Wai Lam Cheung

Three-dimensional nanocomposite scaffolds fabricated via selective laser sintering for bone tissue engineering.

Bionanocomposites formed by combining biodegradable polymers and nanosized osteoconductive inorganic solids have been regarded as promising biomimetic systems which possess much improved structural and functional properties for bone tissue regeneration. In this study three-dimensional nanocomposite scaffolds based on calcium phosphate (Ca-P)/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and carbonated hydroxyapatite (CHAp)/poly(l-lactic acid) (PLLA) nanocomposite microspheres were successfully fabricated using selective laser sintering, which is a rapid prototyping technology. The sintered scaffolds had controlled material microstructure, totally interconnected porous structure and high porosity. The morphology and mechanical properties of Ca-P/PHBV and CHAp/PLLA nanocomposite scaffolds as well as PHBV and PLLA polymer scaffolds were studied. In vitro biological evaluation showed that SaOS-2 cells had high cell viability and normal morphology and phenotype after 3 and 7 days culture on all scaffolds. The incorporation of Ca-P nanoparticles significantly improved cell proliferation and alkaline phosphatase activity for Ca-P/PHBV scaffolds, whereas CHAp/PLLA nanocomposite scaffolds exhibited a similar level of cell response compared with PLLA polymer scaffolds. The nanocomposite scaffolds provide a biomimetic environment for osteoblastic cell attachment, proliferation and differentiation and have great potential for bone tissue engineering applications.

Optimized fabrication of Ca-P/PHBV nanocomposite scaffolds via selective laser sintering for bone tissue engineering.

Biomaterials for scaffolds and scaffold fabrication techniques are two key elements in scaffold-based tissue engineering. Nanocomposites that consist of biodegradable polymers and osteoconductive bioceramic nanoparticles and advanced scaffold manufacturing techniques, such as rapid prototyping (RP) technologies, have attracted much attention for developing new bone tissue engineering strategies. In the current study, poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) microspheres and calcium phosphate (Ca-P)/PHBV nanocomposite microspheres were fabricated using the oil-in-water (O/W) and solid-in-oil-in-water (S/O/W) emulsion solvent evaporation methods. The microspheres with suitable sizes were then used as raw materials for scaffold fabrication via selective laser sintering (SLS), which is a mature RP technique. A three-factor three-level complete factorial design was applied to investigate the effects of the three factors (laser power, scan spacing, and layer thickness) in SLS and to optimize SLS parameters for producing good-quality PHBV polymer scaffolds and Ca-P/PHBV nanocomposite scaffolds. The plots of the main effects of these three factors and the three-dimensional response surface were constructed and discussed. Based on the regression equation, optimized PHBV scaffolds and Ca-P/PHBV scaffolds were fabricated using the optimized values of SLS parameters. Characterization of optimized PHBV scaffolds and Ca-P/PHBV scaffolds verified the optimization process. It has also been demonstrated that SLS has the capability of constructing good-quality, sophisticated porous structures of complex shape, which some tissue engineering applications may require.

RuO4 staining and lamellar structure of α- and β-PP

Monoclinic (α) and hexagonal (β) polypropylene (α- and β-PP) were stained in the vapor of a ruthenium tetroxide solution prepared in situ. The effect of staining on the fusion behavior was investigated using a DSC. A staining duration between 10 and 24 h was found suitable for obtaining a good electron contrast between the crystalline and amorphous regions for TEM examination without causing severe damage to the crystals. The spherulites of the water-quenched α-PP were found to be composed of very fine cross-hatched lamellae whose long period was about 10 nm. In comparison, the β-PP spherulites crystallized isothermally at 130°C had a category 2 morphology and the lamellae have a long period of 20 nm. The morphology of the spherulite boundary varied depending on the contact angle between the lamellae of the neighboring spherulites.

