The osteogenic differentiation of adipose tissue-derived precursor cells in A 3D scaffold/matrix environment

Abstract: This paper details an in-vitro study using human adipose tissue-derived precursor/stem cells (ADSCs) in three-dimensional (3D) tissue culture systems. ADSCs from 3 donors were seeded onto NaOH-treated medical grade polycaprolactone-tricalcium phosphate (mPCL-TCP) scaffolds with two different matrix components; fibrin glue and lyophilized collagen. ADSCs within these scaffolds were then induced to differentiate along the osteogenic lineage for a 28-day period and various assays and imaging techniques were performed at Day 1, 7, 14, 21 and 28 to assess and compare the ADSC’s adhesion, viability, proliferation, metabolism and differentiation along the osteogenic lineage when cultured in the different scaffold/matrix systems. The ADSC cells were proliferative in both collagen and fibrin mPCL-TCP scaffold systems with a consistently higher cell number (by comparing DNA amounts) in the induced group over the non-induced groups for both scaffold systems. In response to osteogenic induction, these ADSCs expressed elevated osteocalcin, alkaline phosphatase and osteonectin levels. Cells were able to proliferate within the pores of the scaffolds and form dense cellular networks after 28 days of culture and induction. The successful cultivation of osteogenic by FDM process manufactured ADSCs within a 3D matrix comprising fibrin glue or collagen, immobilized within a robust synthetic scaffold is a promising technique which should enhance their potential usage in the regenerative medicine arena, such as bone tissue engineering.

Design and evaluation of an MRI compatible axial compression device for 3D assessment of spinal deformity and flexibility

Magnetic Resonance Imaging (MRI) offers a valuable research tool for the assessment of 3D spinal deformity in AIS, however the horizontal patient position imposed by conventional scanners removes the axial compressive loading on the spine which is an important determinant of deformity shape and magnitude in standing scoliosis patients. The objective of this study was to design, construct and test an MRI compatible compression device for research into the effect of axial loading on spinal deformity using supine MRI scans. The compression device was designed and constructed, consisting of a vest worn by the patient, which was attached via straps to a pneumatically actuated footplate. An applied load of 0.5 x bodyweight was remotely controlled by a unit in the scanner operator’s console. The entire device was constructed using non-metallic components for MRI compatibility. The device was evaluated by performing unloaded and loaded supine MRI scans on a series of 10 AIS patients. The study concluded that an MRI compatible compression device had been successfully designed and constructed, providing a research tool for studies into the effect of axial loading on 3D spinal deformity in scoliosis. The 3D axially loaded MR imaging capability developed in this study will allow future research investigations of the effect of axial loading on spinal rotation, and for imaging the response of scoliotic spinal tissues to axial loading.

Creation of a validated 3D finite element model of an ovine tibia

Introduction Ovine models are widely used in orthopaedic research. To better understand the impact of orthopaedic procedures computer simulations are necessary. 3D finite element (FE) models of bones allow implant designs to be investigated mechanically, thereby reducing mechanical testing. Hypothesis We present the development and validation of an ovine tibia FE model for use in the analysis of tibia fracture fixation plates. Material & Methods Mechanical testing of the tibia consisted of an offset 3-pt bend test with three repetitions of loading to 350N and return to 50N. Tri-axial stacked strain gauges were applied to the anterior and posterior surfaces of the bone and two rigid bodies – consisting of eight infrared active markers, were attached to the ends of the tibia. Positional measurements were taken with a FARO arm 3D digitiser. The FE model was constructed with both geometry and material properties derived from CT images of the bone. The elasticity-density relationship used for material property determination was validated separately using mechanical testing. This model was then transformed to the same coordinate system as the in vitro mechanical test and loads applied. Results Comparison between the mechanical testing and the FE model showed good correlation in surface strains (difference: anterior 2.3%, posterior 3.2%). Discussion & Conclusion This method of model creation provides a simple method for generating subject specific FE models from CT scans. The use of the CT data set for both the geometry and the material properties ensures a more accurate representation of the specific bone. This is reflected in the similarity of the surface strain results.

Understanding the Aquifier : 3D Visualisation and Groundwater Time-Series Animation in Lockyer Valley

The Lockyer Valley, southeast Queensland, hosts intensive irrigated agriculture using groundwater from over 5000 alluvial bores. A current project is considering introduction of PRW (purified recycled water) to augment groundwater supplies. To assess this, a valley-wide MODFLOW simulation model is being developed plus a new unsaturated zone flow model. To underpin these models and provide a realistic understanding of the aquifer framework a 3D visualisation model has been developed using Groundwater Visualisation System (GVS) software produced at QUT.

