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

A dual-layer silk fibroin scaffold for reconstructing the human corneal limbus

Membranes prepared from Bombyx mori silk fibroin have shown potential as a substrate for human limbal epithelial (L-EC) and stromal cell cultivation. Here we present fibroin as a dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. We have compared the growth and phenotype of L-EC on non-porous versus porous fibroin membranes. Furthermore, we have compared the growth of limbal mesenchymal stromal cells (L-MSC) in either serum-supplemented medium or the MesenCult-XF® culture system within fibroin fibrous mats. The co-culture of L-EC and L-MSC in fibroin dual-layer constructs was also examined. L-EC on porous membranes displayed a squamous monolayer; in contrast, L-EC on non-porous fibroin appeared cuboidal and stratified. Both constructs maintained evidence of corneal phenotype (cytokeratin 3/12) and distribution of ΔNp63+ progenitor cells. L-MSC cultivated within fibroin fibrous mats in serum-supplemented medium contained less than 64% of cells expressing the characteristic MSC phenotype of CD73+CD90+CD105+ after two weeks, compared with over 81% in MesenCult-XF® medium. Dual-layer fibroin scaffolds consisting of L-EC and L-MSC maintained a similar phenotype as on the separate layers. These results support the feasibility of a 3D engineered limbus constructed from B. mori silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration.

The stimulation of proliferation and differentiation of periodontal ligament cells by the ionic products from Ca7Si2P2O16 bioceramics

The ultimate goal of periodontal tissue engineering is to produce predictable regeneration of alveolar bone, root cementum, and periodontal ligament, which are lost as a result of periodontal diseases. To achieve this goal, it is of great importance to develop novel bioactive materials which could stimulate the proliferation, differentiation and osteogenic/cementogenic gene expression of periodontal ligament cells (PDLCs) for periodontal regeneration. In this study, we synthesized novel Ca7Si2P2O16 ceramic powders for the first time by the sol–gel method and investigated the biological performance of PDLCs after exposure to different concentrations of Ca7Si2P2O16 extracts. The original extracts were prepared at 200 mg ml-1 and further diluted with serum-free cell culture medium to obtain a series of diluted extracts (100, 50, 25, 12.5 and 6.25 mg ml–1). Proliferation, alkaline phosphatase(ALP) activity, Ca deposition, and osteogenesis/cementogenesis-related gene expression (ALP, Col I, Runx2 and CEMP1) were assayed for PDLCs on days 7 and 14. The results showed that the ionic products from Ca7Si2P2O16 powders significantly stimulated the proliferation, ALP activity, Ca deposition and osteogenesis/cementogenesisrelated gene expression of PDLCs. In addition, it was found that Ca7Si2P2O16 powders had excellent apatite-mineralization ability in simulated body fluids. This study demonstrated that Ca7Si2P2O16 powders with such a specific composition possess the ability to stimulate the PDLC proliferation and osteoblast/cemenoblast-like cell differentiation, indicating that they are a promising bioactive material for periodontal tissue regeneration application.

Engineering of vascularized adipose constructs

Adipose tissue engineering offers a promising alternative to the current surgical techniques for the treatment of soft tissue defects. It is a challenge to find the appropriate scaffold that not only represents a suitable environment for cells but also allows fabrication of customized tissue constructs, particularly in breast surgery. We investigated two different scaffolds for their potential use in adipose tissue regeneration. Sponge-like polyurethane scaffolds were prepared by mold casting with methylal as foaming agent, whereas polycaprolactone scaffolds with highly regular stacked-fiber architecture were fabricated with fused deposition modeling. Both scaffold types were seeded with human adipose tissuederived precursor cells, cultured and implanted in nude mice using a femoral arteriovenous flow-through vessel loop for angiogenesis. In vitro, cells attached to both scaffolds and differentiated into adipocytes. In vivo, angiogenesis and adipose tissue formation were observed throughout both constructs after 2 and 4 weeks, with angiogenesis being comparable in seeded and unseeded constructs. Fibrous tissue formation and adipogenesis were more pronounced on polyurethane foam scaffolds than on polycaprolactone prototyped scaffolds. In conclusion, both scaffold designs can be effectively used for adipose tissue engineering.

