Eng San Thian

Effect of silver content on the antibacterial and bioactive properties of silver-substituted hydroxyapatite.

The long-term success of a biomaterial used during surgery may be compromised by infection. A possible effective solution is to make the biomaterial osteoconductive and antibacterial. A range of silver-substituted hydroxyapatite (AgHA) of up to 1.1 wt. % of Ag was synthesized. AgHA displayed a rod-like morphology of dimensions ~50 nm in length and ~15 nm in width. Phase-pure AgHA was demonstrated in the X-ray diffraction patterns and Fourier transform infrared spectroscopy spectra. Comparing with hydroxyaptite (HA), 0.5AgHA exhibited a 3-log reduction in the number of bacteria. Diffusion of the entrapped Ag(+) ions towards the crystal structure surface was revealed by an increase of 6 at. % Ag in the X-ray photoelectron spectroscopy results. Furthermore, less than 0.5 ppm of Ag(+) ions being released from 0.5AgHA into the deionized water medium was evidenced from the inductively coupled plasma mass spectrometry results. AgHA produced by co-precipitation gave rise to minimal release of Ag(+) ions. It was hypothesized that the diffused surface Ag(+) ions damaged the bacteria cell membrane and impede its replication. With the culturing time, significant increase in the number of human mesenchymal stem cells (p < 0.05) was demonstrated on 0.5AgHA.

Development of nanosized silver-substituted apatite for biomedical applications: A review

UNLABELLED The favorable biocompatibility of hydroxyapatite (HA) makes it a popular bone graft material as well as a coating layer on metallic implant. To reduce implant-related infections, silver ions were either incorporated into the apatite during co-precipitation process (AgHA-CP) or underwent ion-exchange with the calcium ions in the apatite (AgHA-IE). However, the distribution of silver ions in AgHA-CP and AgHA-IE was different, thus affecting the antibacterial action. Several studies reported that nanosized AgHA-CP containing 0.5 wt.% of silver provided an optimal trade-off between antibacterial properties and cytotoxicity. Nevertheless, nanosized AgHA and AgHA nanocoatings could not function ideally due to the compromise in the bone differentiation of mesenchymal stem cells, as evidenced in the reduced alkaline phosphatase, type I collagen and osteocalcin. Preliminary studies showed that biological responses of nanosized AgHA and AgHA nanocoatings could be improved with the addition of silicon. This review will discuss on nanosized AgHA and AgHA nanocoatings. FROM THE CLINICAL EDITOR In many patients needing bone graft material, hydroxyapatite (HA) has proven to be a popular choice. Nonetheless, implant-related infections remain a major concern. Hence, effective preventive measures are needed. In this review article, the authors discussed the application of incorporating silver nanoparticles in HA and its use as bone graft biomaterials together with the addition of silica.

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.

Controlled delivery of stromal derived factor-1α from poly lactic-co-glycolic acid core–shell particles to recruit mesenchymal stem cells for cardiac regeneration

Stromal derived factor-1α (SDF-1α) has shown promising results in treatment of myocardial infarction (MI), via recruitment of endogenous stem cells into the injured myocardium. However, the bioactivity of this susceptible signalling chemokine is reduced significantly during the common fabrication processes of drug delivery systems, due to the exposure to organic-aqueous interfaces or elevated temperature. In this study, we developed a novel SDF-1α delivery system using coaxial electrospraying, the technique which enables fabrication of core-shell particles with minimized contact of organic-aqueous phases. The SDF-1α incorporated PLGA particles exhibited distinct core-shell structure, confirmed by transmission electron microscopy (TEM). Controlled release of SDF-1α was obtained for at least 40days, and the release rate was tailored by co-encapsulation of bovine serum albumin (BSA) into the core of the particles. The SDF-1α released from PLGA/SDF-1α and PLGA/BSA-SDF-1α particles retained its chemotactic activity, and enhanced the number of migrated mesenchymal stem cells (MSCs) by 38% and 54%, respectively, compared to basal medium used as the control. Moreover, both SDF-1α and BSA supported the proliferation of MSCs within 3days of cell culture. The SDF-1α incorporated core-shell particles developed by electrospraying technique, can be effectively employed as injectable drug delivery system for in situ cardiac regeneration.

