Porous zirconia scaffold modified with mesoporous bioglass coating

Porous yttria-stabilized zirconia (YSZ) has been regarded as a potential candidate for bone substitute as its high mechanical strength. However, porous YSZ bodies are biologically inert to bone tissue. It is therefore necessary to introduce bioactive coatings onto the walls of the porous structures to enhance the bioactivity. In this study, the porous zirconia scaffolds were prepared by infiltration of Acrylonitrile Butadiene Styrene (ABS) scaffolds with 3 mol% yttria stabilized zirconia slurry. After sintering, a method of sol-gel dip coating was involved to make coating layer of mesoporous bioglass (MBGs). The porous zirconia without the coating had high porosities of 60.1% to 63.8%, and most macropores were interconnected with pore sizes of 0.5-0.8mm. The porous zirconia had compressive strengths of 9.07-9.90MPa. Moreover, the average coating thickness was about 7μm. There is no significant change of compressive strength for the porous zirconia with mesoporous biogalss coating. The bone marrow stromal cell (BMSC) proliferation test showed both uncoated and coated zirconia scaffolds have good biocompatibility. The scanning electron microscope (SEM) micrographs and the compositional analysis graphs demonstrated that after testing in the simulated body fluid (SBF) for 7 days, the apatite formation occurred on the coating surface. Thus, porous zirconia-based ceramics were modified with bioactive coating of mesoporous bioglass for potential biomedical applications.

In situ preparation and protein delivery of silicate/alginate composite microspheres with core-shell structure

It is predicted that with increased life expectancy in the developed world, there will be a greater demand for synthetic materials to repair or regenerate lost, injured or diseased bone (Hench & Thompson 2010). There are still few synthetic materials having true bone inductivity, which limits their application for bone regeneration, especially in large-size bone defects. To solve this problem, growth factors, such as bone morphogenetic proteins (BMPs), have been incorporated into synthetic materials in order to stimulate de novo bone formation in the center of large-size bone defects. The greatest obstacle with this approach is that the rapid diffusion of the protein from the carrier material, leading to a precipitous loss of bioactivity; the result is often insufficient local induction or failure of bone regeneration (Wei et al. 2007). It is critical that the protein is loaded in the carrier material in conditions which maintains its bioactivity (van de Manakker et al. 2009). For this reason, the efficient loading and controlled release of a protein from a synthetic material has remained a significant challenge. The use of microspheres as protein/drug carriers has received considerable attention in recent years (Lee et al. 2010; Pareta & Edirisinghe 2006; Wu & Zreiqat 2010). Compared to macroporous block scaffolds, the chief advantage of microspheres is their superior protein-delivery properties and ability to fill bone defects with irregular and complex shapes and sizes. Upon implantation, the microspheres are easily conformed to the irregular implant site, and the interstices between the particles provide space for both tissue and vascular ingrowth, which are important for effective and functional bone regeneration (Hsu et al. 1999). Alginates are natural polysaccharides and their production does not have the implicit risk of contamination with allo or xeno-proteins or viruses (Xie et al. 2010). Because alginate is generally cytocompatible, it has been used extensively in medicine, including cell therapy and tissue engineering applications (Tampieri et al. 2005; Xie et al. 2010; Xu et al. 2007). Calcium cross-linked alginate hydrogel is considered a promising material as a delivery matrix for drugs and proteins, since its gel microspheres form readily in aqueous solutions at room temperature, eliminating the need for harsh organic solvents, thereby maintaining the bioactivity of proteins in the process of loading into the microspheres (Jay & Saltzman 2009; Kikuchi et al. 1999). In addition, calcium cross-linked alginate hydrogel is degradable under physiological conditions (Kibat PG et al. 1990; Park K et al. 1993), which makes alginate stand out as an attractive candidate material for the protein carrier and bone regeneration (Hosoya et al. 2004; Matsuno et al. 2008; Turco et al. 2009). However, the major disadvantages of alginate microspheres is their low loading efficiency and also rapid release of proteins due to the mesh-like networks of the gel (Halder et al. 2005). Previous studies have shown that a core-shell structure in drug/protein carriers can overcome the issues of limited loading efficiencies and rapid release of drug or protein (Chang et al. 2010; Molvinger et al. 2004; Soppimath et al. 2007). We therefore hypothesized that introducing a core-shell structure into the alginate microspheres could solve the shortcomings of the pure alginate. Calcium silicate (CS) has been tested as a biodegradable biomaterial for bone tissue regeneration. CS is capable of inducing bone-like apatite formation in simulated body fluid (SBF) and its apatite-formation rate in SBF is faster than that of Bioglass® and A-W glass-ceramics (De Aza et al. 2000; Siriphannon et al. 2002). Titanium alloys plasma-spray coated with CS have excellent in vivo bioactivity (Xue et al. 2005) and porous CS scaffolds have enhanced in vivo bone formation ability compared to porous β-tricalcium phosphate ceramics (Xu et al. 2008). In light of the many advantages of this material, we decided to prepare CS/alginate composite microspheres by combining a CS shell with an alginate core to improve their protein delivery and mineralization for potential protein delivery and bone repair applications

