Growth Of Osteoblast-like Cells Research Articles

Effects of Porosity and Stiffness of Fibroin-based Scaffolds on Osteoblast-like Cell Growth.

Effects of Porosity and Stiffness of Fibroin-based Scaffolds on Osteoblast-like Cell Growth.

Functionalization of 3D Printed Scaffolds Using Polydopamine and Silver Nanoparticles for Bone-Interfacing Applications.

The prevention of bacterial colonization and the stimulation of osseointegration are two major requirements for bone-interfacing materials to reduce the incidence of complications and promote the restoration of the patient's health. The present investigation developed an effective, two-step functionalization of 3D printed scaffolds intended for bone-interfacing applications using a simple polydopamine (PDA) dip-coating method followed by the formation of silver nanoparticles (AgNPs) after a second coating step in silver nitrate. 3D printed polymeric substrates coated with a ∼20 nm PDA layer and 70 nm diameter AgNPs proved effective in hindering Staphylococcus aureus biofilm formation, with a 3000-8000-fold reduction in the number of bacterial colonies formed. The implementation of porous geometries significantly accelerated osteoblast-like cell growth. Microscopy characterization further elucidated homogeneity, features, and penetration of the coating inside the scaffold. A proof-of-concept coating on titanium substrates attests to the transferability of the method to other materials, broadening the range of applications both in and outside the medical sector. The antibacterial efficiency of the coating is likely to lead to a decrease in the number of bacterial infections developed after surgery in the presence of these coatings on prosthetics, thus translating to a reduction in revision surgeries and improved health outcomes.

Exploring a new approach for regenerative medicine: Ti-doped polycrystalline diamond layers as bioactive platforms for osteoblast-like cells growth

This study explores the feasibility to use electroconductive Ti-doped polycrystalline diamond layers as scaffolds for tissue engineering. The synthesis of the diamond–based materials is accomplished in a HFCVD reactor where Ti(IV) acetyl acetonate powders are delivered by N2 fluxes to the growing diamond phase. In-depth investigations (Raman spectroscopy, SEM, AFM, XRD, XPS) allowed the characterization of the morphological/structural/compositional features and the properties of charge transport (KPFM, I-V) induced in the diamond layers by the incorporation of Ti-species. The bioactivity of the Ti-doped diamond surface was verified investigating the growth of MG-63 osteoblast-like cells by using MTT assays and confocal microscopy. The study evidenced a net increase of cell replication rate on diamond scaffolds after 4 days of incubation. After 6-days incubation, the cell growth on the Ti-doped diamond scaffolds increased up to 150% compared with the reference polystyrene tissue culture vessel, with a dominant presence of cells in active division.The cell behavior is discussed and related to the structural and functional surface properties of the Ti-diamond systems, acting as bioactive platforms able to offer an extremely beneficial environment for cell proliferation and viability.

Lactoferrin Coating Improves the Antibacterial and Osteogenic Properties of Alkali-Treated Titanium with Nanonetwork Structures

Titanium and its alloys are the main dental implant materials used at present. The biological properties of pure titanium can be further improved by surface treatment methods. Alkali treatment of pure titanium at room temperature can form nanonetwork structures (TNS) on the surface, which has better osteoinductive ability than pure titanium. However, TNS does not possess antimicrobial properties, and bacterial infection is one of the main reasons for the failure of dental implant therapy. Therefore, it was the focus of our research to endow TNS with certain antimicrobial properties on the premise of maintaining its osteoinductive ability. Because of its excellent broad-spectrum antimicrobial properties and because it promotes osteoblast-like cell growth, lactoferrin (LF) was considered a promising prospect as a surface biological treatment material. In this study, bovine LF of physiological concentration was successfully coated on the surface of TNS to form the TNS-LF composite material. Results from in vitro and in vivo experiments showed that TNS-LF had better osteoinductive ability than TNS. Bacterial attachment and biofilm formation were also significantly decreased on the surface of TNS-LF. Therefore, this study has provided an experimental basis for the development of osteoinduction-antimicrobial composite implant materials for dental applications.