Fabrication of three‐dimensional porous scaffolds with controlled filament orientation and large pore size via an improved E‐jetting technique

Biodegradable polymeric scaffolds have been widely used in tissue engineering as a platform for cell proliferation and subsequent tissue regeneration. Conventional microextrusion methods for three-dimensional (3D) scaffold fabrication were limited by their low resolution. Electrospinning, a form of electrohydrodynamic (EHD) printing, is an attractive method due to its capability of fabricating high-resolution scaffolds at the nanometer/micrometer scale level. However, the scaffold was composed of randomly orientated filaments which could not guide the cells in a specific direction. Furthermore, the pores of the electrospun scaffold were small, thus preventing cell infiltration. In this study, an alternative EHD jet printing (E-jetting) technique has been developed and employed to fabricate 3D polycaprolactone (PCL) scaffolds with desired filament orientation and pore size. The effect of PCL solution concentration was evaluated. Results showed that solidified filaments were achieved at concentration >70% (w/v). Uniform filaments of diameter 20 μm were produced via the E-jetting technique, and X-ray diffraction and attenuated total reflectance Fourier transform infrared spectroscopic analyses revealed that there was no physicochemical changes toward PCL. Scaffold with a pore size of 450 μm and porosity level of 92%, was achieved. A preliminary in vitro study illustrated that live chondrocytes were attaching on the outer and inner surfaces of collagen-coated E-jetted PCL scaffolds. E-jetted scaffolds increased chondrocytes extracellular matrix secretion, and newly formed matrices from chondrocytes contributed significantly to the mechanical strength of the scaffolds. All these results suggested that E-jetting is an alternative scaffold fabrication technique, which has the capability to construct 3D scaffolds with aligned filaments and large pore sizes for tissue engineering applications.

Enhancing mesenchymal stem cell response using uniaxially stretched poly(ε-caprolactone) film micropatterns for vascular tissue engineering application

Regeneration of tunica media with anisotropic architecture still remains a challenging issue for vascular tissue engineering (TE). Herein, we present the development of flexible poly(ε-caprolactone) (PCL) film micropatterns to regulate mesenchymal stem cells (MSCs) function for tunica media construction. Results showed that uniaxial thermal stretching of PCL films resulted in topographical micropatterns comprising of ridges/grooves, and improved mechanical properties, including yield stress, Youngs modulus, and fracture stress without sacrificing film elasticity. Culturing on such PCL film micropatterns, MSCs self-aligned along the ridges with a more elongated morphology as compared to that of the un-stretched film group. Moreover, MSCs obtained a contractile SMCs-like phenotype, with ordered organization of cellular stress filaments and upregulated expression of the contractile makers, including SM-α-actin, calponin, and SM-MHC. The PCL film micropatterns could be rolled into a small-diameter 3D tubular scaffold with circumferential anisotropy of ridges/grooves, and in the incorporation of MSCs, which facilitated a hybrid sandwich-like vascular wall construction with ordered cell architecture similar to that of the tunica media. These results provide insights of how geometric cues are able to regulate stem cells with desired functions and have significant implications for the designing of a functionalized vascular TE scaffold with appropriate topographical geometries for guiding tunica media regeneration with microscale control of cell alignment and genetic expression.

Synthesis and characterization of silver/silicon‐cosubstituted nanohydroxyapatite

Favorable cell-material interaction and the absence of undesirable reaction from the host body defence system play a critical role in determining the success and long-term survival of the implants. Substitution of various elements into hydroxyapatite (HA) has been done to alter its chemical composition, thereby mimicking that of the bone mineral. In this study, a cosubstituted nanosized apatite (Ag/Si-HA) containing Ag (0.3 wt %) and Si (0.8 wt %) was synthesized by an aqueous precipitation technique. The synthesized Ag/Si-HA displayed a rod-like morphology of dimensions ~50 nm in length and ~15 nm in width, as observed from the transmission electron microscope image. With an increase in temperature, the aspect ratio of nanosized Ag/Si-HA decreased, whilst the size increased. Autoclaving was used to achieve sufficient crystallinity while maintaining the rod-like morphology and size that were comparable to that of the bone apatite. A pure Ag/Si-HA was produced without any undesirable secondary phases, as evidenced from the X-ray diffraction and thermal gravimetric results. The Ag/Si cosubstitution affected the lattice cell parameters, in particularly the a- and c- axes which further led to an expansion of the unit cell volume. In addition, the relative intensity of the hydroxyl vibrational bands was reduced. These results demonstrated that a stable phase-pure Ag/Si-HA was produced using an aqueous precipitation reaction.

