0.05% of calcium, about 500 mg/die, is daily removed (osteoclastic activity) and hidden (osteoblastic activity). This mean that, in the adult, in about six years all bone minerals is changed.
Bone mass level remain the same when deposition rate is equal to reabsorption rate, that is when osteoclastic and osteoblastic activity are in equilibrium. This turnover is most fast in the infant, in this population all bone minerals is changed in about one year. About the total bone mass (Peak Bone Mass, PBM) is stored up within 18-20 years old. An inadequate intake of calcium in this period may lead to a low bone mineral density, which may have implications for bone health, notably risk of osteoporosis, in later life.
The PBM attainment constitutes a very important goal: grater bone mass in adult life means lower fractures in old age.
Calcium is an essential aliment: all calcium in the body comes from diet. Many foods contain calcium, but the amount of calcium, provided per 100 g or per serving, and is bioavailability vary considerably. The major source of calcium is milk and milk products.
The high bioavailability of calcium in milk is essential due to the presence of caseinphosphopeptides, CPPs, bioactive peptides which are formed by proteolytic digestium of casein in the ileum. Thanks to the presence, in their aminoacidic sequence, of an “acidic motive”, constitutes by three phosphorilated serine and two glutammate, Ser(P)-Ser(P)-Ser(P)-Glu-Glu, these peptides are able to prevent the formation of insoluble Ca-phosphate precipitates in the intestinal by complexing ionized calcium in soluble chelates which enhance the amount of intraluminal calcium available for transport across the mucosa by the non-saturable pathway.
Many searchers have proved their ability to increase the amount of calcium absorption and its utilization in bone tissue. Nevertheless, specially in humans searchers have not only obtained positive but also non-significant results. Long term clinical studies are necessary to better investigate CPPs role in humans.
More positive results are obtained in oral health: pre-formed CPPs and amorphous calcium phosphate nanocomplex (CPP-APC) have the potential to provide superior clinical efficacy in preventing dental caries, treating and reparing early stage of disease.
In present study we are demostrated that CPPs are able to iduce calcium uptake in osteoblasts, likewise they do in intestinal cells, both cell types involved in calcium metabolism in vivo. CPPs have instead no action in other cell types, like fibroblasts or neuronal cells. In particular, in osteoblast, kinetics and morphology CPPs cells response are different versus intestinal cells. This difference can be explained by different functions that they are assigned in vivo: intestinal cells are delegated to the calcium absorption instead osteoblast cells are delegated to the right calcemia maintenance.
Moreover, in osteoblasts, CPPs action is mediated by L-type calcium channel activation and this calcium influx in response to CPPs somministration activates cellular function, such as differenziating activity to mature osteoblasts stadium, likewise in the presence of vitamin D3, osteoblasts lineage physiological growth factor.
This results allow to suppose a role for CPPs not only as a functional food, as integration in prevention strategy of these pathologys and life styles characterized by an inadeguate calcium absorption and intake; but also as possible candidates in bone tissue engineering clinical strategies, for example by their integration in scaffolds. Nevertheless this is only the first step and more studies are necessary to better understand CPPs biological role and effect in vivo.
In last years many scientific reports have open the doors to a new research area, in which bioactive components from foods are the protagonists. They not only contribute to as adequate nutritional value, but generate advantages for health, reducing the risk to untle
several pathologys, without the contribution of body foreign chemical substances
Casein phosphopeptides induces intracellular calcium changes and cell function modulation in primary human osteoblasts
No full textCaseinphosphopeptides (CPPs) are a family of peptides originating from in vivo and in vitro hydrolysis of casein. These nutritional compounds display the ability to bind and solubilize minerals such as calcium. The importance to enhance the mineral uptake by the intestine relies on two findings: i) the inverse correlation found between the colon tumor incidence and the absorption of calcium from diet as put in evidence by recent epidemiologic studies; ii) minerals, especially calcium and zinc, are fundamental for improving and maintaining a correct bone mass. On this basis, CPPs were hypothesized to increase the calcium absorption and retention in vivo, with potential effects on bone mineralization (1). Notwithstanding, there are controversial reports on CPP action. The methodological approach used by different laboratories to study calcium absorption and bone mineralization resulted unable to out light whether the peptides have a specific effect on bone metabolism besides the enhancement of calcium availability. We have therefore designed the following study to evaluate a possible direct role of CPPs in bone cell metabolism. Primary human osteoblasts were established in culture using trabecular bone samples obtained from waste materials during orthopedic surgery of patients without metabolic or malignant bone disease. Cytosolic calcium changes were measured by video-microscopy using the fura-2 method on single cells. A mixture of CPPs of commercial origin as well as pure synthetic CPPs were used. The administration of CPPs to human osteoblasts caused an immediate but transient intracellular calcium change in a dose dependent manner. This CPP-induced effect is not cytotoxic and is triggered by an influx of the extracellular ions through the cell plasma membrane. The osteoblast pre-treatment with the active form of vitamin D, known to differentiate human osteoblast, does not affect the cell responsiveness to CPP administration. 4 hours of cell incubation with CPPs induced an increase of the expression of alkaline phosphatase, while 24 hours of cell incubation induced the increase of activity of alkaline phosphatase and of Runx2 expression, both markers of osteoblast differentiation. The same CPP treatment caused a small but significative reduction in cell rate proliferation and a slight increase in apoptosis activity.
These results open the possibility to use CPPs, a nutrient component of the western diet, as tools for improving osteoblast mineralization and differentiation.