Silk fibroin/sericin 3D sponges: The effect of sericin on structural and biological properties of fibroin

Among naturally occurring polymers, silk fibroin and sericin have attracted much attention in the field of tissue engineering; however, clinical application of silk fibroin/sericin scaffolds in a combined form has been questioned due to the possible pro-inflammatory reaction against native silk and fibroin/sericin 3D constructs. The objective of this study was to fabricate 3D spongy fibroin/sericin scaffolds and to explore the structural, biological and immunological properties of different ratios of fibroin and sericin. Structural characterization revealed a highly porous structure (>91%) with a large surface area and water uptake capacity for all different fibroin/sericin scaffolds. Notably, the scaffolds showed enhanced mechanical properties and a higher degradation rate with increasing sericin content. Excellent cell attachment and no significant cytotoxicity were observed in all scaffold types 7 days after seeding of osteoblast-like MG63 cells. Gene expression of pro-inflammatory markers TNF-α, CXCL10 and CD197 as well as TNF-α secretion by THP-1-derived macrophages revealed no significant immune response to all fibroin/sericin scaffold types when compared to sericin-free F1:S0 samples and a TCP (Mɸ) control group. These results demonstrate that spongy fibroin/sericin scaffolds are able to support the growth of osteoblast-like cells without eliciting a pro-inflammatory response, thus being a promising material for bone tissue engineering.

Effect of Anodized TiO2-Nb2O5-ZrO2 Nanotubes with Different Nanoscale Dimensions on the Biocompatibility of a Ti35Zr28Nb Alloy.

Some important factors in the design of biomaterials are surface characteristics such as surface chemistry and topography, which significantly influence the relationship between the biomaterial and host cells. Therefore, nanotubular oxide layers have received substantial attention for biomedical applications due to their potential benefits in the improvement of the biocompatibility of the substrate. In this study, a nanotubular layer of titania-niobium pentoxide-zirconia (TiO2-Nb2O5-ZrO2) was developed via anodization on a β-type Ti35Zr28Nb alloy surface with enhanced biocompatibility. Scanning electron microscopy (SEM) and surface profilometry analysis of the anodized nanotubes indicated that the inner diameter (Di) and wall thicknesses (Wt) increased with an increase in the water content of electrolyte and the applied voltage during anodization, while the nanotube length (Ln) increased with increasing the anodization time. TiO2-Nb2O5-ZrO2 nanotubes with different Di, Wt, and Ln showed different surface roughnesses (Ra) and surface energies (γ), which affected the biocompatibility of the base alloy. MTS assay results showed that the TiO2-Nb2O5-ZrO2 nanotubes with the largest inner diameter (Di) of 75.9 nm exhibited the highest cell viability of 108.55% due to the high γ of the surface, which led to high adsorption of proteins on the top surface of the nanotubes. The second highest cell viability was observed on the nanotubular surface with Di of 33.3 nm, which is believed to result from its high γ as well as the optimum spacing between nanotubes. Ra did not appear to be clearly linked to cellular response; however, there may exist a threshold value of surface energy of ∼70 mJ/m2, below which the cell response is less sensitive and above which the cell viability increases with increasing γ. This indicates that the TiO2-Nb2O5-ZrO2 nanotubes provided a suitable environment for enhanced attachment and growth of osteoblast-like cells as compared to the bare Ti35Zr28Nb alloy surface.

Bioactive Silk Coatings Reduce the Adhesion of Staphylococcus aureus while Supporting Growth of Osteoblast-like Cells.

Orthopedic and dental implants are associated with a substantial risk of failure due to biomaterial-associated infections and poor osseointegration. To prevent such outcomes, a coating can be applied on the implant to ideally both reduce the risk of bacterial adhesion and support establishment of osteoblasts. We present a strategy to construct dual-functional silk coatings with such properties. Silk coatings were made from a recombinant partial spider silk protein either alone (silkwt) or fused with a cell-binding motif derived from fibronectin (FN-silk). The biofilm-dispersal enzyme Dispersin B (DspB) and two peptidoglycan degrading endolysins, PlySs2 and SAL-1, were produced recombinantly. A sortase recognition tag (SrtTag) was included to allow site-specific conjugation of each enzyme onto silkwt and FN-silk coatings using an engineered variant of the transpeptidase Sortase A (SrtA*). To evaluate bacterial adhesion on the samples, Staphylococcus aureus was incubated on the coatings and subsequently subjected to live/dead staining. Fluorescence microscopy revealed a reduced number of bacteria on all silk coatings containing enzymes. Moreover, the bacteria were mobile to a higher degree, indicating a negative influence on the bacterial adhesion. The capability to support mammalian cell interactions was assessed by cultivation of the osteosarcoma cell line U-2 OS on dual-functional surfaces, prepared by conjugating the enzymes onto FN-silk coatings. U-2 OS cells could adhere to silk coatings with enzymes and showed high spreading and viability, demonstrating good cell compatibility.

