Strategy in developing cell based therapy for large bone

Regeneration of osseous defects by tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. The concept of in vitro cultured osteoblasts having an ability to induce new bone formation has been demonstrated in the critical size defects using small animal models. The bone derived cells can be incorporated into bioengineered scaffolds and synthesize bone matrix, which on implantation can induce new bone formation. In search of optimal cell delivery materials, the extracellular matrix as cell carriers for the repair and regeneration of tissues is receiving increased attention. We have investigated extracellular matrix formed by osteoblasts in vitro as a scaffold for osteoblasts transplantation and found a mineralized matrix, formed by human osteoblasts in vitro, can initiate bone formation by activating endogenous mesenchymal cells. To repair the large bone defects, osteogenic or stem cells need to be prefabricated in a large three dimensional scaffold usually made of synthetic biomaterials, which have inadequate interaction with cells and lead to in vivo foreign body reactions. The interstitial extracellular matrix has been applied to modify biomaterials surface and identified vitronectin, which binds the heparin domain and RGD (Arg-Gly-Asp) sequence can modulate cell spreading, migration and matrix formation on biomaterials. We also synthesized a tri-block copolymer, methoxy-terminated poly(ethylene glycol)(MPEG)-polyL-lactide(PLLA)-polylysine(PLL) for human osteoblasts delivery. We identified osteogenic activity can be regulated by the molecular weight and composition of the triblock copolymers. Due to the sequential loss of lineage differentiation potential during the culture of bone marrow stromal cells that hinderers their potential clinical application, we have developed a clonal culture system and established several stem cell clones with fast growing and multi-differentiation properties. Using proteomics and subtractive immunization, several differential proteins have been identified and verified their potential application in stem cell characterization and tissue regeneration

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.

In vitro and in vivo evaluation of adenovirus combined silk fibroin scaffolds for BMP-7 gene delivery

Introduction and aims: For a scaffold material to be considered effective and efficient for tissue engineering it must be biocompatible as well as bioinductive. Silk fiber is a natural biocompatible material suitable for scaffold fabrication; however, silk is tissue-conductive and lacks tissue-inductive properties. One proposed method to make the scaffold tissue-inductive is to introduce plasmids or viruses encoding a specific growth factor into the scaffold. In this study, we constructed adenoviruses encoding bone morphogenetic protein-7 (BMP-7) and incorporated these into silk scaffolds. The osteo-inductive and new bone formation properties of these constructs were assessed in vivo in a critical-sized skull defect animal model. Materials and methods: Silk fibroin scaffolds containing adenovirus particles coding BMP-7 were prepared. The release of the adenovirus particles from the scaffolds was quantified by tissue-culture infective dose (TCID50) and the bioactivity of the released viruses was evaluated on human bone marrow mesenchymal stromal cells (BMSCs). To demonstrate the in vivo bone forming ability of the virus-carrying silk fibroin scaffold, the scaffold constructs were implanted into calvarial defects in SCID mice. Results: In vitro studies demonstrated that the virus-carrying silk fibroin scaffold released virus particles over a 3 week period while preserving their bioactivity. In vivo test of the scaffold constructs in critical-sized skull defect areas revealed that silk scaffolds were capable of delivering the adenovirus encoding BMP-7, resulting significantly enhanced new bone formation. Conclusions: Silk scaffolds carrying BMP-7 encoding adenoviruses can effectively transfect cells and enhance both in vitro and in vivo osteogenesis. The findings of this study indicate silk fibroin is a promising biomaterial for gene delivery to repair critical-sized bone defects.

Die Geographie der Kreativen Klasse in Deutschland [The geography of the Creative Class in Germany]

We analyze the regional distribution of different categories of creative individuals in Germany. Generally, the share of creative people is higher in cities as compared to the rural area The freelancing artists are a kind of exception in this respect; they constitute a relatively high share of the population in some rural area A high share of creative people in a region can be explained by a high level of public provisions and a high share of foreign born population, which can be regarded as an indicator of the “openness” in the local milieu. Good employment opportunities have only a relatively weak impact. Regions with a high share of creatives tend to have an above average level of new business formation, a high level of innovation and a relatively high share of employees in high-tech industries.