Nanostructured mesoporous bioglass coated zirconia scaffolds for bone tissue engineering

For the filling and reconstruction of non-healing bone defects, the application of porous ceramic scaffold as bone substitutes is considered to be a reasonable choice. In bone tissue engineering, an ideal scaffold must satisfy several criterias such as open porosity, having high compressive strength (it depends where in body, and if external fixatures are used) and the practicability for cell migration. Many researchers have focused on enhancing the mechanical properties of hydroxyapatite scaffolds by combining it with other biomaterials, such as bioglass and polymers. Nevertheless, there is still a lack of suitable scaffolds based on porous biomaterials. In this study, zirconia scaffolds from two different templates (polyurethane (PU) and Acrylonitrile Butadiene Styrene (ABS) templates) were successfully fabricated with dissimilar fabrication techniques. The scaffold surfaces were further modified with mesoporous bioglass for the purpose of bone tissue engineering. In the study of PU template scaffold, high porosity (~88%) sol-gel derived yttria-stabilized zirconia (YSZ) scaffold was prepared by a polyurethane (PU) foam replica method using sol-gel derived zirconia for the first time, and double coated with Mesoporous Bioglass (MBGs) coating. For the ABS template scaffold, two types of templates (cube and cylinder) with different strut spacings were used and fabricated by a 3D Rapid Prototyper. Subsequently, zirconia scaffolds with low porosity (63±2.8% to 68±2.5%) were fabricated by embedding the zirconia powder slurry into the ABS templates and burning out the ABS to produce a uniform porous structure. The zirconia scaffolds were double coated with mesoporous bioglass by dip coating for the first time. The porosities of the scaffolds were calculated before and after coating. The microstructures were then examined using scanning electron microscopy and the mechanical properties were evaluated using compressive test. Accordingly, relationships between microstructure, processing and mechanical behaviour of the porous zirconia was discussed. Scaffold biocompatibility and bioactivity was also evaluated using a bone marrow stromal cell (BMSC) proliferation test and a simulated body fluid test.

Preparation of mesoporous bioglass coated zirconia scaffold for bone tissue engineering

Porous yttria-stabilized zirconia (YSZ) has been regarded as a potential candidate for bone substitute due to its high mechanical strength. However, porous YSZ is biologically inert to bone tissue. It is therefore necessary to introduce bioactive coatings onto the walls of the porous structures to enhance its bioactivity. In this study, porous YSZ scaffolds were prepared using a replication technique and then coated with mesoporous bioglass due to its excellent bioactivity. The microstructures were examined using scanning electron microscopy and the mechanical strength was evaluated via compression test. The biocompatibility and bioactivity were also evaluated using bone marrow stromal cell (BMSC) proliferation test and simulated body fluid test.

Preparation of mesoporous bioglass coated zirconia scaffold for bone tissue engineering

Porous yttria-stabilized zirconia (YSZ) has been regarded as a potential candidate for bone substitute due to its high mechanical strength. However, porous YSZ is biologically inert to bone tissue. It is therefore necessary to introduce bioactive coatings onto the walls of the porous structures to enhance its bioactivity. In this study, porous YSZ scaffolds were prepared using a replication technique and then coated with mesoporous bioglass due to its excellent bioactivity. The microstructures were examined using scanning electron microscopy and the mechanical strength was evaluated via compression test. The biocompatibility and bioactivity were also evaluated using bone marrow stromal cell (BMSC) proliferation test and simulated body fluid test.