Optimization of poly(ε-caprolactone) surface properties for apatite formation and improved osteogenic stimulation†

A biodegradable polymer with surface properties that promotes cell attachment and host integration is widely recognized as a useful three-dimensional construct for bone tissue engineering applications. In this work, studies were carried out to correlate surface properties of modified polycaprolactone (PCL) films with cell-material interactions. PCL film substrates were subjected to various degrees of chemical hydrolysis using different pretreatment solutions to introduce different densities of carboxylate groups onto the surfaces. The extent of hydrolysis on the films was optimized to allow the deposition of a dense and uniform bone-like apatite layer by an alternate soak treatment, followed by subsequent incubation in simulated body fluid (SBF). The hydrolyzed and apatite-coated PCL films were investigated using scanning electron microscopy, thin film X-ray diffractometer (TF-XRD), water contact angle, and Alizarin red staining. Surface wettability, roughness, and chemistry of various PCL substrates were correlated with cell attachment, proliferation, viability, and alkaline phosphatase activity. Results demonstrated that cell attachment increased with increasing surface hydrophilicity and roughness. The apatite-coated films showed significantly improved surface wettability and enhanced surface roughness, which subsequently led to better cell attachment potential, high-cell viability, and enhanced bone formation capability. Thus, surface modification with an apatite coating layer is a promising approach for enhancing the efficacy of the polymeric scaffold for bone tissue engineering applications.

Direct E-jet printing of three-dimensional fibrous scaffold for tendon tissue engineering

Tissue engineering (TE) offers a promising strategy to restore diseased tendon tissue. However, a suitable scaffold for tendon TE has not been achieved with current fabrication techniques. Herein, we report the development of a novel electrohydrodynamic jet printing (E-jetting) for engineering 3D tendon scaffold with high porosity and orientated micrometer-size fibers. The E-jetted scaffold comprised tubular multilayered micrometer-size fibrous bundles, with interconnected spacing and geometric anisotropy along the longitudinal direction of the scaffold. Fiber diameter, stacking pattern, and interfiber distance have been observed to affect the structural stability of the scaffold, of which the enhanced mechanical strength can be obtained for scaffolds with thick fibers as the supporting layer. Human tenocytes showed a significant increase in cellular metabolism on the E-jetted scaffolds as compared to that on conventional electrospun scaffolds (2.7-, 2.8-, and 3.1-fold increase for 150, 300, and 600 µm interfiber distance, respectively; p < 0.05). Furthermore, the scaffolds provided structural support for human tenocytes to align with controlled orientation along the longitudinal direction of the scaffold, and promoted the expression of collagen type I. For the first time, E-jetting has been explored as a novel scaffolding approach for tendon TE, and offers a 3D fibrous scaffold to promote organized tissue reconstruction for potential tendon healing.

A scalable approach to obtain mesenchymal stem cells with osteogenic potency on apatite microcarriers

Bone tissue engineering, which relies on the interactions between stem cells and suitable scaffold materials, represents a highly desirable alternative to currently used allograft or autograft strategies for the treatment of bone defects caused by injury or disease, with one of the major challenges being to generate sufficient quantities of stem cells to bring about the intended therapeutic effect. However, conventional cell culture to achieve sufficient cell numbers faces limitations of low efficiency and diminished efficacy of stem cells due to repeated passaging. Furthermore, current microcarriers available may not be suitable for therapeutic implantation. Here, the authors featured an apatite-based microcarrier intended for bone tissue engineering applications. These apatite microcarriers have a diameter of ∼230 µm, and exhibited porous and rough surface morphology. Peaks obtained from X-ray diffractometry (XRD) corresponded to hydroxyapatite (HA) with high crystallinity. Fourier transform infrared spectrophotometry (FTIR) showed that no residues of alginate remained, and all bands observed belong to phosphate and hydroxyl groups of HA. To evaluate the cytocompatibility of these microcarriers, in vitro proliferation studies were conducted and compared with conventional monolayer as well as Cytodex 3. The authors found that human foetal mesenchymal stem cells (hfMSCs) cultured on apatite microcarriers exhibited comparable growth characteristics, achieving 1.4-fold higher live cells than Cytodex 3 over a 9-day culture period. As these microcarriers were hypothesised to offer enhanced osteogenic potency over conventional monolayer culture, alkaline phosphatase (ALP), type I collagen and osteocalcin expression of hfMSCs cultured on the apatite microcarriers were evaluated over a 12-day period. ALP expression for hfMSCs seeded on apatite microcarriers was 2.7-fold higher than that of adherent monolayer culture (p < 0.001). Additionally, type I collagen and osteocalcin expression were 1.8- and 1.5-fold higher than that of adherent monolayer culture on day 12, respectively (p < 0.001).