Enhancing in vivo vascularized bone formation by cobalt chloride-treated bone marrow stromal cells in a tissue engineered periosteum model

The periosteum plays an indispensable role in both bone formation and bone defect healing. In this study we constructed an artificial in vitro periosteum by incorporating osteogenic differentiated bone marrow stromal cells (BMSCs) and cobalt chloride (CoCl(2))-treated BMSCs. The engineered periostea were implanted both subcutaneously and into skull bone defects in SCID mice to investigate ectopic and orthotopic osteogenesis and vascularization. After two weeks in subcutaneous and four weeks in bone defect areas, the implanted constructs were assessed for ectopic and orthotopic osteogenesis and vascularization by micro-CT, histomorphometrical and immunohistochemical methods. The results showed that CoCl(2) pre-treated BMSCs induced higher degree of vascularization and enhanced osteogenesis within the implants in both ectopic and orthotopic areas. This study provided a novel approach using BMSCs sourced from the same patient for both osteogenic and pro-angiogenic purposes in constructing tissue engineered periosteum to enhance vascularized osteogenesis.

Stem cell–related gene expression in clonal populations of mesenchymal stromal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs.

Structure–property relationships of silk-modified mesoporous bioglass scaffolds

Porous mesopore-bioglass (MBG) scaffolds have been proposed as a new class of bone regeneration materials due to their apatite-formation and drug-delivery properties; however, the material’s inherent brittleness and high degradation and surface instability are major disadvantages, which compromise its mechanical strength and cytocompatibility as a biological scaffold. Silk, on the other hand, is a native biomaterial and is well characterized with respect to biocompatibility and tensile strength. In this study we set out to investigate what effects blending silk with MBG had on the physiochemical, drug-delivery and biological properties of MBG scaffolds with a view to bone tissue engineering applications. Transmission electron microscopy (TEM), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were the methods used to analyze the inner microstructure, pore size and morphology, and composition of MBG scaffolds, before and after addition of silk. The effect of silk modification on the mechanical property of MBG scaffolds was determined by testing the compressive strength of the scaffolds and also compressive strength after degradation over time. The drug-delivery potential was evaluated by the release of dexamethasone (DEX) from the scaffolds. Finally, the cytocompatibility of silk-modified scaffolds was investigated by the attachment, morphology, proliferation, differentiation and bone-relative gene expression of bone marrow stromal cells (BMSCs). The results showed that silk modification improved the uniformity and continuity of pore network of MBG scaffolds, and maintained high porosity (94%) and large-pore size (200–400 mm). There was a significant improvement in mechanical strength, mechanical stability, and control of burst release of DEX in silkmodified MBG scaffolds. Silk modification also appeared to provide a better environment for BMSC attachment, spreading, proliferation, and osteogenic differentiation on MBG scaffolds.

Contributions of vision-proprioception interactions to the estimation of time-varying hand and target locations

We investigated the relative importance of vision and proprioception in estimating target and hand locations in a dynamic environment. Subjects performed a position estimation task in which a target moved horizontally on a screen at a constant velocity and then disappeared. They were asked to estimate the position of the invisible target under two conditions: passively observing and manually tracking. The tracking trials included three visual conditions with a cursor representing the hand position: always visible, disappearing simultaneously with target disappearance, and always invisible. The target’s invisible displacement was systematically underestimated during passive observation. In active conditions, tracking with the visible cursor significantly decreased the extent of underestimation. Tracking of the invisible target became much more accurate under this condition and was not affected by cursor disappearance. In a second experiment, subjects were asked to judge the position of their unseen hand instead of the target during tracking movements. Invisible hand displacements were also underestimated when compared with the actual displacement. Continuous or brief presentation of the cursor reduced the extent of underestimation. These results suggest that vision–proprioception interactions are critical for representing exact target–hand spatial relationships, and that such sensorimotor representation of hand kinematics serves a cognitive function in predicting target position. We propose a hypothesis that the central nervous system can utilize information derived from proprioception and/or efference copy for sensorimotor prediction of dynamic target and hand positions, but that effective use of this information for conscious estimation requires that it be presented in a form that corresponds to that used for the estimations.

Porcine bone marrow stromal cell differentiation on heparin-adsorbed poly (e-caprolactone)-tricalcium phosphate-collagen scaffolds

We evaluate the potential of heparin as a substrate component for the fabrication of bone tissue engineering constructs using poly(e- caprolactone)–tricalcium phosphate–collagen type I (PCL–TCP–Col) three-dimensional (3-D) scaffolds. First we explored the ability of porcine bone marrow precursor cells (MPCs) to differentiate down both the adipogenic and osteogenic pathways within 2-D culture systems, with positive results confirmed by Oil-Red-O and Alizarin Red staining, respectively. Secondly, we examined the influence of heparin on the interaction and behaviour of MPCs when seeded onto PCL–TCP–Col 3-D scaffolds, followed by their induction into the osteogenic lineage. Our 3-D findings suggest that cell metabolism and proliferation increased between days 1 and 14, with deposition of extracellular matrix also observed up to 28 days. However, no noticeable difference could be detected in the extent of osteogenesis for PCL–TCP–Col scaffolds groups with the addition of heparin compared to identical control scaffolds without the addition of heparin.