Lamellar structure of POM spherulites imaged by a two‐stage RuO4 staining technique

Ruthenium tetroxide solution was prepared in situ by oxidation of ruthenium compounds at lower oxidation states with an excess of sodium periodate. The solution was able to stain saturated polymer POM in the vapor phase and it remained effective for up to 2 weeks. The melting behavior of POM samples stained for different lengths of time was studied with DSC and the staining process was analyzed. The results indicated that ruthenium tetroxide affected both the amorphous region and the crystals. During the early stage of the staining process, the tetroxide reacted preferentially with the amorphous material near the specimen surface. Much of this affected material was washed away after the rinsing process, thus resulting in an apparent increase in crystallinity. Prolonged staining would cause more crystals to degrade; hence, the crystallinity would drop. A two-step vapor staining technique was developed to improve the contrast between the amorphous and crystalline regions of ultrathin POM sections. The lamellar structure of POM spherulites was revealed and examined under TEM. The results showed that POM spherulites possess a category 2 structure and the thickness of the lamellae is about 7 nm.

Selective Laser Sintering of Tissue Engineering Scaffolds Using Poly(L-Lactide) Microspheres

This paper reports a study on the modification of a commercial selective laser sintering (SLS) machine for the fabrication of tissue engineering scaffolds from small quantities of poly(L-lactide) (PLLA) microspheres. A miniature build platform was designed, fabricated and installed in the build cylinder of a Sinterstation 2000 system. Porous scaffolds in the form of rectangular prism, 12.7×12.7×25.4 mm3, with interconnected square and round channels were designed using SolidWorks. For initial trials, DuraFormTM polyamide powder was used to build scaffolds with a designed porosity of ~70%. The actual porosity was found to be ~83%, which indicated that the sintered regions were not fully dense. PLLA microspheres in the size range of 5-30 μm were made using an oil-in-water emulsion solvent evaporation procedure and they were suitable for the SLS process. A porous scaffold was sintered from the PLLA microspheres with a laser power of 15W and a part bed temperature of 60oC. SEM examination showed that the PLLA microspheres were partially melted to form the scaffold. This study has demonstrated that it is feasible to build tissue engineering scaffolds from small amounts of biomaterials using a commercial SLS machine with suitable modifications.

Selective Laser Sintering of Poly(L-Lactide)/Carbonated Hydroxyapatite Nanocomposite Porous Scaffolds for Bone Tissue Engineering

Wen You Zhou1, Min Wang2, Wai Lam Cheung2,* and Wing Yuk Ip3 1Biomedical and Tissue Engineering Research Group, Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Hong Kong, China 2Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong, China 3Department of Orthopaedics and Traumatology, The University of Hong Kong, Sassoon Road, Hong Kong, China

Isothermal and Non-isothermal Crystallization Kinetics of Poly(L-Lactide)/Carbonated Hydroxyapatite Nanocomposite Microspheres

Medical-profession-accepted and the US Food and Drug Administration (FDA)-approved biodegradable polymers have been used for tissue engineering applications over the last two decades due to their good biocompatibility and acceptable biodegradation properties. Poly(L-lactide) (PLLA) is a linear aliphatic biodegradable polymer and has been widely studied for use as a scaffolding material for human body tissue regeneration (Wei and Ma 2004; Chen, Mak et al. 2006; Wang 2006). The enzymatic and non-enzymatic hydrolysis rate of PLLA strongly depends on its chemical properties (such as molecular weight and weight distribution) and physical properties (such as crystallinity and morphology). Crystallinity plays an important role in the degradation behavior of biodegradable polymers. It is well known that the crystallinity and morphology of semicrystalline polymers such as PLLA are greatly influenced by their thermal history. Therefore, the crystallization kinetics of PLLA should be carefully studied and correlated to its processing method as it forms a basis for the interpretation of the scaffold properties. The isothermal bulk crystallization kinetics of PLLA has been studied by a number of research groups, covering a temperature range from 70 to 165 °C (Marega, Marigo et al. 1992; Iannace and Nicolais 1997; Miyata and Masuko 1998; Di Lorenzo 2005). But only a few studies were conducted on the non-isothermal crystallization kinetics of neat PLLA. Miyata and Masuko (1998) reported that PLLA could not crystallize and remained amorphous when the cooling rate was higher than 10 °C/min. The knowledge on non-isothermal crystallization kinetics is useful for modelling real industrial processes such as cast film extrusion, which generally takes place at a nonconstant cooling rate (Piorkowska, Galeski et al. 2006). Particulate bioceramic reinforced polymer composites can combine the strength and stiffness of bioactive inorganic fillers with the flexibility and toughness of biodegradable organic matrices. Carbonated hydroxyapatite (CHAp) is a desirable bioactive material for bone substitution as it is bioresorbable and also more bioactive in vivo than stoichiometric hydroxyapatite. PLLA/CHAp nanocomposite has been developed and used for constructing bone tissue engineering scaffolds through selective laser sintering (SLS) (Zhou, Lee et al. 2007; Zhou, Lee et al. 2008). In the SLS process, the laser beam selectively fuses

