Response of cells on surface-induced nanopatterns: Fibroblasts and mesenchymal progenitor cells

Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns

Tissue engineered prefabricated vascularized flaps

BACKGROUND.: Microvascular free tissue transfer has become increasingly popular in the reconstruction of head and neck defects, but it also has its disadvantages. Tissue engineering allows the generation of neo-tissue for implantation, but these tissues are often avascular. We propose to combine tissue-engineering techniques together with flap prefabrication techniques to generate a prefabricated vascularized soft tissue flap. METHODS: Human dermal fibroblasts (HDFs) labeled with fluorescein diacetate were static seeded onto polylactic-co-glycolic acid-collagen (PLGA-c) mesh. Controls were plain PLGA-c mesh. The femoral artery and vein of the nude rat was ligated and used as a vascular carrier for the constructs. After 4 weeks of implantation, the constructs were assessed by gross morphology, routine histology, Masson trichrome, and cell viability determined by green fluorescence. RESULTS: All the constructs maintained their initial shape and dimensions. Angiogenesis was evident in all the constructs with neo-capillary formation within the PLGA-c mesh seen. HDFs proliferated and filled the interyarn spaces of the PLGA-c mesh, while unseeded PLGA-c mesh remained relatively acellular. Cell tracer study indicated that the seeded HDFs remained viable and closely associated to remaining PLGA-c fibers. Collagen formation was more abundant in the constructs seeded with HDFs. CONCLUSIONS: PLGA-c, enveloped by a cell sheet composed of fibroblasts, can serve as a suitable scaffold for generation of a soft tissue flap. A ligated arteriovenous pedicle can serve as a vascular carrier for the generation of a tissue engineered vascularized flap.

Highly efficient, stoichiometric radical exchange reactions using isoindoline profluorescent nitroxides

Exchange reactions between the isoindoline profluorescent nitroxide 1,1,3,3-tetramethyldibenzo[e,g]isoindolin-2-yloxyl (TMDBIO) and a TEMPO capped polystyrene were carried out. High conversions to the desired products were achieved using only stoichiometric ratios of nitroxide relative to polymer. The scope of this study was expanded by exploiting a di-nitroxide 9,10-bis(5-[1,1,3,3-tetramethylisoindolin-2-yloxy])anthracene (BTMIOA) as a connector between two polymer chains forming PS–nitroxide–PS systems.

Methacrylate-functionalized oligomers based on lactide, E-caprolactone and trimethylene carbonate for application in stereo-lithography

Photo-curable biodegradable macromers were prepared by ring opening polymerization of D,L-lactide (DLLA), (similar to)-caprolactone (CL) and 1,3-trimethylene carbonate (TMC) in the presence of glycerol or sorbitol as initiator and stannous octoate as catalyst, and subsequent methacrylation of the terminal hydroxyl groups. These methacrylated macromers, ranging in molecular weight from approximately 700 to 6000 g/mol, were cross-linked using ultraviolet (UV) light to form biodegradable networks. Homogeneous networks with high gel contents were prepared. One of the resins based on PTMC was used to prepare three-dimensional structures by stereo-lithography using a commercially available apparatus.

Differences between in vitro viability and differentiation and in vivo bone-forming efficacy of human mesenchymal stem cells cultured on PCL-TCP scaffolds

