Abstract

In this study, the effects of bioactive glass nanoparticles' (nBGs) size and shape incorporated into hydroxyapatite/β-tricalcium phosphate (BCP) scaffolds were investigated. We prepared a highly porous (> 85%) BCP scaffold and coated its surface with a nanocomposite layer consisted of polycaprolactone (PCL) and rod (~ 153 nm in height and ~ 29 nm in width) or spherical (~ 33 nm and 64 nm in diameter) nBGs. Osteogenic gene expression by primary human osteoblast-like cells (HOB) was investigated using quantitative real time polymerase chain reaction (q-RT-PCR). We demonstrated for the first time that in vitro osteogenesis is dramatically affected by the shape of the nBGs, whereby rod shaped nBGs showed the most significant osteogenic induction, compared to spherical particles (regardless of their size). Importantly, the good biological effect observed for the rod shaped nBGs was coupled by a marked increase in the modulus (~ 48 MPa), compressive strength (~ 1 MPa) and failure strain (~ 6%), compared to those for the BCP scaffolds (~ 4 MPa, ~ 1 MPa and ~ 0.5% respectively). The findings of this study demonstrated that the shape of the nBGs is of significant importance when considering bone regeneration.

Highlights

? Coating the different shape and size of glass nanoparticles on the scaffold surface. ? Evaluating the effect of nanoparticles geometry on the behavior of osteoblast cells. ? Proposing the reasons for improved behavior of needle shape glass nanoparticles.

Abstract

Time-dependent phenotypic response of a model osteoblast cell line (hFOB 1.19, ATCC, and CRL-11372) to substrata with varying surface chemistry and topography is reviewed within the context of extant cell-adhesion theory. Cell-attachment and proliferation kinetics are compared using morphology as a leading indicator of cell phenotype. Expression of (α₂,α₃,α₄,α₅,α_v,β₁, andβ₃) integrins, vinculin, as well as secretion of osteopontin (OP) and type I collagen (Col I) supplement this visual assessment of hFOB growth. It is concluded that significant cell-adhesion events—contact, attachment, spreading, and proliferation—are similar on all surfaces, independent of substratum surface chemistry/energy. However, this sequence of events is significantly delayed and attenuated on hydrophobic (poorly water-wettable) surfaces exhibiting characteristically low-attachment efficiency and long induction periods before cells engage in an exponential-growth phase. Results suggest that a ‘time–cell–substratum–compatibility–superposition principle’ is at work wherein similar bioadhesive outcomes can be ultimately achieved on all surface types with varying hydrophilicity, but the time required to arrive at this outcome increases with decreasing cell–substratum–compatibility. Genomic and proteomic tools offer unprecedented opportunity to directly measure changes in the cellular machinery that lead to observed cell responses to different materials. But for the purpose of measuring structure–property relationships that can guide biomaterial development, genomic/proteomic tools should be applied early in the adhesion/spreading process before cells have an opportunity to significantly remodel the cell–substratum interface, effectively erasing cause and effect relationships between cell–substratum–compatibility and substratum properties.

Impact Statement

This review quantifies relationships among cell phenotype, substratum surface chemistry/energy, topography, and cell–substratum contact time for the model osteoblast cell line hFOB 1.19, revealing that genomic/proteomic tools are most useful in the pursuit of understanding cell adhesion if applied early in the adhesion/spreading process.

Abstract

The development of the new technologies of bone tissue engineering requires the production of bioresorbable macroporous scaffolds. Calcium phosphate cements are good candidate materials for the development of these scaffolds, as an alternative to the traditional porous sintered ceramics. In this work a novel two-step method, based in the foaming of anα-tricalcium phosphate (α-TCP) cement paste and its subsequent hydrolysis to a calcium deficient hydroxyapatite (CDHA) is presented. The foaming agent was a hydrogen peroxide (H₂O₂) solution, which decomposes in water and oxygen gas. CDHA foams, which combined an interconnected macroporosity with a high microporosity were obtained. The apatitic phase obtained by the hydrolysis reaction was more similar to the biologic one, in terms of chemical composition, crystallinity and specific surface than the hydroxyapatites obtained by sintering. The percentage of porosity in the foams reached a 66%. It was shown that it was possible to control the porosity, and pore size and shape by different processing parameters such as the liquid-to-powder ratio, the concentration of the H₂O₂ solution and the particle size of the powder.

