Influence of surface topography on osteoblast response to fibronectin coated calcium phosphate thin films

The ability to engineer biomaterial surfaces that are capable of a dynamic interaction with cells and tissues is central to the development of medical implants with improved functionality. An important consideration in this regard is the role played by the extracellular proteins that bind to an implant surface in vivo. Deliberate use of an ad-layer of such proteins on an implant surface has been observed to guide and direct cell response. However, the role that changes in surface topography might play in determining the nature of this cell–protein–surface interaction has not been investigated in detail. In this study, calcium phosphate (CaP) thin films have been deposited onto substrates with varying topography such that this is reflected in the (conformal) CaP surface features. A fibronectin (FN) ad-layer was then deposited from solution onto each surface and the response of MG63 osteoblast-like cells investigated. The results revealed that in all cases, the presence of the adsorbed FN layer on the CaP thin films improved MG63 cell adhesion, proliferation and promoted early onset differentiation. Moreover, the nature and scale of the response were shown to be influenced by the underlying CaP surface topography. Specifically, MG63 cell on FN-coated CaP thin films with regular topographical features in the nanometer range showed statistically significant differences in focal adhesion assembly, osteocalcin expression and alkaline phosphase activity compared to CaP thin films that lacked these topographical features. As such, these data indicate that surface topography can be used to further influence cell adhesion and downstream differentiation by enhancing the effects of a surface adsorbed FN layer.     

Modulation of the functions of osteoblast-like cells on poly(allylamine hydrochloride) and poly(acrylic acid) multilayer films

Deposition of layer-by-layer polyelectrolyte multilayer (PEM) films has been a widely applied surface modification technique to improve the biocompatibility of biomaterials. The objective of this study was to investigate the impact of the deposition of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) multilayer films on adhesion, growth and differentiation of osteoblasts-like MG63 cells. PAH and PAA were deposited sequentially onto tissue culture polystyrene at either pH 2.0 or pH 6.5 with 4-21 layers. While the MG63 cells attached poorly on the PAH/PAA multilayer films deposited at pH 2.0, while the cells adhered to the PEM films deposited at pH 6.5, depending on layer numbers. Cell adhesion, proliferation and osteogenic activities (alkaline phosphatase activity, expression of osteogenic marker genes and mineralization) were highest on the 4-layer PAH/PAA film and decreased with increasing layer numbers. On the other hand, the behavior of MG63 cells did not show any difference on the adjacent even and odd layers, except PEM4 and PEM5, i.e. the surface charges of the PAH/PAA multilayer films with over ten layers seem indifferent to osteoblastic functions. The results in this study suggested that the mechanical properties of PEM films may play a critical role in modulating the behavior of osteoblasts, providing guidance for application of PEM films to osteopaedic implants.     

Mineralization and osteoblast behavior of multilayered films on TiO2 nanotube surfaces assembled by the layer-by-layer technique

In this paper, the multilayer films of poly-L-lysine (PLL) and DNA were created on TiO₂ nanotube surfaces using the layer-by-layer (LBL) self-assembly technique. Chemical compositions of the assembled multilayered films were investigated by X-ray photoelectron spectroscopy. Biological properties of the multilayered films were evaluated by the biomimetic mineralization and osteoblast cell culture experiments. The results indicated that PLL and DNA were successfully assembled onto TiO₂ nanotube surfaces by electrostatic attraction. Moreover, the samples of assembled PLL or/and DNA had better bioactivity in inducing HA formation and promoting osteoblast cells adhesion, proliferation and early differentiation.     

In vitro Evaluation of Osteoblast Response to Sol-Gel Derived Gelatin-Siloxane Hybrids

Cytotoxicity and cytocompatibility of porous gelatin-siloxane hybrids were evaluated due to osteoblastic cell (MC3T3-E1) proliferation, ALP activity, or their responses to the hybrids and their extracts. The hybrids were found intoxic, and appropriate incorporation of calcium ions stimulated proliferation and differentiation in vitro. Cells were seeded into the porous hybrids and the cell morphology was studied. The hybrids involving calcium ions favored osteoblast growth and differentiation.     

