Functional regeneration of tendons using scaffolds with physical anisotropy engineered via microarchitectural manipulation.

Structural and hierarchical anisotropy underlies the structure-function relationship of most living tissues. Attempts to exploit the interplay between cells and their immediate environment have rarely featured macroscale, three-dimensional constructs required for clinical applications. Furthermore, compromises to biomechanical robustness during fabrication often limit the scaffold's relevance in translational medicine. We report a polymeric three-dimensional scaffold with tendon-like mechanical properties and controlled anisotropic microstructures. The scaffold was composed of two distinct portions, which enabled high porosity while retaining tendon-like mechanical properties. When tenocytes were cultured in vitro on the scaffold, phenotypic markers of tenogenesis such as type-I collagen, decorin, and tenascin were significantly expressed over nonanisotropic controls. Moreover, highly aligned intracellular cytoskeletal network and high nuclear alignment efficiencies were observed, suggesting that microstructural anisotropy might play the epigenetic role of mechanotransduction. When implanted in an in vivo micropig model, a neotissue that formed over the scaffold resembled native tendon tissue in composition and structure.

Proposed mechanism of antibacterial action of chemically modified apatite for reduced bone infection.

Surface-bound silver ions were demonstrated to be responsible for the antibacterial action of silver, silicon-containing hydroxyapatite (Ag,Si-HA). X-ray photoelectron spectroscopy, transmission electron microscopy, and induced coupled plasma spectroscopy results suggested that silver ions in the crystal structure diffused toward the crystal surface of Ag,Si-HA, and interacted with adherent Staphylococcus aureus bacteria, thus damaging the cell wall and inducing leakage of potassium ions. All these steps constitute the mechanism of antibacterial action for Ag,Si-HA. Consequently, Ag,Si-HA gave rise to a 7-log reduction of the adherent bacteria as compared to HA and Si-HA at 168 h. Silicon in Ag,Si-HA helped to mitigate the reduced effect of bone differentiation in Ag-HA as shown in the alkaline phosphatase, type I collagen and osteocalcin results, promoting enhanced biological response, without compromising the antibacterial property. On the whole, Ag,Si-HA containing an optimized content of 0.5 wt % silver and 0.7 wt % silicon provides antibacterial properties and enhanced biological response.

Zinc-substituted hydroxyapatite: a biomaterial with enhanced bioactivity and antibacterial properties.

Hydroxyapatite (HA) is a synthetic biomaterial and has been found to promote new bone formation when implanted in a bone defect site. However, its use is often limited due to its slow osteointegration rate and low antibacterial activity, particularly where HA has to be used for long term biomedical applications. This work will describe the synthesis and detailed characterization of zinc-substituted HA (ZnHA) as an alternative biomaterial to HA. ZnHA containing 1.6 wt% Zn was synthesized via a co-precipitation reaction between calcium hydroxide, orthophosphoric acid and zinc nitrate hexahydrate. Single-phase ZnHA particles with a rod-like morphology measuring ~50 nm in length and ~15 nm in width, were obtained and characterized using transmission electron microscopy and X-ray diffraction. The substitution of Zn into HA resulted in a decrease in both the a- and c-axes of the unit cell parameters, thereby causing the HA crystal structure to alter. In vitro cell culture work showed that ZnHA possessed enhanced bioactivity since an increase in the growth of human adipose-derived mesenchymal stem cells along with the bone cell differentiation markers, were observed. In addition, antibacterial work demonstrated that ZnHA exhibited antimicrobial capability since there was a significant decrease in the number of viable Staphylococcus aureus bacteria after in contact with ZnHA.

Collagen-hyaluronic acid scaffolds for adipose tissue engineering.

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen-HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen-HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen-HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen-HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen-HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.

The role of surface wettability and surface charge of electrosprayed nanoapatites on the behaviour of osteoblasts.

A new deposition method is presented, based on electrospraying, that can build bioceramic structures with desirable surface properties. This technology allows nanoapatite crystals, including hydroxyapatite (nHA), carbonate-substituted HA (nCHA) and silicon-substituted HA (nSiHA), to be electrosprayed on glass substrates. Human osteoblast cells cultured on nSiHA showed enhanced cell attachment, proliferation and protein expression, namely alkaline phosphatase, type 1 collagen and osteocalcin, as compared to nHA and nCHA. The modification of nanoapatite by the addition of silicon into the HA lattice structure renders the electrosprayed surface more hydrophilic and electronegatively charged.

Deposition of nano-hydroxyapatite particles utilising direct and transitional electrohydrodynamic processes.