Evolutionary design of bone scaffolds with reference to material selection

The favourable scaffold for bone tissue engineering should have desired characteristic features, such as adequate mechanical strength and three-dimensional open porosity, which guarantee a suitable environment for tissue regeneration. In fact, the design of such complex structures like bone scaffolds is a challenge for investigators. One of the aims is to achieve the best possible mechanical strength-degradation rate ratio. In this paper we attempt to use numerical modelling to evaluate material properties for designing bone tissue engineering scaffold fabricated via the fused deposition modelling technique. For our studies the standard genetic algorithm was used, which is an efficient method of discrete optimization. For the fused deposition modelling scaffold, each individual strut is scrutinized for its role in the architecture and structural support it provides for the scaffold, and its contribution to the overall scaffold was studied. The goal of the study was to create a numerical tool that could help to acquire the desired behaviour of tissue engineered scaffolds and our results showed that this could be achieved efficiently by using different materials for individual struts. To represent a great number of ways in which scaffold mechanical function loss could proceed, the exemplary set of different desirable scaffold stiffness loss function was chosen. © 2012 John Wiley & Sons, Ltd.

3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling

Mesenchymal stem/stromal cells (MSC) are rapidly becoming a leading candidate for use in tissue regeneration, with first generation of therapies being approved for use in orthopaedic repair applications. Capturing the full potential of MSC will likely require the development of novel in vitro culture techniques and devices. Herein we describe the development of a straightforward surface modification of an existing commercial product to enable the efficient study of three dimensional (3D) human bone marrow-derived MSC osteogenic differentiation. Hundreds of 3D microaggregates, of either 42 or 168 cells each, were cultured in osteogenic induction medium and their differentiation was compared with that occurring in traditional two dimensional (2D) monolayer cultures. Osteogenic gene expression and matrix composition was significantly enhanced in the 3D microaggregate cultures. Additionally, BMP-2 gene expression was significantly up-regulated in 3D cultures at day 3 and 7 by approximately 25- and 30-fold, respectively. The difference in BMP-2 gene expression between 2D and 3D cultures was negligible in the more mature day 14 osteogenic cultures. These data support the notion that BMP-2 autocrine signalling is up-regulated in 3D MSC cultures, enhancing osteogenic differentiation. This study provides both mechanistic insight into MSC differentiation, as well as a platform for the efficient generation of microtissue units for further investigation or use in tissue engineering applications.

Preparation, characterisation and in vivo osteogenesis of mesoporous bioactive glasses

Bone defects, especially large bone defects, remain a major challenge in orthopaedic surgery. Autologous bone transplantation is considered the most effective treatment, but insufficient donor tissue, coupled with concerns about donor site morbidity, has hindered this approach in large-scale applications. Alternative approaches include implanting biomaterials such as bioactive glass (BG), which has been widely used for bone defect healing, due to having generally good biocompatibility, and can be gradually biodegraded during the process of new bone formation. Mesoporous bioactive glass (MBG) is a newly developed bioactive glass which has been proven to have enhanced in-vitro bioactivity; however the in-vivo osteogenesis has not been studied. A critical problem in using the bone tissue engineering approach to restore large bone defects is that the nutrient supply and cell viability at the centre of the scaffold is severely hampered since the diffusion distance of nutrients and oxygen for cell survival is limited to 150-200µm. Cobalt ions has been shown to mimic hypoxia, which plays a pivotal role in coupling angiogenesis with osteogenesis in-vivo by activating hypoxia inducing factor-1α (HIF-1α) transcription factor, subsequently initiating the expression of genes associated with tissue regeneration. Therefore, one aim of this study is to investigate the in-vivo osteogenesis of MBG by comparison with BG and β-TCP, which are widely used clinically. The other aim is to explore hypoxia-mimicking biomaterials by incorporating Cobalt into MBG and β-TCP. MBG and β-TCP incorporated with 5% cobalt (5Co-MBG and 5CCP) have also been studied in-vivo to determine whether the hypoxic effect has a beneficial effect on the bone formation. The composition and microstructure of synthesised materials (BG, MBG, 5Co-MBG, 5CCP) were characterised, along with the mesopore properties of the MBG materials. Dissolution and cytotoxicity of the Co-containing materials were also investigated. Femoral samples with defects harvested at 4 and 8 weeks were scanned using micro-CT followed by processing for histology (H&E staining) to determine bone formation. Histology of MBG showed a slower rate of bone formation at 4 weeks than BG, however at 8 weeks it could be clearly seen that MBG had more bone formation. The in-vivo results show that the osteogenesis of MBG reciprocates the enhanced performance shown in-vitro compared to BG. Dissolution study showed that Co ions can be efficiently released from MBG and β-TCP in a controllable way. Low amounts of Co incorporated into the MBG and β-TCP showed no significant cytotoxicity and the Co-MBG powders maintained a mesopore structure although not as highly ordered as pure MBG. Preliminary study has shown that Co incorporated samples showed little to no bone formation, instead incurring high lymphocyte activity. Further studies need to be done on Co incorporated materials to determine the cause for high lymphocyte activity in-vivo, which appear to hinder bone formation. In conclusion, this study demonstrated the osteogenic activity of MBG and provided some valuable information of tissue reaction to Co-incorporated MBG and TCP materials.