Abstract

Porous calcium silicates (CaSiO₃, WT) are regarded as a potential bioactive material for bone tissue engineering. However, their insufficient mechanical strength and high dissolution (degradation) limit their biological applications. The aim of this study is to surface modify WT scaffolds with poly(d,l-lactic acid) (PDLLA) to improve their mechanical and biological properties. The phase composition, microstructure, porosity and interconnectivity of WT and PDLLA-modified WT (WTPL) scaffolds were analyzed by X-ray diffraction, scanning electron microscopy and micro-computerized tomography. The WTPL scaffolds maintained a more uniform and continuous inner network, compared to that of the WT scaffolds, while maintaining the pore size, porosity and interconnectivity of the original materials. The compressive strength, compressive modulus and percentage strain of the WT and WTPL scaffolds were assessed in air and phosphate-buffered saline. PDLLA modification significantly improved the compressive strength and decreased the brittleness of the WT scaffolds. The weight loss and apatite-forming ability of the two scaffolds were evaluated by soaking them in simulated body fluid (SBF) for 1, 3, 7, 14 and 28 days. PDLLA modification decreased the dissolution of the WT scaffolds while maintaining their apatite-forming ability in SBF. In addition, PDLLA modification improved the spreading and viability of human bone-derived cells. Our results indicate that PDLLA-modified CaSiO₃ scaffolds possess improved mechanical and biological properties, suggesting their potential application for bone tissue regeneration.

Abstract

In this study, the effects of bioactive glass nanoparticles' (nBGs) size and shape incorporated into hydroxyapatite/β-tricalcium phosphate (BCP) scaffolds were investigated. We prepared a highly porous (> 85%) BCP scaffold and coated its surface with a nanocomposite layer consisted of polycaprolactone (PCL) and rod (~ 153 nm in height and ~ 29 nm in width) or spherical (~ 33 nm and 64 nm in diameter) nBGs. Osteogenic gene expression by primary human osteoblast-like cells (HOB) was investigated using quantitative real time polymerase chain reaction (q-RT-PCR). We demonstrated for the first time that in vitro osteogenesis is dramatically affected by the shape of the nBGs, whereby rod shaped nBGs showed the most significant osteogenic induction, compared to spherical particles (regardless of their size). Importantly, the good biological effect observed for the rod shaped nBGs was coupled by a marked increase in the modulus (~ 48 MPa), compressive strength (~ 1 MPa) and failure strain (~ 6%), compared to those for the BCP scaffolds (~ 4 MPa, ~ 1 MPa and ~ 0.5% respectively). The findings of this study demonstrated that the shape of the nBGs is of significant importance when considering bone regeneration.

Highlights

? Coating the different shape and size of glass nanoparticles on the scaffold surface. ? Evaluating the effect of nanoparticles geometry on the behavior of osteoblast cells. ? Proposing the reasons for improved behavior of needle shape glass nanoparticles.

Abstract

Reticulated porous silicon carbide ceramics (SiC RPCs) were fabricated by polymeric sponge replication using preset roller method, then recoating by centrifuging technique. Preparation and properties of the SiC RPCs prepared by roller method are presented in this communication. The loading content and pore diameter of the RPCs body greatly depends on the slurry viscosity. At the same time, the strut diameter and cell size of SiC RPCs are modulated by the recoating slurry.

Abstract

Reticulated porous mullite ceramics (mullite RPCs) were produced by the polymer replica method using alpha alumina and kaolin to form in situ mullite. Up to 30% particulate silicon carbide (SiCp) was added to ceramic mixtures to explore its effect on the structure and properties of the sintered products. The ceramic slurry was coated onto polyurethane sponge using preset roller method, then recoating was applied by the spray coating technique. The strength of the mullite RPCs was found to depend greatly on the phase composition of the structures and on the amount of SiCp addition.