Electrohydrodynamic spraying is a well established process used to deposit, coat, analyse and synthesise materials within the biomedical remit. Recently, electrohydrodynamic printing has been developed to afford structures for potential applications in the biomedical and medical engineering fields. Both of these processes rely on the formation of an electrically-induced jet, however the resulting products can be made strikingly different and offer potential in broader applications. Here we show how spraying and printing are linked by elucidating the ease of transition between the processes. Changes in the deposition distance can result in either spray (>10 mm) or print formation (<3 mm), with an overlap of the two in between this range. For the optimal printing distance of 0.5 mm, gradual changes in the applied voltage (0-4.5 kV) encounters transitional printing modes (dripping, micro-dripping, rapid micro-dripping, unstable and stable jetting) which can be utilised for patterning. The results indicate the robustness of the electrohydrodynamic route in the nano-materials processing arena, with emphasis on biomedical materials.

Influence of nanohydroxyapatite patterns deposited by electrohydrodynamic spraying on osteoblast response.

Electrohydrodynamic spraying has been used to produce patterns of line width up to 100 microm in size on glass discs, using nanohydroxyapatite (nHA). A human osteoblast (HOB)-like cell model was then used to study the interaction between the HOB cells and nHA patterns in vitro. Growth of the cells was significantly increased (p < 0.05) on the nHA surfaces. In addition, HOBs attached and spread well, secreting extracellular matrix. It was found that a confluent, aligned cell layer was achieved on nHA patterns by day 9. Immunofluorescent staining indicated that these cells showed elongated nuclei, enhanced adhesion (vinculin adhesion plaques) and a well-aligned cytoskeleton (actin stress fibres). This work suggests that this type of spraying may provide a route for the production of nanoscale features on implants for biomedical applications.

Silicon-substituted hydroxyapatite thin films: effect of annealing temperature on coating stability and bioactivity.

The effect of annealing temperature on the physicochemical and biological characteristics of magnetron cosputtered silicon-substituted hydroxyapatite (SiHA) thin coatings was studied. Annealing is required to transform as-sputtered amorphous films into crystalline coatings. A nanocrystalline, single-phase apatite structure was achieved for coatings heated to 600 or 700 degrees C and, with increasing annealing temperature, the crystallite size increased. Small crystallites were found to be more soluble in the physiological environment but, at the same time, were able to induce early formation of a new apatite layer. A human osteoblast-like (HOB) cell model was used to evaluate the performance of these annealed SiHA coatings. HOB cells attached and grew well on coatings and, after 42 days in culture, a mineralization process was observed to be taking place, with evidence of calcium phosphate minerals throughout the extracellular matrix. Our findings indicated that an annealing temperature of 600 degrees C is sufficient to achieve crystalline SiHA coatings and exhibiting good chemical stability and bioactivity.

Novel silicon-doped hydroxyapatite (Si-HA) for biomedical coatings: an in vitro study using acellular simulated body fluid.

Magnetron co-sputtering was used to produce silicon-doped hydroxyapatite (Si-HA) as coatings intended for potential applications such as orthopedic and dental implants. It was found that the crystallinity of the as-sputtered coatings increased after annealing, resulting in a nanocrystalline apatite structure. Subsequently, the bioactivity of the coatings was evaluated in an acellular simulated body fluid (SBF). Physicochemical evaluation demonstrated that a carbonate-containing apatite layer, which is essential for bonding at the bone/implant interface, was formed on the coating surfaces after immersion in SBF between 4 and 7 days. The annealed coatings exhibited enhanced bioactivity and chemical stability under physiological conditions, as compared with the as-sputtered coatings. It is proposed that the rate at which the carbonate-containing apatite layer forms is dependent on the scale factor of the structure. A nanocrystalline structure can provide a higher number of nucleation sites for the formation of apatite crystallites, leading to a more rapid precipitation of carbonate-containing apatite layer. This work shows that Si-HA coatings offer considerable potential for applications in hard tissue replacement, owing to their ability to form a carbonate-containing apatite layer rapidly.

A new way of incorporating silicon in hydroxyapatite (Si-HA) as thin films.

Bioactive silicon-containing hydroxyapatite (Si-HA) thin films that can be used as coatings for bone tissue replacement have been developed. A magnetron co-sputtering technique was used to deposit Si-HA films up to 700 nm thick on titanium substrates, with a silicon level up to 1.2 wt%. X-ray diffraction demonstrated that annealing transformed the as-deposited Si-HA films which were amorphous, into a crystalline HA structure. A human osteoblast-like (HOB) cell model was used to determine the biocompatibility of these films. HOB cells were seen to attach and grow well on the Si-HA films, and the metabolic activity of HOB cells on these films was observed to increase with culture time. Furthermore, mineralisation of the cell layers was observed after 8 weeks of culture. Based on the present findings, Si-HA of different film compositions demonstrate bioactive properties in-vitro, and indicate the potential as biocoatings for a wide variety of medical implants including load-bearing applications such as the femoral stem of hip replacement implants.

Magnetron co-sputtered silicon-containing hydroxyapatite thin films--an in vitro study.