Design and evaluation of an MRI compatible axial compression device for 3D assessment of spinal deformity and flexibility in adolescent idiopathic scoliosis

Magnetic Resonance Imaging (MRI) offers a valuable research tool for the assessment of 3D spinal deformity in AIS, however the horizontal patient position imposed by conventional scanners removes the axial compressive loading on the spine. The objective of this study was to design, construct and test an MRI compatible compression device for research into the effect of axial loading on spinal deformity using supine MRI scans. The device was evaluated by performing unloaded and loaded supine MRI scans on a series of 10 AIS patients. The patient group had a mean initial (unloaded) major Cobb angle of 43±7º, which increased to 50±9º on application of the compressive load. The 7° increase in mean Cobb angle is consistent with that reported by a previous study comparing standing versus supine posture in scoliosis patients (Torell et al, 1985. Spine 10:425-7).

Image acquisition and manipulation protocols for CT-PET fusion to improve the accuracy of gross tumour volume localisation for 3D conformal radiotherapy for lung cancer

Aims: To develop clinical protocols for acquiring PET images, performing CT-PET registration and tumour volume definition based on the PET image data, for radiotherapy for lung cancer patients and then to test these protocols with respect to levels of accuracy and reproducibility. Method: A phantom-based quality assurance study of the processes associated with using registered CT and PET scans for tumour volume definition was conducted to: (1) investigate image acquisition and manipulation techniques for registering and contouring CT and PET images in a radiotherapy treatment planning system, and (2) determine technology-based errors in the registration and contouring processes. The outcomes of the phantom image based quality assurance study were used to determine clinical protocols. Protocols were developed for (1) acquiring patient PET image data for incorporation into the 3DCRT process, particularly for ensuring that the patient is positioned in their treatment position; (2) CT-PET image registration techniques and (3) GTV definition using the PET image data. The developed clinical protocols were tested using retrospective clinical trials to assess levels of inter-user variability which may be attributed to the use of these protocols. A Siemens Somatom Open Sensation 20 slice CT scanner and a Philips Allegro stand-alone PET scanner were used to acquire the images for this research. The Philips Pinnacle3 treatment planning system was used to perform the image registration and contouring of the CT and PET images. Results: Both the attenuation-corrected and transmission images obtained from standard whole-body PET staging clinical scanning protocols were acquired and imported into the treatment planning system for the phantom-based quality assurance study. Protocols for manipulating the PET images in the treatment planning system, particularly for quantifying uptake in volumes of interest and window levels for accurate geometric visualisation were determined. The automatic registration algorithms were found to have sub-voxel levels of accuracy, with transmission scan-based CT-PET registration more accurate than emission scan-based registration of the phantom images. Respiration induced image artifacts were not found to influence registration accuracy while inadequate pre-registration over-lap of the CT and PET images was found to result in large registration errors. A threshold value based on a percentage of the maximum uptake within a volume of interest was found to accurately contour the different features of the phantom despite the lower spatial resolution of the PET images. Appropriate selection of the threshold value is dependant on target-to-background ratios and the presence of respiratory motion. The results from the phantom-based study were used to design, implement and test clinical CT-PET fusion protocols. The patient PET image acquisition protocols enabled patients to be successfully identified and positioned in their radiotherapy treatment position during the acquisition of their whole-body PET staging scan. While automatic registration techniques were found to reduce inter-user variation compared to manual techniques, there was no significant difference in the registration outcomes for transmission or emission scan-based registration of the patient images, using the protocol. Tumour volumes contoured on registered patient CT-PET images using the tested threshold values and viewing windows determined from the phantom study, demonstrated less inter-user variation for the primary tumour volume contours than those contoured using only the patient’s planning CT scans. Conclusions: The developed clinical protocols allow a patient’s whole-body PET staging scan to be incorporated, manipulated and quantified in the treatment planning process to improve the accuracy of gross tumour volume localisation in 3D conformal radiotherapy for lung cancer. Image registration protocols which factor in potential software-based errors combined with adequate user training are recommended to increase the accuracy and reproducibility of registration outcomes. A semi-automated adaptive threshold contouring technique incorporating a PET windowing protocol, accurately defines the geometric edge of a tumour volume using PET image data from a stand alone PET scanner, including 4D target volumes.