Preparation, characterization and in vitro angiogenic capacity of cobalt substituted β-tricalcium phosphate ceramics

Divalent cobalt ions (Co2+) have been shown to possess the capacity to induce angiogenesis by activating hypoxia inducible factor-1α (HIF-1α) and subsequently inducing the production of vascular endothelial growth factor (VEGF). However, there are few reports about Co-containing biomaterials for inducing in vitro angiogenesis. The aim of the present work was to prepare Co-containing β-tricalcium phosphate (Co-TCP) ceramics with different contents of calcium substituted by cobalt (0, 2, 5 mol%) and to investigate the effect of Co substitution on their physicochemical and biological properties. Co-TCP powders were synthesized by a chemistry precipitation method and Co-TCP ceramics were prepared by sintering the powder compacts. The effect of Co substitution on phase transition and the sintering property of the β-TCP ceramics was investigated. The proliferation and VEGF expression of human bone marrow mesenchymal stem cells (HBMSCs) cultured with both powder extracts and ceramic discs of Co-TCP was further evaluated. The in vitro angiogenesis was evaluated by the tube-like structure formation of human umbilical vein endothelial cells (HUVECs) cultured on ECMatrix™ in the presence of powder extracts. The results showed that Co substitution suppressed the phase transition from β- to α-TCP. Both the powder extracts and ceramic discs of Co-TCP had generally good cytocompatibility to support HBMSC growth. Importantly, the incorporation of Co into β-TCP greatly stimulated VEGF expression of HBMSCs and Co-TCP showed a significant enhancement of network structure formation of HUVECs compared with pure TCP. Our results suggested that the incorporation of Co into bioceramics is a potential viable way to enhance angiogenic properties of biomaterials. Co-TCP bioceramics may be used for bone tissue regeneration with improved angiogenic capacity.

Preparation, characterization, and in vitro bioactivity of nagelschmidtite bioceramics

The aim of this study is to prepare Ca, P and Si-containing ternary oxide nagelschmidtite (NAGEL, Ca7Si2P2O16) bioceramics and explore their in vitro bioactivity for potential bone tissue regeneration. We prepared dense NAGEL ceramics through high-temperature sintering of NAGEL ceramic powders. The apatite-mineralization ability, dissolution rate, and human osteoblast response (including cytotoxicity analysis, attachment, morphology, proliferation, and bone-related gene expression) to NAGEL ceramics have been systematically studied by comparing with conventional β-tricalcium phosphate (β-TCP) ceramics. The results showed that NAGEL ceramics possessed more obvious apatite mineralization and dissolution (degradation) and stimulated bone-related gene expression (OCN and OPN) of osteoblasts than β-TCP ceramics. NAGEL ceramics also showed no significant cytotoxicity. NAGEL ceramics supported osteoblast attachment, proliferation, and osteogenic gene expression, with a comparable cell proliferation activity with β-TCP ceramics. These results indicate that novel NAGEL bioceramics with the specific composition of Ca7Si2P2O16, are a promising biomaterial for bone tissue regeneration application.