Trabecular bones of almost all vertebrate organisms basically consist of macroporous (55–70% interconnected porosity) bone mineral, i.e. calcium hydroxyapatite (HA: Ca₁₀(PO₄)₆(OH)₂). The macroporosity observed in the trabecular bones then allows the ingrowth of the soft tissues and organic cells into the bone matrix. Sub-micron, chemically uniform, and high phase-purity HA powders produced in our laboratory were mixed, under vigorous ultrasonification, with methyl cellulose of appropriate amounts in the form of an aqueous slurry of proper viscosity and thickness. The ceramic cakes produced in this way were then slowly dried in an oven in the temperature range of 50–90 °C. Dried cakes of porous HA were physically cut into various prismatic shapes. These parts were then slowly heated in an air atmosphere to the optimum sintering temperature of 1250 °C. The HA bioceramic parts obtained by this novel ‘foaming technique’ were found to have tractable and controllable interconnected porosity in the range of 60–90%, with typical pore sizes ranging from 100–250 microns. Sample characterization was mainly achieved by scanning electron microscopy (SEM) studies and three-point bending tests.

Cells in theirin vivo microenvironment constantly encounter and respond to a multitude of signals. While the role of biochemical signals has long been appreciated, the importance of biophysical signals has only recently been investigated. Biophysical cues are presented in different forms including topography and mechanical stiffness imparted by the extracellular matrix and adjoining cells. Microfabrication technologies have allowed for the generation of biomaterials with microscale topographies to study the effect of biophysical cues on cellular function at the cell–substrate interface. Topographies of different geometries and with varying microscale dimensions have been used to better understand cell adhesion, migration, and differentiation at the cellular and sub-cellular scales. Furthermore, quantification of cell-generated forces has been illustrated with micropillar topographies to shed light on the process of mechanotransduction. In this review, we highlight recent advances made in these areas and how they have been utilized for neural, cardiac, and musculoskeletal tissue engineering application.

Abstract

Poly(methyl methacrylate) (PMMA) is a biocompatible polymer widely used for bone substitutes. Its surface properties, however, are not favorable for the induction of biological apatite which can be directly related to natural bone formation. In this study, the surface of PMMA was modified by NaOH treatment or sequential treatments with ethanol (EtOH) and NaOH. Results displayed that surface hydrophilicity was improved for increasing treatment time and NaOH concentration. Field-emission scanning electron microscope (FE–SEM) displayed that in vitro formation of calcium phosphate (CaP) coating was significantly promoted by the surface modifications. X-ray photon spectroscopy (XPS) examination elucidated that the films prepared on PMMA consisted of calcium and phosphorus and their values for Ca/P ratio were closed to octacalcium phosphate (OCP). Fourier transform infrared (FT–IR) spectra of the film coated on PMMA revealed a band characteristic of phosphate groups confirming that CaP films were formed and their characteristics were dependent on the surface properties of PMMA. Cellular assay demonstrated that the adhesion of osteoblast-like MG63 cells was significantly promoted on CaP-coated PMMA. Proliferation assay showed that CaP films appeared not to exert any cytotoxic effects on the growth of MG63 cells.

Cell adhesion and spreading of chick embryo vascular and corneal explants grown on rough and smooth poly (methyl methacrylate) (PMMA) were analyzed to test the cell response specificity to substratum surface properties. Different degrees of roughness were obtained by sandblasting PMMA with alumina grains. Hydrophilic and hydrophobic components of the surface free energy (SFE) were calculated according to Good-van Oss's model. Contact angles were determined using a computerized angle meter. The apolar component of the SFE, γs^LW, increased with a slight roughness whereas the basic component, γs⁻, decreased. The acido-basic properties disappeared as roughness increased. Incubation of PMMA in culture medium, performed to test the influence of the biological environment, allowed surface adsorption of medium proteins which annihilated roughness effect and restored hydrophilic properties. An organotypic culture assay was carried out in an attempt to relate the biocompatibility to substratum surface state. Cell migration was calculated from the area of cell layer. Cellular adhesion was determined by measuring the kinetic of release of enzymatically dissociated cells. A slight roughness raised the migration area to an upper extent no matter which cell type. Enhancement of the cell adhesion potential was related to the degree of roughness and the hydrophobicity. © 1997 John Wiley & Sons, Inc.J Biomed Mater Res,36, 99–108, 1997.