Scaffold development using 3D printing with a starch-based polymer

Rapid prototyping (RP) techniques have been utilised by tissue engineers to produce three-dimensional (3D) porous scaffolds. RP technologies allow the design and fabrication of complex scaffold geometries with a fully interconnected pore network. Three-dimensional printing (3DP) technique was used to fabricate scaffolds with a novel micro- and macro-architecture. In this study, a unique blend of starch-based polymer powders (cornstarch, dextran and gelatin) was developed for the 3DP process. Cylindrical scaffolds of five different designs were fabricated and post-processed to enhance the mechanical and chemical properties. The scaffold properties were characterised by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), porosity analysis and compression tests

Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening. © 2010 Landes Bioscience.

Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells

The behaviour of cells cultured within three-dimensional (3D) structures rather than onto two-dimensional (2D) culture plastic more closely reflects their in vivo responses. Consequently, 3D culture systems are becoming crucial scientific tools in cancer cell research. We used a novel 3D culture concept to assess cell-matrix interactions implicated in carcinogenesis: a synthetic hydrogel matrix equipped with key biomimetic features, namely incorporated cell integrin-binding motifs (e.g. RGD peptides) and the ability of being degraded by cell-secreted proteases (e.g. matrix metalloproteases). As a cell model, we chose epithelial ovarian cancer, an aggressive disease typically diagnosed at an advanced stage when chemoresistance occurs. Both cell lines used (OV-MZ-6, SKOV-3) proliferated similarly in 2D, but not in 3D. Spheroid formation was observed exclusively in 3D when cells were embedded within hydrogels. By exploiting the design flexibility of the hydrogel characteristics, we showed that proliferation in 3D was dependent on cell-integrin engagement and the ability of cells to proteolytically remodel their extracellular microenvironment. Higher survival rates after exposure to the anti-cancer drug paclitaxel were observed in cell spheroids grown in hydrogels (40-60%) compared to cell monolayers in 2D (20%). Thus, 2D evaluation of chemosensitivity may not reflect pathophysiological events seen in patients. Because of the design flexibility of their characteristics and their stability in long-term cultures (28 days), these biomimetic hydrogels represent alternative culture systems for the increasing demand in cancer research for more versatile, physiologically relevant and reproducible 3D matrices.

The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds

Our strategy entails investigating the influence of varied concentrations (0, 10, 100 and 1000 ng/ml) of human recombinant bone morphogenetic protein-2 (rhBMP-2) on the osteogenic expression of canine osteoblasts, seeded onto poly-caprolactone 20% tricalcium phosphate (PCL-TCP) scaffolds in vitro. Biochemical assay revealed that groups with rhBMP-2 displayed an initial burst in cell growth that was not dose-dependent. However, after 13 days, cell growth declined to a value similar to control. Significantly less cell growth was observed for construct with 1000 ng/ml of rhBMP-2 from 20 days onwards. Confocal microscopy confirmed viability of osteoblasts and at day 20, groups seeded with rhBMP-2 displayed heightened cell death as compared to control. Phase contrast and scanning electron microscopy revealed that osteoblasts heavily colonized surfaces, rods and pores of the PCL-TCP scaffolds. This was consistent for all groups. Finally, Von Kossa and osteocalcin assays demonstrated that cells from all groups maintained their osteogenic phenotype throughout the experiment. Calcification was observed as early as four days after stimulation for groups seeded with rhBMP-2. In conclusion, rhBMP-2 seems to enhance the differentiated function of canine osteoblasts in a non-dose dependent manner. This resulted in accelerated mineralization, followed by death of osteoblasts as they underwent terminal differentiation. Notably, PCL-TCP scaffolds seeded only with canine osteoblasts could sustain excellent osteogenic expression in vitro. Hence, the synergy of PCL with bioactive TCP and rhBMP-2 in a novel composite scaffold, could offer an exciting approach for bone regeneration.

A research opportunity : “3D-ROSE” : virtual reality preparation for children having radiotherapy or imaging procedures

Professor Christian Langton is a medical physicist at Queensland University of Technology in Brisbane. He has developed a way of preparing children who are about to have either radiotherapy or MRI imaging procedures and is seeking research partners to develop and test these further. This is a great opportunity for nurses interested in research, and who have access to a children’s hospital, to work with Professor Langton on some truly innovative, multidisciplinary research.