Osteoblast-like Cell Growth and Differentiation Behaviors on the Phospholipid Vesicle-Interacted Calcium Phosphate Films

Osteoblast-like Cell Growth and Differentiation Behaviors on the Phospholipid Vesicle-Interacted Calcium Phosphate Films

Novel injectable gellan gum hydrogel composites incorporating Zn- and Sr-enriched bioactive glass microparticles: High-resolution X-ray microcomputed tomography, antibacterial and in vitro testing.

Mineralization of hydrogel biomaterials is desirable to improve their suitability as materials for bone regeneration. In this study, gellan gum (GG) hydrogels were formed by simple mixing of GG solution with bioactive glass microparticles of 45S5 composition, leading to hydrogel formation by ion release from the amorphous bioactive glass microparticles. This resulted in novel injectable, self-gelling composites of GG hydrogels containing 20% bioactive glass. Gelation occurred within 20min. Composites containing the standard 45S5 bioactive glass preparation were markedly less stiff. X-ray microcomputed tomography proved to be a highly sensitive technique capable of detecting microparticles of diameter approximately 8μm, that is, individual microparticles, and accurately visualizing the size distribution of bioactive glass microparticles and their aggregates, and their distribution in GG hydrogels. The widely used melt-derived 45S5 preparation served as a standard and was compared with a calcium-rich, sol-gel derived preparation (A2), as well as A2 enriched with zinc (A2Zn5) and strontium (A2Sr5). A2, A2Zn, and A2Sr bioactive glass particles were more homogeneously dispersed in GG hydrogels than 45S5. Composites containing all four bioactive glass preparations exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. Composites containing A2Zn5 and A2Sr5 bioactive glasses supported the adhesion and growth of osteoblast-like cells and were considerably more cytocompatible than 45S5. All composites underwent mineralization with calcium-deficient hydroxyapatite upon incubation in simulated body fluid. The extent of mineralization appeared to be greatest for composites containing A2Zn5 and 45S5. The results underline the importance of the choice of bioactive glass when preparing injectable, self-gelling composites.

Enzymatic, urease-mediated mineralization of gellan gum hydrogel with calcium carbonate, magnesium-enriched calcium carbonate and magnesium carbonate for bone regeneration applications

Mineralization of hydrogel biomaterials is considered desirable to improve their suitability as materials for bone regeneration. Calcium carbonate (CaCO3 ) has been successfully applied as a bone regeneration material, but hydrogel-CaCO3 composites have received less attention. Magnesium (Mg) has been used as a component of calcium phosphate biomaterials to stimulate bone-forming cell adhesion and proliferation and bone regeneration in vivo, but its effect as a component of carbonate-based biomaterials remains uninvestigated. In the present study, gellan gum (GG) hydrogels were mineralized enzymatically with CaCO3 , Mg-enriched CaCO3 and magnesium carbonate to generate composite biomaterials for bone regeneration. Hydrogels loaded with the enzyme urease were mineralized by incubation in mineralization media containing urea and different ratios of calcium and magnesium ions. Increasing the magnesium concentration decreased mineral crystallinity. At low magnesium concentrations calcite was formed, while at higher concentrations magnesian calcite was formed. Hydromagnesite (Mg5 (CO3 )4 (OH)2 .4H2 O) formed at high magnesium concentration in the absence of calcium. The amount of mineral formed and compressive strength decreased with increasing magnesium concentration in the mineralization medium. The calcium:magnesium elemental ratio in the mineral formed was higher than in the respective mineralization media. Mineralization of hydrogels with calcite or magnesian calcite promoted adhesion and growth of osteoblast-like cells. Hydrogels mineralized with hydromagnesite displayed higher cytotoxicity. In conclusion, enzymatic mineralization of GG hydrogels with CaCO3 in the form of calcite successfully reinforced hydrogels and promoted osteoblast-like cell adhesion and growth, but magnesium enrichment had no definitive positive effect. Copyright © 2017 John Wiley & Sons, Ltd.