Human mesenchymal stem cells (hMSCs) possess great therapeutic potential for the treatment of bone disease and fracture non-union. Too often however, in vitro evidence alone of the interaction between hMSCs and the biomaterial of choice is used as justification for continued development of the material into the clinic. Clearly for hMSC-based regenerative medicine to be successful for the treatment of orthopaedic trauma, it is crucial to transplant hMSCs with a suitable carrier that facilitates their survival, optimal proliferation and osteogenic differentiation in vitro and in vivo. This motivated us to evaluate the use of polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds produced by fused deposition modeling for the delivery of hMSCs. When hMSCs were cultured on the PCL-TCP scaffolds and imaged by a combination of phase contrast, scanning electron and confocal laser microscopy, we observed five distinct stages of colonization over a 21-day period that were characterized by cell attachment, spreading, cellular bridging, the formation of a dense cellular mass and the accumulation of a mineralized extracellular matrix when induced with osteogenic stimulants. Having established that PCL-TCP scaffolds are able to support hMSC proliferation and osteogenic differentiation, we next tested the in vivo efficacy of hMSC-loaded PCL-TCP scaffolds in nude rat critical-sized femoral defects. We found that fluorescently labeled hMSCs survived in the defect site for up to 3 weeks post-transplantation. However, only 50% of the femoral defects treated with hMSCs responded favorably as determined by new bone volume. As such, we show that verification of hMSC viability and differentiation in vitro is not sufficient to predict the efficacy of transplanted stem cells to consistently promote bone formation in orthotopic defects in vivo.

Low frequency of CHEK2 1100delC allele in Australian multiple-case breast cancer families : functional analysis in heterozygous individuals

A protein-truncating variant of CHEK2, 1100delC, is associated with a moderate increase in breast cancer risk. We have determined the prevalence of this allele in index cases from 300 Australian multiple-case breast cancer families, 95% of which had been found to be negative for mutations in BRCA1 and BRCA2. Only two (0.6%) index cases heterozygous for the CHEK2 mutation were identified. All available relatives in these two families were genotyped, but there was no evidence of co-segregation between the CHEK2 variant and breast cancer. Lymphoblastoid cell lines established from a heterozygous carrier contained approximately 20% of the CHEK2 1100delC mRNA relative to wild-type CHEK2 transcript. However, no truncated CHK2 protein was detectable. Analyses of expression and phosphorylation of wild-type CHK2 suggest that the variant is likely to act by haploinsufficiency. Analysis of CDC25A degradation, a downstream target of CHK2, suggests that some compensation occurs to allow normal degradation of CDC25A. Such compensation of the 1100delC defect in CHEK2 might explain the rather low breast cancer risk associated with the CHEK2 variant, compared to that associated with truncating mutations in BRCA1 or BRCA2.

A Modified inverse integer Cholesky decorrelation method and the performance on ambiguity resolution

One of the research focuses in the integer least squares problem is the decorrelation technique to reduce the number of integer parameter search candidates and improve the efficiency of the integer parameter search method. It remains as a challenging issue for determining carrier phase ambiguities and plays a critical role in the future of GNSS high precise positioning area. Currently, there are three main decorrelation techniques being employed: the integer Gaussian decorrelation, the Lenstra–Lenstra–Lovász (LLL) algorithm and the inverse integer Cholesky decorrelation (IICD) method. Although the performance of these three state-of-the-art methods have been proved and demonstrated, there is still a potential for further improvements. To measure the performance of decorrelation techniques, the condition number is usually used as the criterion. Additionally, the number of grid points in the search space can be directly utilized as a performance measure as it denotes the size of search space. However, a smaller initial volume of the search ellipsoid does not always represent a smaller number of candidates. This research has proposed a modified inverse integer Cholesky decorrelation (MIICD) method which improves the decorrelation performance over the other three techniques. The decorrelation performance of these methods was evaluated based on the condition number of the decorrelation matrix, the number of search candidates and the initial volume of search space. Additionally, the success rate of decorrelated ambiguities was calculated for all different methods to investigate the performance of ambiguity validation. The performance of different decorrelation methods was tested and compared using both simulation and real data. The simulation experiment scenarios employ the isotropic probabilistic model using a predetermined eigenvalue and without any geometry or weighting system constraints. MIICD method outperformed other three methods with conditioning improvements over LAMBDA method by 78.33% and 81.67% without and with eigenvalue constraint respectively. The real data experiment scenarios involve both the single constellation system case and dual constellations system case. Experimental results demonstrate that by comparing with LAMBDA, MIICD method can significantly improve the efficiency of reducing the condition number by 78.65% and 97.78% in the case of single constellation and dual constellations respectively. It also shows improvements in the number of search candidate points by 98.92% and 100% in single constellation case and dual constellations case.