Investigation of the effect of organics on the water uptake of marine aerosols

Water uptake refers to the ability of atmospheric particles to take up water vapour from the surrounding atmosphere. This is an important property that affects particle size and phase and therefore influences many characteristics of aerosols relevant to air quality and climate. However, the water uptake properties of many important atmospheric aerosol systems, including those related to the oceans, are still not fully understood. Therefore, the primary aim of this PhD research program was to investigate the water uptake properties of marine aerosols. In particular, the effect of organics on marine aerosol water uptake was investigated. Field campaigns were conducted at remote coastal sites on the east coast of Australia (Agnes Water; March-April 2007) and west coast of Ireland (Mace Head; June 2007), and laboratory measurements were performed on bubble-generated sea spray aerosols. A combined Volatility-Hygroscopicity-Tandem Differential Mobility Analyser (VH-TDMA) was employed in all experiments. This system probes the changes in the hygroscopic properties of nanoparticles as volatile organic components are progressively evaporated. It also allows particle composition to be inferred from combined volatility-hygroscopicity measurements. Frequent new particle formation and growth events were observed during the Agnes Water campaign. The VH-TDMA was used to investigate freshly nucleated particles (17-22.5 nm) and it was found that the condensation of sulphate and/or organic vapours was responsible for driving particle growth during the events. Aitken mode particles (~40 nm) were also measured with the VH-TDMA. In 3 out of 18 VH-TDMA scans evaporation of a volatile, organic component caused a very large increase in hygroscopicity that could only be explained by an increase in the absolute water uptake of the particle residuals, and not merely an increase in their relative hygroscopicity. This indicated the presence of organic components that were suppressing the hygroscopic growth of mixed particles on the timescale of humidification in the VH-TDMA (6.5 secs). It was suggested that the suppression of water uptake was caused by either a reduced rate of hygroscopic growth due to the presence of organic films, or organic-inorganic interactions in solution droplets that had a negative effect on hygroscopicity. Mixed organic-inorganic particles were rarely observed by the VH-TDMA during the summer campaign conducted at Mace Head. The majority of particles below 100 nm in clean, marine air appeared to be sulphates neutralised to varying degrees by ammonia. On one unique day, 26 June 2007, particularly large concentrations of sulphate aerosol were observed and identified as volcanic emissions from Iceland. The degree of neutralisation of the sulphate aerosol by ammonia was calculated by the VH-TDMA and found to compare well with the same quantity measured by an aerosol mass spectrometer. This was an important verification of the VH-TMDA‘s ability to identify ammoniated sulphate aerosols based on the simultaneous measurement of aerosol volatility and hygroscopicity. A series of measurements were also conducted on sea spray aerosols generated from Moreton Bay seawater samples in a laboratory-based bubble chamber. Accumulation mode sea spray particles (38-173 nm) were found to contain only a minor organic fraction (< 10%) that had little effect on particle hygroscopicity. These results are important because previous studies have observed that accumulation mode sea spray particles are predominantly organic (~80% organic mass fraction). The work presented here suggests that this is not always the case, and that there may be currently unknown factors that are controlling the transfer of organics to the aerosol phase during the bubble bursting process. Taken together, the results of this research program have significantly improved our understanding of organic-containing marine aerosols and the way they interact with water vapour in the atmosphere.

Genetic evidence for the vital function of osterix in cementogenesis

To date, attempts to regenerate a complete tooth, including the critical periodontal tissues associated with the tooth root, have not been successful. Controversy still exists regarding the origin of the cell source for cellular cementum (epithelial or mesenchymal). This disagreement may be partially due to a lack of understanding of the events leading to the initiation and development of the tooth roots and supportive tissues, such as the cementum. Osterix (OSX) is a transcriptional factor essential for osteogenesis, but its role in cementogenesis has not been addressed. In the present study, we first documented a close relationship between the temporal- and spatial-expression pattern of OSX and the formation of cellular cementum. We then generated 3.6 Col 1-OSX transgenic mice, which displayed accelerated cementum formation vs. WT controls. Importantly, the conditional deletion of OSX in the mesenchymal cells with two different Cre systems (the 2.3 kb Col 1 and an inducible CAG-CreER) led to a sharp reduction in cellular cementum formation (including the cementum mass and mineral deposition rate) and gene expression of dentin matrix protein 1 (DMP1) by cementocytes. However, the deletion of the OSX gene after cellular cementum formed did not alter the properties of the mature cementum as evaluated by backscattered SEM and resin-cast SEM. Transient transfection of Osx in the cementoblasts in vitro significantly inhibited cell proliferation and increased cell differentiation and mineralization. Taken together, these data support 1) the mesenchymal origin of cellular cementum (from PDL progenitor cells); 2) the vital role of OSX in controlling the formation of cellular cementum; and 3) the limited remodeling of cellular cementum in adult mice.