Abstract

Objectives

This study aims to investigate the effects of anatase-structured titania surface on the response of osteoblast-like cells.

Methods

Three kinds of titanium (Ti) disks were prepared: the polished Ti (PT), the roughened Ti (RT) formed by sandblasting and acid etching, and the anatase-coated roughened Ti (ART) formed on the RT via H₂O₂/HCl solution treatment followed by heating at 400 °C. The surface topography of Ti disks was observed under a scanning electron microscope. MC3T3-E1 cells were cultured on the Ti disks. Cell morphology was observed by fluorescence microscopy. Cell adhesion and proliferation were assessed by MTT testing. To evaluate the cell differentiation on RT and ART, the expressions of core-binding factor α, osterix, bone sialoprotein, collagen type I????, osteocalcin and alkaline phosphatase-2 were measured by real-time reverse transcriptase polymerase chain reaction.

Results

The three kinds of Ti disks had distinct surface topographic characteristics. The cells on the ART more rapidly displayed well-defined stress fibers and numerous processes and pseudopodi than the cells on the PT and RT. At 1 and 3 h after culture, there were more cells adhering to the ART than to PT and RT. Cell proliferation did not differ between groups after 1–5 days of culture. In addition, ART generally enhanced the osteogenic gene expression in a time-dependent manner compared with RT.

Significance

Anatase-structured titania promotes initial adhesion and spreading of osteoblast-like cells, but does not influence cell proliferation. Anatase could also enhance cell differentiation by enhancing osteogenic gene expression.

Abstract

This paper presents a technique to independently form mechanical topography and surface chemical patterns on polymer cell substrates, and studies the response of osteoblast cells to these surface patterns. The patterns were formed in two separate steps: hot embossing imprint lithography formed the mechanical topography and microcontact printing created the chemical pattern. The resulting substrate had surface features consisting of embossed grooves 4 μm deep and 8 μm wide spaced by 16 μm wide mesas and microcontact printed adhesive lanes 10 μm wide with spacings that ranged from 10 to 100 μm. When presented with either mechanical topography or chemical patterns alone, the cells significantly aligned to the pattern presented. When presented with mechanical topography overlaid with an orthogonal chemical pattern, the cells aligned to the mechanical topography. As the chemical pattern spacing was increased, osteoblasts remained aligned to the mechanical topography. Unlike traditional microfabrication approaches based on photolithography and wet chemistry, the patterning technique presented is compatible with a large number of biomaterials, could form patterns with features much smaller than 1 μm, and is highly scalable to large substrates.

Human serum albumin was adsorbed into porous silicon layers with thickness up to 3 μm and with different mean pore radius in the range 4.5–10 nm. The adsorbed amount of protein was quantified by I¹²⁵ radioactive labeling techniques and ellipsometry. The results show that albumin penetrated into the pores when the mean pore radius was larger than 5.5 nm, but could not totally occupy the available surface area when the layer thickness was larger than 1 μm. Loading of albumin both into porous layers and onto plane silicon as a function of albumin concentration was also investigated. These measurements show that loading of protein increased with protein concentration at least up to 10 mg/ml for porous silicon and up to 1 mg/ml for plane silicon. The maximum deposition into the type of porous layers used here was 28 μg/cm², compared to 0.36 μg/cm² for plane silicon.

Abstract

Protein adsorption affects the function of cells and determines the bioactivity of biomaterial implants. Surface structure and properties of materials determine the behavior of protein adsorption. In the present study, two biphasic calcium-phosphate ceramics (BCPs) with different surface structures were fabricated by pressing and H₂O₂ foaming methods. Their surface characteristics were analyzed and the in vitro and in vivo protein adsorption on them was investigated. Porous BCP showed higher ability to adsorb proteins, and transforming growth factor-β1 (TGF-β1) adsorption notably increased with increasing in vivo implantation time. The strong affinity of BCP to TGF-β1 might provide important information for exploring the mechanism of the osteoinduction of calcium phosphates.