Further studies are in progress to develop the way to use CPPs in bone tissue repairing and associated pathology
Caseinphosphopeptides induce intracellular calcium changes and cell function modulation in primary human osteoblasts
No full textThe influence of caseinphosphopeptides on intracellular calcium changes in primary human osteoblasts : a nutrient dependent modulation of bone cell metabolism
Get PDFCaseinphosphopeptides (CPPs) are a family of peptides originating from in vivo and in vitro hydrolysis of casein. They possess a sequence of three phosphorylated serines followed by two glutamic acids, the acidic motif, able to bind minerals such as calcium. These nutritional compounds display the ability to increase calcium solubility in the digestive tract. Thus, CPPs were hypothesized to increase the calcium absorption and retention in vivo, with potential effects on bone mineralization. Notwithstanding, there are controversial reports on CPP action. The methodological approach used by different laboratories to study calcium absorption and bone mineralization resulted unable to out light whether the peptides have a specific effect on bone metabolism besides the enhancement of calcium availability. We have therefore designed the following study to evaluate a possible direct role of CPPs in bone cell metabolism. Primary human osteoblasts were established in culture using trabecular bone samples obtained from waste materials during orthopedic surgery of patients without metabolic or malignant bone disease. Cytosolic calcium changes were measured by video-microscopy using the fura-2 method on single cells. A mixture of CPPs of commercial origin as well as pure synthetic CPPs were used. The administration of CPPs to human osteoblasts caused an immediate but transient intracellular calcium change in a dose dependent manner. This CPP-induced effect, analogous to that reported for human intestinal cells, is not cytotoxic and is triggered by an influx of the extracellular ions through the cell plasma membrane. The osteoblast pre-treatment with the active form of vitamin D, known to differentiate human osteoblast, does not affect the cell responsiveness to CPP administration. The 24 hours cell incubation with CPPs induced the increase of the activity of alkaline phosphatase, a marker of osteoblast differentiation, reaching a level similar to that produced by vitamin D. The same CPP treatment caused a small but significative reduction in cell rate proliferation and a slight increase in apoptosis activity. Taken together these results indicate that CPPs are endowed of a bone specific effect which underlying mechanism requires further evaluation. CPPs may act not only as a mere carrier for improving calcium absorption and utilization, but also as a trophyc compound for bone health by enhancing osteoblast differentiation and activity
Casein phosphopeptides and cellular calcium uptake : biological activity and supramolecular structure
No full textCaseinphosphopeptide-induced calcium uptake in human intestinal cell lines HT-29 and Caco2 is correlated to cellular differentiation
No full textCaseinphosphopeptides (CPPs) are considered as mineral carriers because of their ability to bind and solubilize calcium ions, with the possible role, yet to be
definitely assessed, of improving calcium absorption at the intestinal level. Previous works demonstrated that CPPs improve calcium uptake, with increasing
intracellular calcium concentration, by human differentiated tumor HT-29 cells, and that this effect correlates with the supramolecular structure of CPPs in the
presence of calcium ions. The aim of the present study was to establish whether the CPP effect on calcium uptake is specific for HT-29 cells and depends on the
differentiated state of the cells. To this purpose, HT-29 and Caco2 cells, two models of intestinal cells, were differentiated following appropriate protocols,
including treatment with 1,25-(OH)2 vitamin D3. The CPP-dependent intracellular calcium rises were monitored at the single-cell level through fura2-
fluorescence assays, and cell differentiation was assessed by biochemical and morphological methods. Results clearly showed that the ability to take up
extracellular calcium ions under CPP stimulation is exhibited by both HT-29 and Caco2 cells, but only upon cell differentiation. This evidence adds novel support
to the notion that CPPs favour calcium absorption, thus possibly acting as cellular bio-modulators and carrying a nutraceutical potential
Standard and innovative methodological approaches to induce a differentiated phenotype in human colon adenocarcinoma HT-29 and CaCo-2 cell lines
No full textHuman colon adenocarcinoma HT-29 and Caco2 cell lines are extensively used to gain insight not only into mechanisms underlying cancer development, but also into the main intestinal functions. This statement relies on the possibility to use the two cell lines at different degree of differentiation, thus mimicking the heterogeneity of the intestinal epithelium. On this basis, herein we analyzed well studied and alternative approaches for obtaining a differentiated phenotype for each cell line. Cell differentiation was evaluated by means of cell morphology and biochemical markers. In the case of HT-29 cells, the substitution of the standard culture medium (DMEM) with RPMI 1640, the addition of sodium butyrate and the replacement of glucose with galactose were studied (Gravaghi et al, 2007). In the case of Caco2 cells, the adoption of a new methodology consisting of pre-confluent subcultures for at least 50 passages without altering culture conditions generate a population at various stages of differentiation (Ferraretto et al, 2007). Finally, since the active form of vitamin D3 (VitD3), 1,25-(OH)2D3, is both a differentiation agent and a modulator of the active calcium transport in the intestinal cells, we pretreated undifferentiated and differentiated HT-29 and Caco2 cells with the vitamer. The VitD3 pre treatment in HT-29 cells cultured in DMEM resulted in a slightly more differentiated cells, while in RPMI HT-29 cells does not induce any significant morphological changes. The overall effect of VitD3 in Caco2 cells at early passages is promoting differentiation, while at higher passages the effect of VitD3 on cell differentiation was not so evident. Two fundamental conclusions can be derived: i) it is possible to differentiate human adenocarcinoma cell lines without altering their culture conditions; ii) VitD3 can act either inducing a cell differentiation or altering the differentiated phenotype in dependence of the preexisting cell differentiation degree
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