Influence of surface pre-treatment on the cytocompatibility of a novel biodegradable ZnMg alloy

Degradable zinc-based alloys with an appropriate corrosion rate are promising materials for the preparation of temporary orthopaedic implants. Previously, we prepared and characterised a novel Zn1.5Mg alloy. This paper is focused on the characterisation of this alloy after a surface pre-treatment, which should mimic processes occurring in vivo.The samples of the Zn1.5Mg alloy were immersed in a simulated body fluid (SBF) at 37°C for 14days in order to form a protective layer of corrosion products. Thereafter, these samples were used for the corrosion rate determination, an indirect in vitro cytotoxicity test, as well as for a direct contact test and were compared with the non-treated samples. The protective layer was characterized by SEM and its chemical composition was determined by EDS and XPS analysis.The corrosion rate was significantly decreased after the pre-incubation. The protective layer of corrosion products was rich in Ca and P. The pre-incubated samples exhibited increased cytocompatibility in the indirect test (metabolic activity of L929 cells was above 70%) and we also observed osteoblast-like cell growth directly on the samples during the contact tests. Thus, the pre-incubation in SBF leading to improved cytocompatibility could represent more appropriate model to in vivo testing.

Chemically Modified Diatoms Biosilica for Bone Cell Growth with Combined Drug-Delivery and Antioxidant Properties.

Nanostructured biosilica produced by Thalassiosira weissflogii diatoms is covalently functionalized with the cyclic nitroxide 2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), an efficient scavenger of reactive oxygen species (ROS) in biological systems. Drug delivery properties of the TEMPO-functionalized biosilica are studied for Ciprofloxacin, an antimicrobial thoroughly employed in orthopedic or dental implant related infections. The resulting TEMPO-biosilica, combining Ciprofloxacin drug delivery with anti-oxidant properties, is demonstrated to be a suitable material for fibroblasts and osteoblast-like cells growth.

Physicochemical and biological properties of electrodeposited graphene oxide/chitosan films with drug-eluting capacity

In this study, novel graphene oxide/chitosan nanocomposite coatings with long term drug-eluting potential are presented. The coatings are fabricated by the facile and reproducible electrophoretic deposition technique. Analysis of the prepared films shows that the graphene oxide nanosheets are exfoliated in the chitosan matrix. Fourier-transform infrared spectrometry reveals polymer attachment to the carboxylic bonds of graphene oxide, providing a strong interaction and exfoliation of the nanolayers. In vitro viability assay by human osteosarcoma cells (MG-63) demonstrates that the nanocomposite films are highly biocompatible up to 30wt% graphene oxide, but at higher concentrations a slight cytotoxicity is noticed. Alkaline phosphates enzyme assay also reveals that the presence of graphene oxide nanosheets moderately hampers osteogenesis of the cultured cells. It is shown that vancomycin-loaded nanocomposite coatings gradually release the drug macromolecules for relatively long period of time (up to 4weeks). The electrodeposited films also exhibit a high bactericidal potential against Gram-positive Staphylococcus aureus. Effects of graphene oxide nanosheets on the physicochemical, biological, antimicrobial and drug-eluting properties of electrodeposited chitosan films are presented and discussed. It is shown that the GO/CS films support the initial attachment, proliferation and growth of osteoblast-like cells.

Immobilization of RGD peptide onto the surface of apatite-wollastonite ceramic for enhanced osteoblast adhesion and bone regeneration

The arginine-glycine-aspartic (RGD) acid peptide was grafted to the surface of apatite-wollastonite (AW) ceramic in an effort to improve its cell adhesion, proliferation and osteoinduction. RGD peptide was covalently immobilized onto the surface of AW ceramic via the synthetic cross linker AAPTS-E and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The modified surfaces were characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). The chemical analysis indicated that RGD peptide had been immobilized onto the AW surface successfully. The growth of osteoblast-like cells (MG63) showed that modifying the AW surface with RGD peptide enhanced the cell adhesion and proliferation. And the histological evaluation of RGD-AW showed that the bone regeneration and remodeling process were significantly enhanced compared to the original AW ceramics after 2, 4 and 8 weeks implantation in rabbit’s femoral condyles.