Influence of medium range transport of particles from nucleation burst on particle number concentration within the urban airshed

An elevated particle number concentration (PNC) observed during nucleation events could play a significant contribution to the total particle load and therefore to the air pollution in the urban environments. Therefore, a field measurement study of PNC was commenced to investigate the temporal and spatial variations of PNC within the urban airshed of Brisbane, Australia. PNC was monitored at urban (QUT), roadside (WOO) and semi-urban (ROC) areas around the Brisbane region during 2009. During the morning traffic peak period, the highest relative fraction of PNC reached about 5% at QUT and WOO on weekdays. PNC peaks were observed around noon, which correlated with the highest solar radiation levels at all three stations, thus suggesting that high PNC levels were likely to be associated with new particle formation caused by photochemical reactions. Wind rose plots showed relatively higher PNC for the NE direction, which was associated with industrial pollution, accounting for 12%, 9% and 14% of overall PNC at QUT, WOO and ROC, respectively. Although there was no significant correlation between PNC at each station, the variation of PNC was well correlated among three stations during regional nucleation events. In addition, PNC at ROC was significantly influenced by upwind urban pollution during the nucleation burst events, with the average enrichment factor of 15.4. This study provides an insight into the influence of regional nucleation events on PNC in the Brisbane region and it the first study to quantify the effect of urban pollution on semi-urban PNC through the nucleation events. © 2012 Author(s).

Vertical particle concentration profiles around urban office buildings

Despite its role in determining both indoor and outdoor human exposure to anthropogenic particles, there is limited information describing vertical profiles of particle concentrations in urban environments, especially for ultrafine particles. Furthermore, the results of the few studies performed have been inconsistent. As such, this study aimed to assess the influence of vehicle emissions and nucleation formation on particle characteristics (particle number size distribution-PNSD and PM 2.5 concentration) at different heights around three urban office buildings located next to busy roads in Brisbane, Australia, and place these results in the broader context of the existing literature. Two sets of instruments were used to simultaneously measure PNSD, particle number (PN) and PM 2.5 concentrations, respectively, for up to three weeks at each building. The results showed that both PNSD and PM 2.5 concentration around building envelopes were influenced by vehicle emissions and new particle formation, and that they exhibited variability across the three different office buildings. During nucleation events, PN concentration in size range of <30 nm and total PN concentration increased (7-65% and 5-46%, respectively), while PM 2.5 concentration decreased (36-52%) with height. This study has shown an under acknowledged role for nucleation in producing particles that can affect large numbers of people, due to the high density and occupancy of urban office buildings and the fact that the vast majority of people's time is spent indoors. These findings highlight important new information related to the previously overlooked role of particle formation in the urban atmosphere and its potential effects on selection of air intake locations and appropriate filter types when designing or upgrading mechanical ventilation systems in urban office buildings. The results also serve to better define particle behaviour and variability around building envelopes, which has implications for studies of both human exposure and particle dynamics. © 2012 Author(s).

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.

Implantation of osteogenic differentiated donor mesenchymal stem cells causes recruitment of host cells

The interaction between host and donor cells is believed to play an important role in osteogenesis. However, it is still unclear how donor osteogenic cells behave and interact with host cells in vivo. The purpose of this study was to track the interactions between transplanted osteogenic cells and host cells during osteogenesis. In vitro migration assay was carried out to investigate the ability of osteogenic differentiated humanmesenchymal stemcells (O-hMSCs) to recruit MSCs. At the in vivo level, O-hMSCs were implanted subcutaneously or into skull defects in severe combined immunodeficient (SCID) mice. New bone formation was observed bymicro-CT and histological procedures. In situ hybridization (ISH) against human Alu sequences was performed to distinguish donor osteogenic cells from host cells. In vitro migration assay revealed an increased migration potential of MSCs by co-culturing with O-hMSCs. In agreement with the results of in vitro studies, ISH against human Alu sequences showed that host mouse MSCs migrated in large numbers into the transplantation site in response to O-hMSCs. Interestingly, host cells recruited by O-hMSCs were the major cell populations in newly formed bone tissues, indicating that O-hMSCs can trigger and initiate osteogenesis when transplanted in orthotopic sites. The observations fromthis study demonstrated that in vitro induced O-hMSCs were able to attract hostMSCs in vivo andwere involved inosteogenesis togetherwith host cells,whichmay be of importance for bone tissue-engineering applications.