Adhesion and dispersion of salmeterol xinofoate from lactose interactive mixtures for inhalation - can surface roughness of carriers determine performance

The objective was to understand the influence of the surface roughness of lactose carriers on the adhesion and dispersion of salmeterol xinafoate (SX) from interactive mixtures. The surface roughness of lactose carriers was determined by confocal microscopy. Particle images and adhesion forces between SX and lactose particles were determined by Atomic Force Microscopy. The dispersion of SX (2.5%) from interactive mixtures with lactose was determined using a twin-stage impinger (TSI) with a Rotahaler® at an airflow rate of 60L/min. SX was analysed using a validated HPLC assay. The RMS Rq of lactose carriers ranged from 0.93-2.84μm, the Fine Particle Fraction (FPF) of SX ranged between 4 and 24 percent and average adhesion force between a SX and lactose particles ranged between 49 and 134 nN. No direct correlation was observed between the RMS Rq of lactose carriers and either the FPF of SX for the interactive mixtures or the adhesion force of a SX on the lactose particles; however, the presence of fine lactose associated with the carrier surface increased the FPF of SX. Dispersion through direct SX detachment from the carrier surface was not consistent with the poor correlations described and was more likely to occur through complex particulate interactions involving fine lactose.

Novel synthetic bio-mimic polymers for potential cell delivery therapy

Cell-based therapy is one of the major potential therapeutic strategies for cardiovascular, neuronal and degenerative diseases in recent years. Synthetic biodegradable polymers have been utilized increasingly in pharmaceutical, medical and biomedical engineering. Control of the interaction of living cells and biomaterials surfaces is one of the major goals in the design and development of new polymeric biomaterials in tissue engineering. The aims of this study is to develop a novel bio-mimic polymeric materials which will facilitate the delivery cells, control cell bioactivities and enhance the focal integration of graft cells with host tissues.

A comparative study of mesoporous-glass/silk and non-mesoporous-glass/silk scaffolds : physiochemistry and in vivo osteogenesis

Mesoporous bioactive glass (MBG) is a new class of biomaterials with a well-ordered nanochannel structure, whose in vitro bioactivity is far superior than that of non-mesoporous bioactive glass (BG); the material's in vivo osteogenic properties are, however, yet to be assessed. Porous silk scaffolds have been used for bone tissue engineering, but this material's osteoconductivity is far from optimal. The aims of this study were to incorporate MBG into silk scaffolds in order to improve their osteoconductivity and then to compare the effect of MBG and BG on the in vivo osteogenesis of silk scaffolds. MBG/silk and BG/silk scaffolds with a highly porous structure were prepared by a freeze-drying method. The mechanical strength, in vitro apatite mineralization, silicon ion release and pH stability of the composite scaffolds were assessed. The scaffolds were implanted into calvarial defects in SCID mice and the degree of in vivo osteogenesis was evaluated by microcomputed tomography (μCT), hematoxylin and eosin (H&E) and immunohistochemistry (type I collagen) analyses. The results showed that MBG/silk scaffolds have better physiochemical properties (mechanical strength, in vitro apatite mineralization, Si ion release and pH stability) compared to BG/silk scaffolds. MBG and BG both improved the in vivo osteogenesis of silk scaffolds. μCT and H&E analyses showed that MBG/silk scaffolds induced a slightly higher rate of new bone formation in the defects than did BG/silk scaffolds and immunohistochemical analysis showed greater synthesis of type I collagen in MBG/silk scaffolds compared to BG/silk scaffolds.