Investigation on bioactivity of titanium dioxide thin films fabricated by electron cyclotron resonance plasma aided pulsed laser deposition

Titanium dioxide thin films with a rutile crystallinite size around 20 nm were fabricated by pulsed laser deposition (PLD) aided with an electron cyclotron resonance (ECR) plasma. With annealing treatment, the crystal size of the rutile crystallinite increased to 100 nm. The apatite-forming ability of the films as deposited and after annealing was investigated in a kind of simulated body fluid with ion concentrations nearly equal to those of human blood plasma. The results indicate that ECR aided PLD is an effective way both to fabricate bioactive titanium dioxide thin films and to optimize the bioactivity of titanium dioxide, with both crystal size and defects of the film taken into account.

Reactive plasma-aided RF sputtering deposition of hydroxyapatite bio-implant coatings

The plasma-assisted RF sputtering deposition of a biocompatible, functionally graded calcium phosphate bioceramic on a Ti6A14 V orthopedic alloy is reported. The chemical composition and presence of hydroxyapatite (HA), CaTiO3, and CaO mineral phases can be effectively controlled by the process parameters. At higher DC biases, the ratio [Ca]/[P] and the amount of CaO increase, whereas the HA content decreases. Optical emission spectroscopy suggests that CaO+ is the dominant species that responds to negative DC bias and controls calcium content. Biocompatibility tests in simulated body fluid confirm a positive biomimetic response evidenced by in-growth of an apatite layer after 24 h of immersion.

RF plasma sputtering deposition of hydroxyapatite bioceramics : synthesis, performance, and biocompatibility

Hydroxyapatite (HA) coatings have numerous applications in orthopedics and dentistry, owing to their excellent ability to promote stronger implant fixation and faster bone tissue ingrowth and remodeling. Thermal plasma spray and other plasma-assisted techniques have recently been used to synthesize various calcium phosphate-based bioceramics. Despite notable recent achievements in the desired stoichiometry, phase composition, mechanical, structural, and bio-compatible properties, it is rather difficult to combine all of the above features in a single coating. For example, many existing plasma-sprayed HA coatings fall short in meeting the requirements of grain size and crystallinity, and as such are subject to enhanced resorption in body fluid. On the other hand, relatively poor interfacial bonding and stability is an obstacle to the application of the HA coatings in high load bearing Ti6Al4V knee joint implants. Here, we report on an alternative: a plasma-assisted, concurrent, sputtering deposition technique for high performance biocompatible HA coatings on Ti6Al4V implant alloy. The plasma-assisted RF magnetron co-sputtering deposition method allows one to simultaneously achieve most of the desired attributes of the biomimetic material and overcome the aforementioned problems. This article details the film synthesis process specifications, extensive analytical characterization of the material's properties, mechanical testing, simulated body fluid assessments, biocompatibility and cytocompatibility of the HA-coated Ti6Al4V orthopedic alloy. The means of optimization of the plasma and deposition process parameters to achieve the desired attributes and performance of the HA coating, as well as future challenges in clinical applications are also discussed.

Development and characterization of a novel, antimicrobial, sterile hydrogel dressing for burn wounds : single-step production with gamma irradiation creates silver nanoparticles and radical polymerization

Patients with burn wounds are susceptible to wound infection and sepsis. This research introduces a novel burn wound dressing that contains silver nanoparticles (SNPs) to treat infection in a 2-acrylamido-2-methylpropane sulfonic acid sodium salt (AMPS-Na(+) ) hydrogel. Silver nitrate was dissolved in AMPS-Na(+) solution and then exposed to gamma irradiation to form SNP-infused hydrogels. The gamma irradiation results in a cross-linked polymeric network of sterile hydrogel dressing and a reduction of silver ions to form SNPs infused in the hydrogel in a one-step process. About 80% of the total silver was released from the hydrogels after 72 h immersion in simulated body fluid solution; therefore, they could be used on wounds for up to 3 days. All the hydrogels were found to be nontoxic to normal human dermal fibroblast cells. The silver-loaded hydrogels had good inhibitory action against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Results from a pilot study on a porcine burn model showed that the 5-mM silver hydrogel was efficient at preventing bacterial colonization of wounds, and the results were comparable to the commercially available silver dressings (Acticoat(TM) , PolyMem Silver(®) ). These results support its use as a potential burn wound dressing.