Effect of Composition of Lung Biomimetic Niche on the Mesenchymal Stem Cell Differentiation toward Alveolar Type II Pneumocytes

Pulmonary niche dynamically orchestrates the signals, such as proliferation or differentiation of mesenchymal stem cells (MSCs), which allows inducing tissue repair. Lung niche includes extracellular matrix (ECM), comprising hyaluronic acid (HA) and collagen (COLL), and several types of MSCs. Impaired ECM, in lung pathologies, makes the promising therapies based on MSCs ineffective, as it results in a reduced attachment and homing of MSCs, precluding their differentiation and viability. To overcome this problem, in this study a pulmonary biomimetic niche based on HA and COLL hydrogel is developed, with the specific aim to elucidate the role of COLL and HA/COLL semi-interpenetrating polymer networks (SIPNs) in directing the differentiation of MSCs into Alveolar Type II (ATII) cells. The effect of low (L), medium (M), and high (H) molecular weight (MW) HA is investigated, both like structural component of the SIPNs hydrogel and like trophic factor in cell culture media solution. HA in the culture media significantly improves surfactant protein (SP)-C expression (≈2 ng mL⁻¹), without showing difference in the MW tested, compared to control only (≈1 ng mL⁻¹). Furthermore, LMWHA/COLL hydrogel promotes the SPC expression (approximately two times) compared to COLL, MMWHA/COLL, and HMWHA/COLL hydrogels.     

Human Serum Albumin Conjugated Nanoparticles for pH and Redox‐Responsive Delivery of a Prodrug of Cisplatin

Platinum anticancer drugs are particularly in need of controlled drug delivery because of their severe side effects. Platinum(IV) agents are designed as prodrugs to reduce the side effects of platinum(II) drugs; however, premature reduction could limit the effect as a prodrug. In this work, a highly biocompatible, pH and redox dual‐responsive delivery system is prepared by using hybrid nanoparticles of human serum albumin (HSA) and calcium phosphate (CaP) for the Pt^IV prodrug of cisplatin. This conjugate is very stable under extracellular conditions, so that it protects the platinum(IV) prodrug in HSA. Upon reaching the acidic and hypoxic environment, the platinum drug is released in its active form and is able to bind to the target DNA. The Pt–HSA/CaP hybrid inhibits the proliferation of various cancer cells more efficiently than cisplatin. Different cell cycle arrests suggest different cellular responses of the Pt^IV prodrug in the CaP nanocarrier. Interestingly, this delivery system demonstrates enhanced cytotoxicity to tumor cells, but not to normal cells.     

Calcium Phosphate‐Reinforced Photosensitizer‐Loaded Polymer Nanoparticles for Photodynamic Therapy

Calcium phosphate‐reinforced photosensitizer‐loaded polymer nanoparticles have been developed for photodynamic therapy. Chlorin e6 (Ce6)‐loaded core–shell–corona polymer micelles of poly(ethylene glycol)‐b‐poly(L ‐aspartic acid)‐b‐poly(L ‐phenylalanine) ( PEG-PAsp-PPhe ) were employed as template nanoparticles for mineralization with calcium phosphate (CaP). CaP deposition was performed by the electrostatic localization of calcium ions at the anionic PAsp middle shells and the subsequent addition of phosphate anions. CaP‐reinforced nanoparticles exhibited enhanced stability. The CaP mineral layer effectively inhibited Ce6 release from the Ce6‐loaded mineralized nanoparticles (Ce6‐NP‐CaP) at physiological pH value. At an acidic endosomal pH value of 5.0, Ce6 release was enhanced, owing to rapid dissolution of the CaP minerals. Upon irradiation of Ce6‐NP‐CaP‐treated MCF‐7 breast‐tumor cells, the cell viability dramatically decreased with increasing irradiation time. The phototoxicity of Ce6‐NP‐CaP was much higher than that of free Ce6. Non‐invasive optical‐imaging results indicated that Ce6‐NP‐CaP exhibited enhanced tumor specificity compared with free Ce6 and Ce6‐loaded non‐mineralized polymer nanoparticles (Ce6‐NP).     