Orthopaedics and Trauma Queensland Annual Report 2011

Orthopaedics and Trauma Queensland, a Centre for Research and Education in Musculoskeletal Disorders, is an internationally recognised research group that is developing into an international leader in research and education. It provides a stimulus for research, education and clinical application within the international orthopaedic and trauma communities. Orthopaedics and Trauma Queensland develops and promotes the innovative use of engineering and technology, in collaboration with surgeons, to provide new techniques, materials, procedures and medical devices. Its integration with clinical practice and strong links with hospitals ensure that the research will be translated into practical outcomes for patients. The group undertakes clinical practice in orthopaedics and trauma and applies core engineering skills to challenges in medicine. The research is built on a strong foundation of knowledge in biomedical engineering, and incorporates expertise in cell biology, mathematical modelling, human anatomy and physiology and clinical medicine in orthopaedics and trauma. New knowledge is being developed and applied to the full range of orthopaedic diseases and injuries, such as knee and hip replacements, fractures and spinal deformities.

Experimental investigation of the governing parameters in the electrospinning of poly(3-hydroxybutyrate) scaffolds : structural characteristics of the pores

We sought to determine the impact of electrospinning parameters on a trustworthy criterion that could evidently improve the maximum applicability of fibrous scaffolds for tissue regeneration. We used an image analysis technique to elucidate the web permeability index (WPI) by modeling the formation of electrospun scaffolds. Poly(3-hydroxybutyrate) (P3HB) scaffolds were fabricated according to predetermined conditions of levels in a Taguchi orthogonal design. The material parameters were the polymer concentration, conductivity, and volatility of the solution. The processing parameters were the applied voltage and nozzle-to-collector distance. With a law to monitor the WPI values when the polymer concentration or the applied voltage was increased, the pore interconnectivity was decreased. The quality of the jet instability altered the pore numbers, areas, and other structural characteristics, all of which determined the scaffold porosity and aperture interconnectivity. An initial drastic increase was observed in the WPI values because of the chain entanglement phenomenon above a 6 wt % P3HB content. Although the solution mixture significantly (p < 0.05) changed the scaffold architectural characteristics as a function of the solution viscosity and surface tension, it had a minor impact on the WPI values. The solution mixture gained the third place of significance, and the distance was approved as the least important factor.

Electrospraying of polymers with therapeutic molecules : state of the art

The encapsulation and release of bioactive molecules from polymeric vehicles represents the holy grail of drug and growth factor delivery therapies, whereby sustained and controlled release is crucial in eliciting a positive therapeutic effect. To this end, electrospraying is rapidly emerging as a popular technology for the production of polymeric particles containing bioactive molecules. Compared with traditional emulsion fabrication techniques, electrospraying has the potential to reduce denaturation of protein drugs and affords tighter regulation over particle size distribution and morphology. In this article, we review the importance of the electrospraying parameters that enable reproducible tailoring of the particles' physical and in vitro drug release characteristics, along with discussion of existing in vivo data. Controlled morphology and monodispersity of particles can be achieved with electrospraying, with high encapsulation efficiencies and without unfavorable denaturation of bioactive molecules throughout the process. Finally, the combination of electrospraying with electrospun scaffolds, with an emphasis on tissue regeneration is reviewed, depicting a technique in its relative infancy but holding great promise for the future of regenerative medicine.

Mussel-inspired bioceramics with self-assembled Ca-P/polydopamine composite nanolayer : preparation, formation mechanism, improved cellular bioactivity and osteogenic differentiation of bone marrow stromal cells