Engineering tubular bone constructs

Cell-sheet techniques have been proven effective in various soft tissue engineering applications. In this experiment, we investigated the feasibility of bone tissue engineering using a hybrid of mesenchymal stem cell (MSC) sheets and PLGA meshes. Porcine MSCs were cultured to a thin layer of cell sheets via osteogenic induction. Tube-like long bones were constructed by wrapping the cell sheet on to PLGA meshes resulting in constructs which could be cultured in spinner flasks, prior to implantation in nude rats. Our results showed that the sheets were composed of viable cells and dense matrix with a thickness of about 80–120 mm, mineral deposition was also observed in the sheet. In vitro cultures demonstrated calcified cartilage-like tissue formation and most PLGA meshes were absorbed during the 8-week culture period. In vivo experiments revealed that dense mineralized tissue was formed in subcutaneous sites and the 8- week plants shared similar micro-CT characteristics with native bone. The neo tissue demonstrated histological markers for both bone and cartilage, indicating that the bone formation pathway in constructs was akin to endochondral ossification, with the residues of PLGA having an effect on the neo tissue organization and formation. These results indicate that cell-sheet approaches in combination with custom-shaped scaffolds have potential in producing bone tissue.

Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts

In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone–calcium phosphate (mPCL–CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (554mm3) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10104mm3) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.

Improved image contrast of the bone-muscle interface with 3T MRI compared to 1.5T MRI [Abstract]

Virtual 3D models of long bones are increasingly being used for implant design and research applications. The current gold standard for the acquisition of such data is Computed Tomography (CT) scanning. Due to radiation exposure, CT is generally limited to the imaging of clinical cases and cadaver specimens. Magnetic Resonance Imaging (MRI) does not involve ionising radiation and therefore can be used to image selected healthy human volunteers for research purposes. The feasibility of MRI as alternative to CT for the acquisition of morphological bone data of the lower extremity has been demonstrated in recent studies [1, 2]. Some of the current limitations of MRI are long scanning times and difficulties with image segmentation in certain anatomical regions due to poor contrast between bone and surrounding muscle tissues. Higher field strength scanners promise to offer faster imaging times or better image quality. In this study image quality at 1.5T is quantitatively compared to images acquired at 3T. --------- The femora of five human volunteers were scanned using 1.5T and 3T MRI scanners from the same manufacturer (Siemens) with similar imaging protocols. A 3D flash sequence was used with TE = 4.66 ms, flip angle = 15° and voxel size = 0.5 × 0.5 × 1 mm. PA-Matrix and body matrix coils were used to cover the lower limb and pelvis respectively. Signal to noise ratio (SNR) [3] and contrast to noise ratio (CNR) [3] of the axial images from the proximal, shaft and distal regions were used to assess the quality of images from the 1.5T and 3T scanners. The SNR was calculated for the muscle and bone-marrow in the axial images. The CNR was calculated for the muscle to cortex and cortex to bone marrow interfaces, respectively. --------- Preliminary results (one volunteer) show that the SNR of muscle for the shaft and distal regions was higher in 3T images (11.65 and 17.60) than 1.5T images (8.12 and 8.11). For the proximal region the SNR of muscles was higher in 1.5T images (7.52) than 3T images (6.78). The SNR of bone marrow was slightly higher in 1.5T images for both proximal and shaft regions, while it was lower in the distal region compared to 3T images. The CNR between muscle and bone of all three regions was higher in 3T images (4.14, 6.55 and 12.99) than in 1.5T images (2.49, 3.25 and 9.89). The CNR between bone-marrow and bone was slightly higher in 1.5T images (4.87, 12.89 and 10.07) compared to 3T images (3.74, 10.83 and 10.15). These results show that the 3T images generated higher contrast between bone and the muscle tissue than the 1.5T images. It is expected that this improvement of image contrast will significantly reduce the time required for the mainly manual segmentation of the MR images. Future work will focus on optimizing the 3T imaging protocol for reducing chemical shift and susceptibility artifacts.

Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

Typical accuracy and quality control of a process for creating CT-Based virtual bone models

A pragmatic method for assessing the accuracy and precision of a given processing pipeline required for converting computed tomography (CT) image data of bones into representative three dimensional (3D) models of bone shapes is proposed. The method is based on coprocessing a control object with known geometry which enables the assessment of the quality of resulting 3D models. At three stages of the conversion process, distance measurements were obtained and statistically evaluated. For this study, 31 CT datasets were processed. The final 3D model of the control object contained an average deviation from reference values of −1.07±0.52 mm standard deviation (SD) for edge distances and −0.647±0.43 mm SD for parallel side distances of the control object. Coprocessing a reference object enables the assessment of the accuracy and precision of a given processing pipeline for creating CTbased 3D bone models and is suitable for detecting most systematic or human errors when processing a CT-scan. Typical errors have about the same size as the scan resolution.