Scaffold preferences of mesenchymal stromal cells and adipose-derived stem cells from green fluorescent protein transgenic mice influence the tissue engineering of bone

We have analysed the growth and differentiation of mesenchymal stromal cells (MSC) from bone marrow, and of adipose derived stem cells (ASC) from murine abdominal fat tissue, of green fluorescent protein (GFP) transgenic animals grown directly on two types of hydroxyapatite ceramic bone substitutes. BONITmatrix® and NanoBone® have specific mechanical and physiochemical properties such as porosity and an inner surface that influence cellular growth. Both MSC and ASC were separately seeded on 200mg of each biomaterial and cultured for 3 weeks under osteogenic differentiation conditions. The degree of mineralisation was assessed by alizarin red dye and the specific alkaline phosphatase activity of the differentiated cells. The morphology of the cells was examined by scanning electron microscopy and confocal microscopy. The osteoblastic phenotype of the cells was confirmed by analysing the expression of bone-specific genes (Runx2, osteocalcin, osteopontin, and osteonectin) by semiquantitative reverse transcriptase polymerase chain reaction (PCR).Comparison of BONITmatrix® and NanoBone® showed cell type-specific preferences in terms of osteogenic differentiation. MSC-derived osteoblast-like cells spread optimally on the surface of NanoBone® but not BONITmatrix® granules. In contrast BONITmatrix® granules conditioned the growth of osteoblast-like cells derived from ASC. The osteoblastic phenotype of the cultured cells on all matrices was confirmed by specific gene expression.Our results show that the in vitro growth and osteogenic differentiation of murine MSC or ASC of GFP transgenic mice are distinctly influenced by the ceramic substratum. While NanoBone® granules support the proliferation and differentiation of murine MSC isolated from bone marrow, the growth of murine ASC is supported by BONITmatrix® granules. NanoBone® is therefore recommended for use as scaffold in tissue engineering that requires MSC, whereas ASC can be combined with BONITmatrix® for in vitro bone engineering.

Synthesis of biomedical composite scaffolds by laser sintering: Mechanical properties and in vitro bioactivity evaluation

In this study, biomedical composite materials were employed to fabricate bone scaffolds using a self-developed rapid prototyping (RP) apparatus. The slurry formed by combining hydroxyapatite (HA), silica sol, and sodium tripolyphosphate (STPP) was heated by a CO2 laser. Under appropriate processing parameters, a biocomposite green body was subsequently fabricated. Its mechanical properties, including surface roughness, bending and compression strengths, volume shrinkage rate, and surface microstructure, were analyzed after heat treatment to 1200°C, 1300°C, and 1400°C. The results showed that after heating the specimen to 1200°C, its compression and bending strengths increased significantly to 43.26MPa and 1.28MPa, respectively; the surface roughness was 12μm; and surface pores were of size 5–25μm. Furthermore, the results of WST-1 and LDH assay indicate that the biocomposites showed no cytotoxicity on 3T3 fibroblast. An optical density (OD) of 1.1 was also achieved, and the specimen was suitable for the adhesion and growth of osteoblast-like cells (MG63). Therefore, the biocomposite bone scaffolds fabricated in this study have potential to be bone implants for developing hard tissue.

In vitro study of vancomycin release and osteoblast-like cell growth on structured calcium phosphate-collagen

A drug delivery vehicle consisting of spherical calcium phosphate-collagen particles covered by flower-like (SFCaPCol) blossoms composed of nanorod building blocks and their cellular response is studied. The spherical structure was achieved by a combination of sonication and freeze-drying. The SFCaPCol blossoms have a high surface area of approximately 280m2g−1. The blossom-like formation having a high surface area allows a drug loading efficiency of 77.82%. The release profile for one drug, vancomycin (VCM), shows long term sustained release in simulated body fluid (SBF), in a phosphate buffer saline (PBS, pH 7.4) solution and in culture media over 2weeks with a cumulative release ~53%, 75% and 50%, respectively, over the first 7days. The biocompatibility of the VCM-loaded SFCaPCol scaffold was determined by in vitro cell adhesion and proliferation tests of rat osteoblast-like UMR-106 cells. MTT tests indicated that UMR-106 cells were viable after exposure to the VCM loaded SFCaPCol, meaning that the scaffold (the flower-like blossoms) did not impair the cell's viability. The density of cells on the substrate was seen to increase with increasing cultured time.