The nanostructured surface of biomaterials plays an important role in improving their in vitro cellular bioactivity as well as stimulating in vivo tissue regeneration. Inspired by the mussel’s adhesive versatility, which is thought to be due to the plaque–substrate interface being rich in 3,4-dihydroxy-L-phenylalamine (DOPA) and lysine amino acids, in this study we developed a self-assembly method to prepare a uniform calcium phosphate (Ca-P)/polydopamine composite nanolayer on the surface of b-tricalcium phosphate (b-TCP) bioceramics by soaking b-TCP bioceramics in Tris–dopamine solution. It was found that the addition of dopamine, reaction temperature and reaction time are three key factors inducing the formation of a uniform Ca-P/polydopamine composite nanolayer. The formation mechanism of a Ca-P/polydopamine composite nanolayer involved two important steps: (i) the addition of dopamine to Tris–HCl solution decreases the pH value and accelerates Ca and P ionic dissolution from the crystal boundaries of b-TCP ceramics; (ii) dopamine is polymerized to form self-assembled polydopamine film and, at the same time, nanosized Ca-P particles are mineralized with the assistance of polydopamine, in which the formation of polydopamine occurs simultaneously with Ca-P mineralization (formation of nanosized microparticles composed of calcium phosphate-based materials), and finally a self-assembled Ca-P/polydopamine composite nanolayer forms on the surface of the b-TCP ceramics. Furthermore, the formed self-assembled Ca-P/polydopamine composite nanolayer significantly enhances the surface roughness and hydrophilicity of b-TCP ceramics, and stimulates the attachment, proliferation, alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, COL1 and Runx2) of human bone marrow stromal cells. Our results suggest that the preparation of self-assembled Ca-P/polydopamine composite nanolayers is a viable method to modify the surface of biomaterials by significantly improving their surface physicochemical properties and cellular bioactivity for bone regeneration application.

Flapless dental implant surgery : a retrospective study of 1,241 consecutive implants

Purpose: The purpose of this study was to identify retrospectively the predictors of implant survival when the flapless protocol was used in two private dental practices. Materials and Methods: The collected data were initially computer searched to identify the patients; later, a hand search of patient records was carried out to identify all flapless implants consecutively inserted over the last 10 years. The demographic information gathered on statistical predictors included age, sex, periodontal and peri-implantitis status, smoking, details of implants inserted, implant locations, placement time after extraction, use of simultaneous guided hard and soft tissue regeneration procedures, loading protocols, type of prosthesis, and treatment outcomes (implant survival and complications). Excluded were any implants that required flaps or simultaneous guided hard and soft tissue regeneration procedures, and implants narrower than 3.25 mm. Results: A total of 1,241 implants had been placed in 472 patients. Life table analysis indicated cumulative 5-year and 10-year implant survival rates of 97.9% and 96.5%, respectively. Most of the failed implants occurred in the posterior maxilla (54%) in type 4 bone (74.0%), and 55.0% of failed implants had been placed in smokers. Conclusion: Flapless dental implant surgery can yield an implant survival rate comparable to that reported in other studies using traditional flap techniques.

A stimulatory effect of Ca3ZrSi2O9 bioceramics on cementogenic/osteogenic differentiation of periodontal ligament cells

The regeneration of periodontal tissues to cure periodontitis remains a medical challenge. Therefore, it is of great importance to develop a novel biomaterial that could induce cementogenesis and osteogenesis in periodontal tissue engineering. Calcium silicate (Ca–Si) based ceramics have been found to be potential bioactive materials due to their osteostimulatory effect. Recently, it is reported that zirconium modified calcium-silicate-based (Ca3ZrSi2O9) ceramics stimulate cell proliferation and osteogenic differentiation of osteoblasts. However, it is unknown whether Ca3ZrSi2O9 ceramics possess specific cementogenic stimulation for human periodontal ligament cells (hPDLCs) in periodontal tissue regeneration in vitro. The purpose of this study was to investigate whether Ca3ZrSi2O9 ceramic disks and their ionic extracts could stimulate cell growth and cementogenic/osteogenic differentiation of hPDLCs; the possible molecular mechanism involved in this process was also explored by investigating the Wnt/β-catenin signalling pathway of hPDLCs. Our results showed that Ca3ZrSi2O9 ceramic disks supported cell adhesion, proliferation and significantly up-regulated relative alkaline phosphatase (ALP) activity, cementogenic/osteogenic gene expression (CEMP1, CAP, ALP and OPN) and Wnt/β-catenin signalling pathway-related genes (AXIN2 and CTNNB) for hPDLCs, compared to that of β-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic extracts from Ca3ZrSi2O9 powders also significantly enhanced relative ALP activity, cementogenic/osteogenic and Wnt/β-catenin-related gene expression of hPDLCs. The present results demonstrate that Ca3ZrSi2O9 ceramics are capable of stimulating cementogenic/osteogenic differentiation of hPDLCs possibly via activation of the Wnt/β-catenin signalling pathway, suggesting that Ca3ZrSi2O9 ceramics have the potential to be used for periodontal tissue regeneration.