The use of visual analogue scales to assess motivation to eat in human subjects: a review of their reliability and validity with an evaluation of new hand-held computerized stsytems for temporal tracking of appetite ratings.

This present paper reviews the reliability and validity of visual analogue scales (VAS) in terms of (1) their ability to predict feeding behaviour, (2) their sensitivity to experimental manipulations, and (3) their reproducibility. VAS correlate with, but do not reliably predict, energy intake to the extent that they could be used as a proxy of energy intake. They do predict meal initiation in subjects eating their normal diets in their normal environment. Under laboratory conditions, subjectively rated motivation to eat using VAS is sensitive to experimental manipulations and has been found to be reproducible in relation to those experimental regimens. Other work has found them not to be reproducible in relation to repeated protocols. On balance, it would appear, in as much as it is possible to quantify, that VAS exhibit a good degree of within-subject reliability and validity in that they predict with reasonable certainty, meal initiation and amount eaten, and are sensitive to experimental manipulations. This reliability and validity appears more pronounced under the controlled (but more arti®cial) conditions of the laboratory where the signal : noise ratio in experiments appears to be elevated relative to real life. It appears that VAS are best used in within-subject, repeated-measures designs where the effect of different treatments can be compared under similar circumstances. They are best used in conjunction with other measures (e.g. feeding behaviour, changes in plasma metabolites) rather than as proxies for these variables. New hand-held electronic appetite rating systems (EARS) have been developed to increase reliability of data capture and decrease investigator workload. Recent studies have compared these with traditional pen and paper (P&P) VAS. The EARS have been found to be sensitive to experimental manipulations and reproducible relative to P&P. However, subjects appear to exhibit a signi®cantly more constrained use of the scale when using the EARS relative to the P&P. For this reason it is recommended that the two techniques are not used interchangeably

An examination of the factors that influence drivers’ willingness to use hand-held mobile phones

Hand-held mobile phone use while driving is illegal throughout Australia yet many drivers persist with this behaviour. This study aims to understand the internal, driver-related and external, situational-related factors influencing drivers’ willingness to use a hand-held mobile phone while driving. Sampling 160 university students, this study utilised the Theory of Planned Behaviour (TPB) to examine a range of belief-based constructs. Additionally, drivers’ personality traits of neuroticism and extroversion were measured with the Neuroticism Extroversion Openness-Five Factor Inventory (NEO-FFI). In relation to the external, situational-related factors, four different driving-related scenarios, which were intended to evoke differing levels of drivers’ reported stress, were devised for the study and manipulated drivers’ time urgency (low versus high) and passenger presence (alone versus with friends). In these scenarios, drivers’ willingness to use a mobile phone in general was measured. Hierarchical regression analyses across the four different driving scenarios found that, overall, the TPB components significantly accounted for drivers’ willingness to use a mobile phone above and beyond the demographic variables. Subjective norms, however, was only a significant predictor of drivers’ willingness in situations where the drivers were driving alone. Generally, neuroticism and extroversion did not significantly predict drivers’ willingness above and beyond the TPB and demographic variables. Overall, the findings broaden our understanding of the internal and external factors influencing drivers’ willingness to use a hand-held mobile phone while driving despite the illegality of this behaviour. The findings may have important practical implications in terms of better informing road safety campaigns targeting drivers’ mobile phone use which, in turn, may contribute to a reduction in the extent that mobile phone use contributes to road crashes.

The effect of friction on indenter force and pile-up in numerical simulations of bone nanoindentation

Nanoindentation is a useful technique for probing the mechanical properties of bone, and finite element (FE) modeling of the indentation allows inverse determination of elasto-plastic constitutive properties. However, FE simulations to date have assumed frictionless contact between indenter and bone. The aim of this study was to explore the effect of friction in simulations of bone nanoindentation. Two dimensional axisymmetric FE simulations were performed using a spheroconical indenter of tip radius 0.6m and angle 90°. The coefficient of friction between indenter and bone was varied between 0.0 (frictionless) and 0.3. Isotropic linear elasticity was used in all simulations, with bone elastic modulus E=13.56GPa and Poisson’s ratio =0.3. Plasticity was incorporated using both Drucker-Prager and von Mises yield surfaces. Friction had a modest effect on the predicted force-indentation curve for both von Mises and Drucker-Prager plasticity, reducing maximum indenter displacement by 10% and 20% respectively as friction coefficient was increased from zero to 0.3 (at a maximum indenter force of 5mN). However, friction has a much greater effect on predicted pile-up after indentation, reducing predicted pile-up from 0.27m to 0.11m with a von Mises model, and from 0.09m to 0.02m with Drucker-Prager plasticity. We conclude that it is important to include friction in nanoindentation simulations of bone.