New phosphorylated derivatives of carboxymethylcellulose with osteogenic activity

New phosphorylated derivatives of carboxymethylcellulose (CMC) and amidic CMC were realized using trisodium trimetaphosphate (STMP) as the phosphating agent. The new polysaccharides were characterized by infrared spectroscopy and elemental analysis. The characterized polysaccharides were then crosslinked and their rheological and swelling properties determined. The presence of phosphate groups made the three‐dimensional structures more compact and harder than the corresponding non‐phosphated hydrogels. Evaluation of the bioactivity of phosphorylated hydrogels toward osteoblast‐like cells (MG63) showed a significant increase in the osteocalcin production. Copyright © 2008 John Wiley & Sons, Ltd.     

微图案化钙磷盐膜层的电化学构筑及其生物性能

基于表面分子自组装和光催化转印技术,在TiO2膜层表面获得超亲/超疏水阵列微图案模板,结合电化学沉积技术,成功制备了微图案化钙磷盐膜(CaP)层.扫描电子显微镜(SEM)和电子探针分析(EPMA)结果表明,通过超亲/超疏水阵列微图案模板可构筑高空间分辨的微图案化钙磷盐膜层.微图案化钙磷盐膜层的体外MG-63细胞培养证实,细胞对钙磷盐膜层微单元有强烈的选择性粘附作用,从而可望控制细胞在微单元中的贴壁生长,实现高通量评价细胞行为.     

Activated protein C differentially regulates both viability and differentiation of osteoblasts mediated by bisphosphonates

Activated protein C (APC) is a cytoprotective anticoagulant that can promote cutaneous healing. We examined the effect of APC on viability and differentiation of the osteoblastic line, MG63, in the presence and absence of bisphosphonates (BPs). Osteoblasts were cultured and treated for 24 or 48 h with Alendronate (Aln), Zoledronate (Zol) or Pamidronate (Pam) at concentrations ranging from 10⁻⁴ to 10⁻⁶ ℳ. Cell differentiation was measured using type 1 collagen production, Alizarin red staining and alkaline phosphatase activity, whereas cell viability was assessed using MTT and crystal violet assays. All three BPs induced MG63 cell death in a dose- and time-dependent manner. Pam- and Zol-related cell death was prevented by APC treatment; however, cell death induced by Aln was accelerated by APC. APC induced MG63 cell differentiation that was enhanced by Aln, but inhibited by Pam or Zol. Endothelial protein C receptor (EPCR) was expressed by MG63 cells and mediated the protective effect of APC on Zol-induced viability. In summary, we have demonstrated that (1) APC favorably regulates MG63 viability and differentiation toward bone growth, (2) APC differentially regulates the effects of specific BPs and (3) at least part of the effects of APC is mediated through EPCR. These findings highlight the potential importance of the PC pathway in bone physiology and provide strong evidence that APC may influence bone cells and has potential to be a therapeutic drug for bone regeneration, depending on concurrent BP treatment.     

碳纳米管/壳聚糖复合微球的原位仿生矿化及表征

对碳纳米管(CNTs)进行酸化处理, 采用乳化交联法制备CNTs/壳聚糖(CS)复合微球, 在其表面诱导羟基磷灰石仿生合成, 研究了CNTs对复合微球仿生矿化的影响, 并与纯CS微球的仿生矿化进行了对比. 利用扫描电子显微镜(SEM)、 X射线衍射仪(XRD)、 溶胀率和含水率测试等考察了复合微球矿化前后的形貌特征、 物相结构及稳定性. 结果表明, 在相同时间下, CNTs/CS复合微球表面纳米羟基磷灰石的形成能力明显优于纯CS微球, 且形态稳定性更高. 细胞实验结果表明, 与MG63细胞共培养7 d时, 矿化复合微球细胞增殖明显.     