Polymer encapsulation of magnesium to control biodegradability and biocompatibility

Clinical utility of biodegradable magnesium implants is undermined by the untimely degradation of these materials in vivo. Their high corrosion rate leads to loss of mechanical integrity, peri–implant alkalization and localised accumulation of hydrogen gas. Biodegradable coatings were produced on pure magnesium using RF plasma polymerisation. A monoterpene alcohol with known anti-inflammatory and antibacterial properties was used as a polymer precursor. The addition of the polymeric layer was found to reduce the degradation rate of magnesium in simulated body fluid. The in vitro studies indicated good cytocompatibility of non-adherent THP–1 cells and mouse macrophage cells with the polymer, and the polymer coated sample. The viability of THP–1 cells was significantly improved when in contact with polymer encapsulated magnesium compared to unmodified samples. Collectively, these results suggest plasma enhanced polymer encapsulation of magnesium as a suitable method to control degradation kinetics of this biomaterial.

Additively manufactured device for dynamic culture of large arrays of 3D tissue engineered constructs

The ability to test large arrays of cell and biomaterial combinations in 3D environments is still rather limited in the context of tissue engineering and regenerative medicine. This limitation can be generally addressed by employing highly automated and reproducible methodologies. This study reports on the development of a highly versatile and upscalable method based on additive manufacturing for the fabrication of arrays of scaffolds, which are enclosed into individualized perfusion chambers. Devices containing eight scaffolds and their corresponding bioreactor chambers are simultaneously fabricated utilizing a dual extrusion additive manufacturing system. To demonstrate the versatility of the concept, the scaffolds, while enclosed into the device, are subsequently surface-coated with a biomimetic calcium phosphate layer by perfusion with simulated body fluid solution. 96 scaffolds are simultaneously seeded and cultured with human osteoblasts under highly controlled bidirectional perfusion dynamic conditions over 4 weeks. Both coated and noncoated resulting scaffolds show homogeneous cell distribution and high cell viability throughout the 4 weeks culture period and CaP-coated scaffolds result in a significantly increased cell number. The methodology developed in this work exemplifies the applicability of additive manufacturing as a tool for further automation of studies in the field of tissue engineering and regenerative medicine.

Evaluation of the in vitro bioactivity of bioceramics

Two common methods have been used to evaluate the in vitro bioactivity of bioceramics for the application of bone repair. One is to evaluate the ability of apatite formation by soaking ceramics in simulated body fluids (SBF); the other method is to evaluate the effect of ceramics on osteogenic differentiation using cell experiments. Both methods have their own drawbacks in evaluating the in vitro bioactivity of bioceramics. In this commentary paper we review the application of both methods in bioactivity of bioceramics and conclude that (i) SBF method is an efficient method to investigate the in vitro bioactivity of silicate-based bioceramics, (ii) cellular bioactivity of bioceramics should be investigated by evaluating their stimulatory ability using standard bioceramics as controls; and (iii) the combination of these two methods to evaluate the in vitro bioactivity of bioceramics can improve the screening efficiency for the selection of bioactive ceramics for bone regeneration.

Bioactive mesopore-glass microspheres with controllable protein-delivery properties by biomimetic surface modification

Microsphere systems with the ideal properties for bone regeneration need to be bioactive, and at the same time possess the capacity for controlled protein/drug-delivery; however, the current crop of microsphere system fails to fulfill these properties. The aim of this study was to develop a novel protein-delivery system of bioactive mesoporous glass (MBG) microspheres by a biomimetic method through controlling the density of apatite on the surface of microspheres, for potential bone tissue regeneration. MBG microspheres were prepared by using the method of alginate cross-linking with Ca2+ ions. The cellular bioactivity of MBG microspheres was evaluated by investigating the proliferation and attachment of bone marrow stromal cell (BMSC). The loading efficiency and release kinetics of bovine serum albumin (BSA) on MBG microspheres were investigated after coprecipitating with biomimetic apatite in simulated body fluids (SBF). The results showed that MBG microspheres supported BMSC attachment and the Si containing ionic products from MBG microspheres stimulated BMSCs proliferation. The density of apatite on MBG microspheres increased with the length of soaking time in SBF. BSA-loading efficiency of MBG was significantly enhanced by co-precipitating with apatite. Furthermore, the loading efficiency and release kinetics of BSA could be controlled by controlling the density of apatite formed on MBG microspheres. Our results suggest that MBG microspheres are a promising protein-delivery system as a filling material for bone defect healing and regeneration.