Identification of bone morphogenetic proteins 2 and 4 in commercial demineralized freeze-dried bone allograft preparations : pilot study

BACKGROUND: Demineralized freeze-dried bone allografts (DFDBAs) have been proposed as a useful adjunct in periodontal therapy to induce periodontal regeneration through the induction of new bone formation. The presence of bone morphogenetic proteins (BMPs) within the demineralized matrix has been proposed as a possible mechanism through which DFDBA may exert its biologic effect. However, in recent years, the predictability of results using DFDBA has been variable and has led to its use being questioned. One reason for the variability in tissue response may be attributed to differences in the processing of DFDBA, which may lead to loss of activity of any bioactive substances within the DFDBA matrix. Therefore, the purpose of this investigation was to determine whether there are detectable levels of bone morphogenetic proteins in commercial DFDBA preparations. METHODS: A single preparation of DFDBA was obtained from three commercial sources. Each preparation was studied in triplicate. Proteins within the DFDBA samples were first extracted with 4M guanidinium HCI for seven days at 40 degrees celsius and the residue was further extracted with 4M guanidinium HCL/EDTA for seven days at 40 degrees celsius. Two anti-human BMP-2 and -4 antibodies were used for the detection of the presence of BMP's in the extracts. RESULTS: Neither BMP-2 nor BMP-4 was detected in any of the extracts. When recombinant human BMP-2 and -4 were added throughout the extraction process of DFDBA extraction, not only were intact proteins detected but smaller molecular weight fragments were also noted in the extract. CONCLUSIONS: These results indicate that all of the DFDBA samples tested had no detectable amounts of BMP-2 and -4. In addition, an unknown substance present in the DFDBA may be responsible for degradation of whatever BMPs might be present.

Particle nucleation in urban subtropical atmosphere

New particle formation (NPF) and growth have been observed in different environments all around the world and NPF affects the environment by forming cloud condensation nuclei (CCN). Detailed characterisation of NPF events in a subtropical urban environment is the main aim of this study. Particle size distribution (PSD) of atmospheric aerosol particles in range 9-414 nm were measured using a Scanning Mobility Particle Sizer (SMPS), within the framework of the “Ultrafine Particles from Traffic Emissions and Children’s Health” (UPTECH) study, which seeks to determine the relationship between exposure to traffic related ultrafine particles and children’s health (http://www.ilaqh.qut. edu.au/Misc/UPTECH%20Home.htm). The UPTECH study includes measurements of air quality, meteorological and traffic parameters in 25 randomly selected state primary school within the Brisbane metropolitan area, in Queensland, Australia. Measurements at 17 schools have been completed so far.

Alternative modes of entrepreneurial opportunity exploitation : the case on inter-organizational project

While entrepreneurship research has taken firm formation to be the predominant mode of opportunity exploitation, entrepreneurship can take place through many other types of organizational arrangements. In the present article, we consider one such alternative arrangement, namely the formation of inter-organizational projects (IOPs). We propose a multi-level contingency model that suggests that uncertainty both at the level of the firm and at the level of the environment makes the exploitation of opportunities through IOPs more likely. The model is tested by telephone survey data collected amongst a panel of 1725 SMEs and longitudinal industry data. Our findings provide strong support for the industry-level part of the model, but interestingly, only partial support for the firm level part of the model. This indicates that the effects of uncertainty need to be dissected into different levels of analysis to understand the conditions under which alternative modes of opportunity exploitation can be a prominent entrepreneurial alternative to new firm formation.

Effects of scaffold architecture on cranial bone healing

Scaffolds for bone tissue engineering should be designed to optimize cell migration, enhance new bone formation and give mechanical support. In the present study, we used polycaprolactone-tricalciumphosphate (PCL/TCP) scaffolds with two different fibre lay down patterns which were coated with hydroxyapatite and gelatine as an approach for optimizing bone regeneration in a critical sized calvarial defect. After 12 weeks bone regeneration was quantified using microCT analysis, biomechanical testing and histological evaluation. Notably, the experimental groups containing coated scaffolds showed lower bone formation and lower biomechanical properties within the defect compared to the uncoated scaffolds. Surprisingly, the different lay down pattern of the fibres resulted in different bone formation and biomechanical properties; namely 0/60/120° scaffolds revealed lower bone formation and biomechanical properties compared to the 0/90° scaffolds in all the experimental groups. The different architecture of the scaffold fibres may have an effect on nutrition supply as well as the attachment of the newly formed matrix to the scaffold. Therefore, future bone regeneration strategies utilising scaffolds should consider scaffold architecture as an important factor during the scaffold optimisation stages in order to move closer to a clinical application.

Turnover in entrepreneurial teams : are spousal teams different?

Entrepreneurial teams are increasingly prevalent. An important stage in new venture formation involves the recruitment and retention of team members, characterised by changes in the membership of the ET. ET turnover involves members joining or leaving the team, which could have positive or negative effects on the team and the firm. Spousal Entrepreneurial Teams (SET) are a frequently occurring, yet unique type of ET, with very distinct characteristics and dynamics. In an SET turnover is likely to be have different meanings and effects than in Other Entrepreneurial Teams. This research asks: What influences members joining and leaving entrepreneurial teams? And are SET different? Using data from 561 young firms over X years, we find significant differences between SET and NSET. Specifically, we find X.