Poly(ethylene imine)-controlled calcium phosphate mineralization

The current paper shows that poly(ethylene imine) (PEI) is an efficient template for the fabrication of spherical calcium phosphate (CaP)/polymer hybrid particles at pH values above 8. The polymer forms spherical entities, which contain one or a few CaP particles with diameters of ca. 6 nm. The samples contain up to 20 wt % polymer, which appears to be wrapped around the small CaP particles. The particles form via a mineralization-trapping pathway, where at the beginning of the precipitation small CaP particles form. Further particle growth is then prevented by precipitation of the PEI onto these particles at pH values of ca. 8. Stabilization of the particles is provided by the re-protonation of the PEI, which is adsorbed on the CaP particles, during the remainder of the mineralization process. At low pH, much larger particles form. They most likely grow via heterogeneous nucleation and growth on existing, polymer-modified CaP surfaces.     

Enhanced effects of nano-scale topography on the bioactivity and osteoblast behaviors of micron rough ZrO2 coatings

Implant surface topography is one of the most important factors affecting the rate and extent of osseointegration. Randomly micron-roughened surfaces have been documented to support osteoblast adhesion, differentiation, and mineralized phenotype, and thus favoring bone fixation of implants to host tissues. However, few studies have been done yet to investigate whether their effects on osteoblast behaviors can be enhanced by incorporation of nano-scale topographic cues. To validate this hypothesis, zirconia coatings with micron roughness (about 6.6 μm) superimposed by nano-sized grains (<50 nm) were fabricated by plasma spraying. To validate the impact of nano-sized grains, post-treatments of surface polishing (SP) and heat treatment (HT) were performed on the as-sprayed (AS) coatings to change the surface topographies but keep the chemical and phase composition similar. Results of in vitro bioactivity test showed that apatite was formed only on coating surfaces with nano-sized grains (AS coatings), indicating the significance of nano-topographic cues on the in vitro bioactivity. Enhanced osteoblast adhesion and higher cell proliferation rate were observed on coatings with both micron-roughness and nano-sized grains (AS-coatings), compared to coatings with smooth surfaces (SP-coatings) and coatings with only micron-scale roughness (heat-treated coatings), indicating the significant effects of nano-size grains on osteoblast responses. As the micron rough surfaces have been well-documented to enhance bone fixation, results of this work suggest that a combination of surface modifications at both micron and nano-scale is required for enhanced osseointegration of orthopedic implants.     

The chiral effects on the responses of osteoblastic cells to the polymeric substrates

In order to study the chiral effects of polylactides on responses of osteoblastic cells, poly(l-lactide) (PLLA), poly(d-lactide) (PDLA), poly(dl-lactide) (PDLLA) and the stereocomplex of PLLA and PDLLA (SC) films with different stereoforms were prepared. The surface properties of the four polylactide films were tested and the osteoblastic ROS 17/2.8 cells were cultured on the films. The protein adsorption behaviors of fibrinogen and bovine serum albumin on films were studied. The cell proliferation, total protein amount, DNA content and alkaline phosphatase activity of osteoblastic ROS 17/2.8 cells were evaluated. The results showed that the protein adsorption was dependant on the type of proteins. The observation of cell morphologies revealed that the PDLA film provide an unfavorable surface for cell attachment. The total protein amount, DNA content and ALP activity were closely related to the stereoforms of polylactide films. All the levels of total protein amount, DNA content and ALP activity of ROS 17/2.8 cells on PDLA film were decreased. The racemic stereocomplex of PLLA and PDLA showed relatively higher positive effects on both cell growth and proliferation.     