Bioactive SrO–SiO2 glass with well-ordered mesopores : characterization, physiochemistry and biological properties

For a biomaterial to be considered suitable for bone repair it should ideally be both bioactive and have a capacity for controllable drug delivery; as such, mesoporous SiO2 glass has been proposed as a new class of bone regeneration material by virtue of its high drug-loading ability and generally good biocompatibility. It does, however, have less than optimum bioactivity and controllable drug delivery properties. In this study, we incorporated strontium (Sr) into mesoporous SiO2 in an effort to develop a bioactive mesoporous SrO–SiO2 (Sr–Si) glass with the capacity to deliver Sr2+ ions, as well as a drug, at a controlled rate, thereby producing a material better suited for bone repair. The effects of Sr2+ on the structure, physiochemistry, drug delivery and biological properties of mesoporous Sr–Si glass were investigated. The prepared mesoporous Sr–Si glass was found to have an excellent release profile of bioactive Sr2+ ions and dexamethasone, and the incorporation of Sr2+ improved structural properties, such as mesopore size, pore volume and specific surface area, as well as rate of dissolution and protein adsorption. The mesoporous Sr–Si glass had no cytotoxic effects and its release of Sr2+ and SiO44− ions enhanced alkaline phosphatase activity – a marker of osteogenic cell differentiation – in human bone mesenchymal stem cells. Mesoporous Sr–Si glasses can be prepared to porous scaffolds which show a more sustained drug release. This study suggests that incorporating Sr2+ into mesoporous SiO2 glass produces a material with a more optimal drug delivery profile coupled with improved bioactivity, making it an excellent material for bone repair applications. Keywords: Mesoporous Sr–Si glass; Drug delivery; Bioactivity; Bone repair; Scaffolds

Three-dimensional printing of hierarchical and tough mesoporous bioactive glass scaffolds with a controllable pore architecture, excellent mechanical strength and mineralization ability

New-generation biomaterials for bone regenerations should be highly bioactive, resorbable and mechanically strong. Mesoporous bioactive glass (MBG), as a novel bioactive material, has been used for the study of bone regeneration due to its excellent bioactivity, degradation and drug-delivery ability; however, how to construct a 3D MBG scaffold (including other bioactive inorganic scaffolds) for bone regeneration still maintains a significant challenge due to its/their inherit brittleness and low strength. In this brief communication, we reported a new facile method to prepare hierarchical and multifunctional MBG scaffolds with controllable pore architecture, excellent mechanical strength and mineralization ability for bone regeneration application by a modified 3D-printing technique using polyvinylalcohol (PVA), as a binder. The method provides a new way to solve the commonly existing issues for inorganic scaffold materials, for example, uncontrollable pore architecture, low strength, high brittleness and the requirement for the second sintering at high temperature. The obtained 3D-printing MBG scaffolds possess a high mechanical strength which is about 200 times for that of traditional polyurethane foam template-resulted MBG scaffolds. They have highly controllable pore architecture, excellent apatite-mineralization ability and sustained drug-delivery property. Our study indicates that the 3D-printed MBG scaffolds may be an excellent candidate for bone regeneration.

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

Adhesion of perichondrial cells to a polylactic acid scaffold

The number of chondrogenic cells available locally is an, important factor in the repair process for cartilage defects. Previous studies demonstrated that the number of transplanted rabbit perichondrial cells (PC) remaining in a cartilage defect in vivo, after being carried into the site in a polylactic acid (PLA) scaffold, declined markedly within two days. This study examined the ability of in vitro culture of PC/PLA constructs to enhance subsequent biomechanical stability of the cells and the matrix content in an in vitro screening assay. PC/PLA constructs were analyzed after 1 h, 1 and 2 weeks of culture. The biomechanical adherence of PC to the PLA scaffold was tested by subjecting the PC/PLA constructs to a range of flow velocities (0.25-25 mm/s), spanning the range estimated to occur under conditions of construct insertion in vivo. The adhesion of PC to the PLA carrier was increased significantly by 1 and 2 weeks of incubation, with 25 mm/s flow causing a 57% detachment of cells after 1 h of seeding, but only 7% and 16% after I and 2 weeks of culture, respectively (p < 0.001). This adherence was associated with marked deposition of glycosaminoglycan and collagen. These findings suggest that pre-incubation of PC-laden PLA scaffolds markedly enhances the stability of the indwelling cells. (C) 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.