Microstructure is essential for inductive bone formation in calcium phosphate materials after soft tissue implantation. We hereby evaluated activities (cell attachment, proliferation, ALP/DNA and protein/DNA) of three types of cells cultured on three kinds of calcium phosphate ceramic discs to study how microstructure takes its role in inductive bone formation. Three kinds of biphasic calcium phosphate (BCP) ceramic discs with the same chemistry and the same dimension of ∅10.0 × 1.0 mm³ (BCP1150-P, BCP1150-D and BCP1300), either having similar micropore sizes and surface roughness but different surface area (BCP1150-P vs BCP1150-D) or having similar surface area but different micropore sizes and different roughness (BCP1150-D vs BCP1300), were prepared. Conventionally Culturing C2C12, human bone marrow stromal cells (HBMSC) and MC3T3-E1 cells on BCP discs showed that, surface roughness did not affect cell attachment, cell proliferation and ALP expression of all cell types evaluated, while surface area did affect cell functions. ALP/DNA of C2C12 on BCP1150-P, having larger surface area, was significantly higher than on BCP1300 and BCP1150-D. Furthermore, all cells cultured on all of the three kinds of BCPs pre-soaked in culture medium having additional rhBMP-2 had a higher ALP expression than the conventional cell culture. Comparing with on BCP1300 and BCP1150-D, ALP/DNA of all cells tested increased more on BCP1150-P after the discs were pre-soaked in culture medium with rhBMP-2. The results indicated that increasing surface areas, microstructured calcium phosphate materials might concentrate more proteins (including bone-inducing proteins) that differentiate inducible cells to osteogenic cells that form inductive bone.

Abstract

The purpose of this study is to investigate the biocompatibility and osteoconduction of active porous calcium–phosphate films on the novel Ti–3Zr–2Sn–3Mo–25Nb biomedical alloy. The active porous calcium–phosphate films were prepared by the micro-arc oxidation method on the surface of a near β biomedical Ti–3Zr–2Sn–3Mo–25Nb alloy, and then activated in a hydroxyl solution followed by an aminated solution. The phase composition, surface micro-topography and elemental characteristics of the active porous calcium–phosphate films were investigated with XRD, SEM, EDS and XPS. The biocompatibility was assessed using corrosion testing, the in vitro osteoblast cultivation test and implantation in soft tissue (subcutaneous and musculature). The osteoconduction was evaluated using the simulated body fluid test and by implantation in hard tissue. The results show that the active porous films are mainly composed of TiO₂ anatase and rutile. The oxide layer is a kind of porous ceramic intermixture containing Ca and P. Immersion in simulated body fluid can induce apatite formation on the porous calcium–phosphate films resulting in excellent bioactivity. Cell cultures revealed that MC3T3-E1 cells grew on the surface exhibiting favorable morphologies. These results indicate that the Ti–3Zr–2Sn–3Mo–25Nb biomedical alloy coated with an active porous calcium–phosphate film has been shown to have excellent corrosion resistance, good biocompatibility and osteoconduction, which can promote cell proliferation and bone formation.

Graphical abstract

The purpose of this study is to investigate the biocompatibility and osteoconduction of active porous calcium–phosphate films on the novel Ti-3Zr–2Sn–3Mo–25Nb biomedical alloy. The results indicate that the Ti–3Zr–2Sn–3Mo–25Nb biomedical alloy coated with an active porous calcium–phosphate film has been shown to have excellent corrosion resistance, good biocompatibility and osteoconduction, which can promote cell proliferation and bone formation, and the biocompatibility and osteoconduction properties of the Ti–3Zr–2Sn–3Mo–25Nb alloy with a surface modified to incorporate an active porous calcium–phosphate film are better than that of the Ti–6Al–4V(ELI) alloy and bare TLM.