Development of a biologically relevant calcium phosphate substrate for sum frequency generation vibrational spectroscopy

A novel biologically relevant composite substrate has been prepared consisting of a calcium phosphate (CaP) layer formed by magnetron sputter-coating from a hydroxyapatite (HA) target onto a gold-coated silicon substrate. The CaP layer is intended to mimic tooth and bone surfaces and allows polymers used in oral care to be deposited in a procedure analogous to that used for dental surfaces. The polymer cetyl dimethicone copolyol (CDC) was deposited onto the CaP surface of the substrate by Langmuir Blodgett deposition, and the structure of the adsorbed layer was investigated by the surface specific technique of sum frequency generation (SFG) vibrational spectroscopy. The gold sublayer provides enhancement of the SFG signal arising from the polymer but plays no part in the adsorption of the polymer. The surface morphology of the substrate was investigated using SEM and AFM. The surface roughness was commensurate with that of the thermally evaporated gold sublayer and uniform over areas of at least 36 mum(2). The chemical composition of the CaP-coated surface was determined by FTIR and TOF-SIMS. It was concluded that the surface is primarily calcium phosphate present as a mixture of amorphous, non-hydroxylated phases rather than solely stoichiometric hydroxyapatite. The SFG spectra from CDC on CaP were closely similar, both in resonance wavenumbers and in their relative intensities, with spectra of thin films of CDC recorded directly on gold. Application of previous analysis of the spectra of CDC on gold therefore enabled interpretation of the polymer orientation and conformation on the CaP substrate.     

Nanoscale Confinement Controls the Crystallization of Calcium Phosphate: Relevance to Bone Formation

A key feature of biomineralization processes is that they take place within confined volumes, in which the local environment can have significant effects on mineral formation. Herein, we investigate the influence of confinement on the formation mechanism and structure of calcium phosphate (CaP). This is of particular relevance to the formation of dentine and bone, structures of which are based on highly mineralized collagen fibrils. CaP was precipitated within 25–300 nm diameter, cylindrical pores of track etched and anodised alumina membranes under physiological conditions, in which this system enables systematic study of the effects of the pore size in the absence of a structural match between the matrix and the growing crystals. Our results show that the main products were polycrystalline hydroxapatite (HAP) rods, together with some single crystal octacalcium phosphate (OCP) rods. Notably, we demonstrate that these were generated though an intermediate amorphous calcium phosphate (ACP) phase, and that ACP is significantly stabilised in confinement. This effect may have significance to the mineralization of bone, which can occur through a transient ACP phase. We also show that orientation of the HAP comparable, or even superior to that seen in bone can be achieved through confinement effects alone. Although this simple experimental system cannot be considered, a direct mimic of the in vivo formation of ultrathin HAP platelets within collagen fibrils, our results show that the effects of physical confinement should not be neglected when considering the mechanisms of formation of structures, such as bones and teeth.     

Amorphous calcium phosphate/poly(D,L-lactic acid) composite nanofibers: electrospinning preparation and biomineralization

Amorphous calcium phosphate (ACP) has been recognized as an attractive biomaterial due to its bioactivity and biocompatibility. Electrospinning is a simple and low-cost way to fabricate polymer fibers. In this study, ACP nanoparticles with diameters ranging from 20 to 80 nm were synthesized using a simple precipitation method. ACP nanoparticles were hybridized with poly(D,L-lactic acid) (PDLLA) to form ACP/PDLLA composite nanofibers by electrospinning, and different architectures including the nanofibrous mesh and tube consisting of ACP/PDLLA composite nanofibers were obtained and characterized. The biomineralization and cytocompatibility of as-prepared ACP/PDLLA composite nanofibers were evaluated in vitro. Osteoblast-like MG63 cells were seeded on the ACP/PDLLA composite nanofiber meshes to perform the cytocompatibility evaluation. The ACP/PDLLA composite nanofibers exhibited a fast mineralization behavior in the simulated body fluid. The attachment of MG63 cells and cytotoxicity of ACP/PDLLA composite nanofibers were also evaluated, and the experiments indicated good biocompatibility and bioactivity of ACP/PDLLA composite nanofibers.     