The effects of angiotensin II and the oxidative stress mediator 4-hydroxynonenal on human osteoblast-like cell growth: possible relevance to otosclerosis

Otosclerosis is a complex disease characterized by an abnormal bone turnover of the otic capsule resulting in conductive hearing loss. Recent findings have shown that angiotensin II (Ang II), a major effector peptide of the renin–angiotensin system, plays an important role in the pathophysiology of otosclerosis, most likely by its proinflammatory effects on the bone cells. Because reactive oxygen species play a role both in inflammation and in the cellular signaling pathway of Ang II, the appearance of protein adducts of the “second messenger of free radicals,” the aldehyde 4-hydroxynonenal (HNE), in otosclerotic bone has been analyzed. Immunohistochemical analysis of HNE-modified proteins in tissue samples of the stapedial bones performed on 15 otosclerotic patients and 6 controls revealed regular HNE–protein adducts present in the subperiosteal parts of control bone specimens, whereas irregular areas of a pronounced HNE–protein adduct presence were found within stapedial bone in cases of otosclerosis. To study possible interference by HNE and Ang II in human bone cell proliferation, differentiation, and induction of apoptosis we used an in vitro model of osteoblast-like cells. HNE interacted with Ang II in a dose-dependent manner, both by forming HNE–Ang II adducts, as revealed by immunoblotting, and by modifying its effects on cultured cells. Namely, treatment with 0.1nM Ang II and 2.5μM HNE stimulated proliferation, whereas treatment with 10μM HNE or in combination with Ang II (0.1, 0.5, and 1nM) decreased cell proliferation. Moreover, 10μM HNE alone and with Ang II (except if 1nM Ang II was used) increased cellular differentiation and apoptosis. HNE at 5μM did not affect differentiation nor significantly change apoptosis. On the other hand, when cells were treated with lower concentrations of HNE and Ang II we observed a decrease in cellular differentiation (combination of 1.0 or 2.5μM HNE with 0.1nM Ang II) and decrease in apoptosis (0.1 and 0.5nM Ang II). Cellular necrosis was increased with 5 and 10μM HNE if given alone or combined with Ang II, whereas 0.5nM Ang II and combination of 1μM HNE with Ang II (0.1 and 0.5nM) reduced necrosis. These results indicate that HNE and Ang II might act mutually dependently in the regulation of bone cell growth and in the pathophysiology of otosclerosis.

In Vitro Evaluation of Osteoblast-Like Cell Adhesion and Proliferation on Titanium Oxide Film Formed by Sand-Blasted Titanium Anode Treatment Process

The aim of the present study was to evaluate the in vitro cyto-compatibility of titanium oxide layers which were formed by electrochemical anode treatment on sand-blasted titanium plates. The in vitro biological response was characterized by cell morphology, adhesion, proliferation activity. We investigate those responses of MG-63 cells, one kind of human osteoblast-like cell lines, cultivated on these TiO2 films of sandblasted titanium plates formed by different electrochemical anode treatment conditions. The results showed that surface roughness was affected by the sandblasting condition majorly. The higher voltages of anodic treatment were applied, the thicker titanium oxide layers were formed. These thicker titanium oxide layers also reduced the surface roughness slightly. The cell adhesion and proliferation performance of MG63 osteoblast-like cells revealed that the surface which was sandblasted by φ106-125 μm alumina was the better condition for osteoblast-like cell growth.

Direct effects of casein phosphopeptides on growth and differentiation of in vitro cultured osteoblastic cells (MC3T3-E1)

Casein phosphopeptides (CPPs) obtained by enzymatic hydrolysis in vitro of caseins, have been shown to enhance calcium solubility and to increase the calcification of embryonic rat bones in their diaphyseal area. Little is known about the direct effects of CPPs on cultured osteoblastic cells. Calcium in the microenvironment surrounding bone cells is not only important for the mineralization of the extracellular matrix, but it is believed to provide preosteblasts with a signal that modulates their proliferation and differentiation. The aim of the present study was to investigate the direct effects of four selected casein phosphopeptides on osteoblastic cell (MC3T3-E1 cells) viability and differentiation. The selected peptides have been obtained by chemical synthesis and differed in the number of phosphorylated sites and in the amino acid spacing out two phosphorylated sites, in order to further characterize the relationship between structure and function. The results obtained in this work demonstrated that CPPs may directly affect osteoblast-like cell growth, calcium uptake and ultimately calcium deposition in the extracellular matrix. The effects exerted by distinct CPPs on osteogenesis in vitro can be either stimulatory or inhibitory. Differential short amino acid sequences in their molecules, like the -SpEE- and the -SpTSpEE-motifs, are likely the molecular determinants for their biological activities on osteoblastic cells. Moreover, two genetic variants of CPPs showing one amino acid change in their sequence may profoundly differ in their biological activities. Finally, our data may also suggest important clues about the role of intrinsic phosphorylated peptides derived from endogenous phosphorylated proteins in bone metabolism, apart from extrinsic CPPs.