Fabrication and characterization of composite microspheres containing carbonated hydroxyapatite nanoparticles

Nano-sized carbonated hydroxyapatite (CHAp) particles were firstly synthesized using a nanoemulsion method. TEM analyses revealed that as-synthesized nanoparticles were calcium-deficient and spherical in shape (diameter: 16.8±2.6nm). Biocomposite microspheres comprising CHAp nanoparticles and poly(L-lactide) (PLLA) were fabricated using the single emulsion solvent evaporation technique. SEM images showed that composite microspheres were mainly 5-30 μm in size despite the change of CHAp nanoparticle content. When the CHAp nanoparticle content in composite microspheres was below 10 wt%, all nanoparticles were encapsulated within the microspheres which possessed a nanocomposite structure. DSC results showed that the crystallinity of the PLLA matrix of microspheres increased from 38 to 42% when the CHAp nanoparticle content was increased from 0 to 20 wt%. The biocomposite microspheres should be a suitable material for constructing bone tissue engineering scaffolds.

Selective Laser Sintering of Poly(L-Lactide) Porous Scaffolds for Bone Tissue Engineering

The aim of this study was to investigate the feasibility of utilizing selective laser sintering (SLS) to build 3D porous tissue engineering scaffolds from small quantities of poly(L-lactide) (PLLA). PLLA microspheres with suitable particle sizes for the SLS process were produced by the oil-in-water emulsion solvent evaporation technique. A miniature build platform was designed, fabricated and incorporated in an existing Sinterstation® 2000 system to enable small quantities of polymer powder to be used for the production of 3D porous scaffolds. Trial runs were first performed using the DuraForm™ polyamide powder and interfacing problems between the miniature build platform and the existing machine were solved. Then 3D porous scaffolds were successfully built from the PLLA microspheres using the modified SLS machine. This study paved the way for further comprehensive studies on selective laser sintering of tissue engineering scaffolds using expensive biopolymers and their composites.

Selective laser sintered Poly(L-Lactide)/Carbonated hydroxyapatite nanocomposite scaffolds: a bottom-up approach

The main objective of this research is to study the feasibility of using the selective laser sintering (SLS) technology to fabricate 3D porous scaffolds from poly(L-lactide) (PLLA) and poly(L-lactide)/carbonated hydroxyapatite (PLLA/CHAp) nanocomposite for bone tissue engineering applications. There are great demands for tissue engineering (TE) and ideal tissue engineering scaffolds should possess physical, mechanical, chemical and biological properties to fulfill the requirements for tissue regeneration. These properties basically depend on two key factors; namely, material composition and scaffold architecture. To address the first issue, biocomposites seem to be a better choice than single matrix. In this study, a biocomposite, which consists of PLLA microspheres filled with CHAp nanoparticles, is developed. PLLA is chosen because it is an FDA-approved, biocompatible and biodegradable polymer which has been widely used in many biomedical applications. Meanwhile carbonated hydroxyapatite is a promising material for bone substitution as it is bioresorbable and also more bioactive in vivo than stoichiometric hydroxyapatite. In terms of scaffold architecture, modern rapid prototyping (RP) technologies such as stereolithography apparatus (SLA), fused deposition modeling (FDM), 3D printing and SLS offer excellent flexibility. However, materials used for SLA are typically acrylics and epoxies, which are non-biodegradable. At present, only very limited choices of materials are available for FDM because the materials have to be in the form of filament. 3D printing is also limited by the availability of suitable binders to meet the biological and strength requirements of tissue engineering scaffolds. In contrast, SLS has already been used to produce porous poly(ecaprolactone) (PCL) bone tissue engineering scaffolds based on actual model of minipig and human condyle (Partee, Hollister et al. 2006), therefore it has a great potential for tissue engineering scaffold fabrication and has been chosen for this project. However, SLS has been developed primarily for industrial applications. At present, it is not financially viable to process most biopolymers or their composites in commercial SLS machines because the amount of material required is quite substantial and the costs of biopolymers are very high.