The use of silicon-substituted hydroxyapatite (Si-HA) as a biomaterial has been reported recently. In vivo testing has shown that Si-HA promotes early bonding of the bone/implant interface. In order to extend its usage to major load-bearing applications such as artificial hip replacement implants, it has been proposed that the material could be used in the form of a coating on implant surfaces. This paper reports a preliminary study of the biocompatibility of magnetron co-sputtered silicon-containing hydroxyapatite (Si-HA) coatings on a metallic substrate. Magnetron co-sputtered Si-HA films of thickness 600 nm with a Si content of approximately 0.8 wt% were produced on titanium substrates. X-ray diffraction analysis showed that the as-deposited Si-HA films were either amorphous or made up of very small crystals. The crystallinity of Si-HA films was increased after post-deposition heat treatment at 700 degrees C for 3 h, and the principal peaks were attributable to HA. The formation of nano-scale silicon-calcium phosphate precipitates was noted on the heat-treated films. In vitro cell culture has demonstrated that human osteoblast-like cells attached and grew well on all films, with the highest cell growth and signs of mineralisation observed on the heat-treated Si-HA films. In addition, many focal contacts were produced on the films and the cells had well-defined actin cytoskeletal organisation. This work shows that as-deposited and heat-treated Si-HA films have excellent bioactivity and are good candidates when rapid bone apposition is required. Furthermore, heat-treated Si-HA films have improved biostability compared to as-deposited films under physiological conditions.

Microstructures and mechanical properties of powder injection molded Ti-6Al-4V/HA powder.

Taguchi method with an L9 orthogonal array was employed to investigate the sintered properties of Ti-6Al-4V/HA tensile bars produced by powder injection molding. The effects of sintering factors at the 90% significance level: sintering temperature (1050 degrees C, 1100 degrees C and 1150 degrees C), heating rate (5 degrees C/min, 7.5 degrees C/min and 10 degrees C/min), holding time (30, 45 and 60 min) and cooling rate (5 degrees C/min, 20 degrees C/min and 40 degrees C/min) were investigated. Results showed that sintering temperature, heating rate and cooling rate have significant effects on sintered properties, whereas the influence of holding time was insignificant. It was found that a sintering temperature of 1100 degrees C, a heating rate of 7.5 degrees C/min and a cooling rate of 5 degrees C/min increased the relative density, Vicker's microhardness, flexural strength and flexural modulus. However, a further increment of sintering temperature to 1150 degrees C did not show any discernable improvement in the relative density and Vicker's microhardness, but there was a slight increase of 0.6% and 0.9% in the flexural strength and flexural modulus, respectively. Mechanically strong Ti-6Al-4V/HA parts with an open porosity of around 50% were developed.

In vitro behavior of sintered powder injection molded Ti-6Al-4V/HA.

This article reports the morphology and mechanical properties of sintered powder injection molded Ti-6Al-4V/HA parts in a simulated physiological environment. Sintered Ti-6Al-4V/HA parts were immersed in a simulated body fluid (SBF) with ion concentrations that were comparable to those of human blood plasma for a total period of 12 weeks. At intervals of 2 weeks, the immersed Ti-6Al-4V/HA parts were analyzed with the use of scanning electron microscopy (SEM), X-ray diffractometry (XRD), and inductively coupled plasma-atomic emission spectroscopy (ICP-AES). Mechanical properties such as flexural strength, flexural modulus, compressive strength, and compressive modulus were also evaluated. Results showed that complete dissolution of the more soluble phases such as tricalcium phosphate (TCP), tetracalcium phosphate (TTCP), and calcium oxide (CaO) were found after 2 weeks of immersion in SBF. ICP analysis showed that high calcium concentration release of around 200 ppm was observed in the SBF solution after 2-4 weeks of immersion, indicating that dissolution has taken place. Next, a gradual decrease in calcium concentration release in the SBF solution was observed after immersion for 4-6 weeks, with increasing amounts of calcium phosphate precipitates being observed on the Ti-6Al-4V/HA surface. Mechanical properties such as strength and modulus were found to deteriorate during 2-4 weeks of immersion, followed by gradual increment as the immersion period increased. This study also showed that parts sintered at 1150 C exhibited faster dissolution and precipitation rates than parts sintered at 1050 C in a physiological environment.

Processing of HA-coated Ti-6Al-4V by a ceramic slurry approach: an in vitro study.

Hydroxyapatite-coated titanium alloy composite powders (Ti-6Al-4V/HA) was produced by a ceramic slurry approach. The aim was to evaluate the stability of the coating when subjected to a physiological medium simulated body fluid (SBF). Three consolidation conditions (700 degrees C for 5 h, 700 degrees C for 8 h and 700 degrees C for 11 h) were used in the production of the Ti-6Al-4V/HA composite powders. Results showed that biodissolution followed by apatite precipitation had taken place after soaking in SBF. In addition, it was found that consolidation at 700 degrees C for 5 h resulted in a weak mechanical locking of calcium phosphate on the Ti-6Al-4V surfaces; and the formation of small crystallites, which would increase the dissolution rate.