Swept frequency biompedance analysis for the determination of body water compartments

Bioelectrical impedance analysis, (BIA), is a method of body composition analysis first investigated in 1962 which has recently received much attention by a number of research groups. The reasons for this recent interest are its advantages, (viz: inexpensive, non-invasive and portable) and also the increasing interest in the diagnostic value of body composition analysis. The concept utilised by BIA to predict body water volumes is the proportional relationship for a simple cylindrical conductor, (volume oc length2/resistance), which allows the volume to be predicted from the measured resistance and length. Most of the research to date has measured the body's resistance to the passage of a 50· kHz AC current to predict total body water, (TBW). Several research groups have investigated the application of AC currents at lower frequencies, (eg 5 kHz), to predict extracellular water, (ECW). However all research to date using BIA to predict body water volumes has used the impedance measured at a discrete frequency or frequencies. This thesis investigates the variation of impedance and phase of biological systems over a range of frequencies and describes the development of a swept frequency bioimpedance meter which measures impedance and phase at 496 frequencies ranging from 4 kHz to 1 MHz. The impedance of any biological system varies with the frequency of the applied current. The graph of reactance vs resistance yields a circular arc with the resistance decreasing with increasing frequency and reactance increasing from zero to a maximum then decreasing to zero. Computer programs were written to analyse the measured impedance spectrum and determine the impedance, Zc, at the characteristic frequency, (the frequency at which the reactance is a maximum). The fitted locus of the measured data was extrapolated to determine the resistance, Ro, at zero frequency; a value that cannot be measured directly using surface electrodes. The explanation of the theoretical basis for selecting these impedance values (Zc and Ro), to predict TBW and ECW is presented. Studies were conducted on a group of normal healthy animals, (n=42), in which TBW and ECW were determined by the gold standard of isotope dilution. The prediction quotients L2/Zc and L2/Ro, (L=length), yielded standard errors of 4.2% and 3.2% respectively, and were found to be significantly better than previously reported, empirically determined prediction quotients derived from measurements at a single frequency. The prediction equations established in this group of normal healthy animals were applied to a group of animals with abnormally low fluid levels, (n=20), and also to a group with an abnormal balance of extra-cellular to intracellular fluids, (n=20). In both cases the equations using L2/Zc and L2/Ro accurately and precisely predicted TBW and ECW. This demonstrated that the technique developed using multiple frequency bioelectrical impedance analysis, (MFBIA), can accurately predict both TBW and ECW in both normal and abnormal animals, (with standard errors of the estimate of 6% and 3% for TBW and ECW respectively). Isotope dilution techniques were used to determine TBW and ECW in a group of 60 healthy human subjects, (male. and female, aged between 18 and 45). Whole body impedance measurements were recorded on each subject using the MFBIA technique and the correlations between body water volumes, (TBW and ECW), and heighe/impedance, (for all measured frequencies), were compared. The prediction quotients H2/Zc and H2/Ro, (H=height), again yielded the highest correlation with TBW and ECW respectively with corresponding standard errors of 5.2% and 10%. The values of the correlation coefficients obtained in this study were very similar to those recently reported by others. It was also observed that in healthy human subjects the impedance measured at virtually any frequency yielded correlations not significantly different from those obtained from the MFBIA quotients. This phenomenon has been reported by other research groups and emphasises the need to validate the technique by investigating its application in one or more groups with abnormalities in fluid levels. The clinical application of MFBIA was trialled and its capability of detecting lymphoedema, (an excess of extracellular fluid), was investigated. The MFBIA technique was demonstrated to be significantly more sensitive, (P<.05), in detecting lymphoedema than the current technique of circumferential measurements. MFBIA was also shown to provide valuable information describing the changes in the quantity of muscle mass of the patient during the course of the treatment. The determination of body composition, (viz TBW and ECW), by MFBIA has been shown to be a significant improvement on previous bioelectrical impedance techniques. The merit of the MFBIA technique is evidenced in its accurate, precise and valid application in animal groups with a wide variation in body fluid volumes and balances. The multiple frequency bioelectrical impedance analysis technique developed in this study provides accurate and precise estimates of body composition, (viz TBW and ECW), regardless of the individual's state of health.