Graphene‐Oxide‐Decorated Microporous Polyetheretherketone with Superior Antibacterial Capability and In Vitro Osteogenesis for Orthopedic Implant

Due to its similar elastic modulus of human bones, polyetheretherketone (PEEK) has been considered as an excellent cytocompatible material. However, the bioinertness, poor osteoconduction, and weak antibacterial activity of PEEK limit its wide applications in clinics. In this study, a facile strategy is developed to prepare graphene oxide (GO) modified sulfonated polyetheretherketone (SPEEK) (GO‐SPEEK) through a simple dip‐coating method. After detailed characterization, it is found that the GO closely deposits on the surface of PEEK, which is attributed to the π–π stacking interaction between PEEK and GO. Antibacterial tests reveal that the GO‐SPEEK exhibits excellent suppression toward Escherichia coli. In vitro cell attachment, growth, differentiation, alkaline phosphatase activity, quantitative real‐time polymerase chain reaction analyses, and calcium mineral deposition all illustrate that the GO‐SPEEK substrate can significantly accelerate the proliferation and osteogenic differentiation of osteoblast‐like MG‐63 cells compared with those on PEEK and SPEEK groups. These results suggest that the GO‐SPEEK has an improved antibacterial activity and cytocompatibility in vitro, showing that the developed GO‐SPEEK has a great potential as the bioactive implant material in bone tissue engineering.     

Does the nanometre scale topography of titanium influence protein adsorption and cell proliferation?

To investigate the influence of titanium films with nanometre scale topography on protein adsorption and cell growth, three different model titanium films were utilized in the present study. The chemical compositions, surface topographies and wettability were investigated by using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and water contact angle measurement, respectively. The films share the same surface chemistry but exhibit different topographies on a nanometre scale. Thus, they act as model systems for biological studies regarding surface topography effects. The films were obtained by varying the deposition rate and the film thickness, respectively. These films displayed nanometre scale surface roughness (root mean square roughness, R_rms) from 2 to 21 nm over areas of 50 μm × 50 μm, with different grain sizes at their surfaces. Albumin and fibrinogen adsorption on these model titanium films were performed in this study. Bicinchoninic acid assay was employed to determine the amount of adsorbed protein on titanium film surfaces. No statistically significant differences, however, were observed for either albumin or fibrinogen adsorption between the different groups of titanium films. No statistically significant influence of surface roughness on osteoblast proliferation and cell viability was detected in the present study.     

Electrochemical Monitoring of Saos‐2 Cell Differentiation on Pyrolytic Carbon Electrodes

In this study we establish an electrochemical platform based on two dimensional (2D) pyrolytic carbon electrodes for in vitro analysis of osteoblast differentiation. Electrochemical impedance spectroscopy (EIS) was used to monitor cell adhesion and proliferation, while an electrochemical assay based on square wave voltammetry (SWV) was applied to measure the activity of the differentiation marker alkaline phosphatase (ALP). 2D pyrolytic carbon electrodes were fabricated and used to monitor Saos‐2 cell differentiation for a period of up to 21 days. With this method it was possible to detect a faster increase of ALP activity for cells cultured in medium supplemented with differentiation factors compared to cells cultured in growth medium. This was confirmed by the results obtained with Alizarin Red staining, showing that cells subjected to osteogenic medium went through the entire differentiation process, from proliferation to mineralization. Finally, for the first time, real‐time monitoring of ALP activity combined with continuous EIS monitoring of the same cell culture was achieved using the pyrolytic carbon electrodes.