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.

Mathematical modelling of muscle recruitment and function in the lumbar spine

Low back pain is an increasing problem in industrialised countries and although it is a major socio-economic problem in terms of medical costs and lost productivity, relatively little is known about the processes underlying the development of the condition. This is in part due to the complex interactions between bone, muscle, nerves and other soft tissues of the spine, and the fact that direct observation and/or measurement of the human spine is not possible using non-invasive techniques. Biomechanical models have been used extensively to estimate the forces and moments experienced by the spine. These models provide a means of estimating the internal parameters which can not be measured directly. However, application of most of the models currently available is restricted to tasks resembling those for which the model was designed due to the simplified representation of the anatomy. The aim of this research was to develop a biomechanical model to investigate the changes in forces and moments which are induced by muscle injury. In order to accurately simulate muscle injuries a detailed quasi-static three dimensional model representing the anatomy of the lumbar spine was developed. This model includes the nine major force generating muscles of the region (erector spinae, comprising the longissimus thoracis and iliocostalis lumborum; multifidus; quadratus lumborum; latissimus dorsi; transverse abdominis; internal oblique and external oblique), as well as the thoracolumbar fascia through which the transverse abdominis and parts of the internal oblique and latissimus dorsi muscles attach to the spine. The muscles included in the model have been represented using 170 muscle fascicles each having their own force generating characteristics and lines of action. Particular attention has been paid to ensuring the muscle lines of action are anatomically realistic, particularly for muscles which have broad attachments (e.g. internal and external obliques), muscles which attach to the spine via the thoracolumbar fascia (e.g. transverse abdominis), and muscles whose paths are altered by bony constraints such as the rib cage (e.g. iliocostalis lumborum pars thoracis and parts of the longissimus thoracis pars thoracis). In this endeavour, a separate sub-model which accounts for the shape of the torso by modelling it as a series of ellipses has been developed to model the lines of action of the oblique muscles. Likewise, a separate sub-model of the thoracolumbar fascia has also been developed which accounts for the middle and posterior layers of the fascia, and ensures that the line of action of the posterior layer is related to the size and shape of the erector spinae muscle. Published muscle activation data are used to enable the model to predict the maximum forces and moments that may be generated by the muscles. These predictions are validated against published experimental studies reporting maximum isometric moments for a variety of exertions. The model performs well for fiexion, extension and lateral bend exertions, but underpredicts the axial twist moments that may be developed. This discrepancy is most likely the result of differences between the experimental methodology and the modelled task. The application of the model is illustrated using examples of muscle injuries created by surgical procedures. The three examples used represent a posterior surgical approach to the spine, an anterior approach to the spine and uni-lateral total hip replacement surgery. Although the three examples simulate different muscle injuries, all demonstrate the production of significant asymmetrical moments and/or reduced joint compression following surgical intervention. This result has implications for patient rehabilitation and the potential for further injury to the spine. The development and application of the model has highlighted a number of areas where current knowledge is deficient. These include muscle activation levels for tasks in postures other than upright standing, changes in spinal kinematics following surgical procedures such as spinal fusion or fixation, and a general lack of understanding of how the body adjusts to muscle injuries with respect to muscle activation patterns and levels, rate of recovery from temporary injuries and compensatory actions by other muscles. Thus the comprehensive and innovative anatomical model which has been developed not only provides a tool to predict the forces and moments experienced by the intervertebral joints of the spine, but also highlights areas where further clinical research is required.

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.

A subject-specific model of human buttocks and thighs in a seated posture

Finite element analyses of the human body in seated postures requires digital models capable of providing accurate and precise prediction of the tissue-level response of the body in the seated posture. To achieve such models, the human anatomy must be represented with high fidelity. This information can readily be defined using medical imaging techniques such as Magnetic Resonance Imaging (MRI) or Computed Tomography (CT). Current practices for constructing digital human models, based on the magnetic resonance (MR) images, in a lying down (supine) posture have reduced the error in the geometric representation of human anatomy relative to reconstructions based on data from cadaveric studies. Nonetheless, the significant differences between seated and supine postures in segment orientation, soft-tissue deformation and soft tissue strain create a need for data obtained in postures more similar to the application posture. In this study, we present a novel method for creating digital human models based on seated MR data. An adult-male volunteer was scanned in a simulated driving posture using a FONAR 0.6T upright MRI scanner with a T1 scanning protocol. To compensate for unavoidable image distortion near the edges of the study, images of the same anatomical structures were obtained in transverse and sagittal planes. Combinations of transverse and sagittal images were used to reconstruct the major anatomical features from the buttocks through the knees, including bone, muscle and fat tissue perimeters, using Solidworks® software. For each MR image, B-splines were created as contours for the anatomical structures of interest, and LOFT commands were used to interpolate between the generated Bsplines. The reconstruction of the pelvis, from MR data, was enhanced by the use of a template model generated in previous work CT images. A non-rigid registration algorithm was used to fit the pelvis template into the MR data. Additionally, MR image processing was conducted to both the left and the right sides of the model due to the intended asymmetric posture of the volunteer during the MR measurements. The presented subject-specific, three-dimensional model of the buttocks and thighs will add value to optimisation cycles in automotive seat development when used in simulating human interaction with automotive seats.

Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment

Prostate cancer metastasis is reliant on the reciprocal interactions between cancer cells and the bone niche/micro-environment. The production of suitable matrices to study metastasis, carcinogenesis and in particular prostate cancer/bone micro-environment interaction has been limited to specific protein matrices or matrix secreted by immortalised cell lines that may have undergone transformation processes altering signaling pathways and modifying gene or receptor expression. We hypothesize that matrices produced by primary human osteoblasts are a suitable means to develop an in vitro model system for bone metastasis research mimicking in vivo conditions. We have used a decellularized matrix secreted from primary human osteoblasts as a model for prostate cancer function in the bone micro-environment. We show that this collagen I rich matrix is of fibrillar appearance, highly mineralized, and contains proteins, such as osteocalcin, osteonectin and osteopontin, and growth factors characteristic of bone extracellular matrix (ECM). LNCaP and PC3 cells grown on this matrix, adhere strongly, proliferate, and express markers consistent with a loss of epithelial phenotype. Moreover, growth of these cells on the matrix is accompanied by the induction of genes associated with attachment, migration, increased invasive potential, Ca2+ signaling and osteolysis. In summary, we show that growth of prostate cancer cells on matrices produced by primary human osteoblasts mimics key features of prostate cancer bone metastases and thus is a suitable model system to study the tumor/bone micro-environment interaction in this disease.

Cell interactions of osteoarthritic subchondral bone osteoblasts and articular chondrocytes aggravate MMP-2 and MMP-9 production through the mediation of ERK1/2 and JNK phosphorylation

Matrix Metalloproteinases (MMP) play a key role in osteoarthritis (OA) development. The aim of the present study was to investigate whether, the cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of MMPs, and also to test the potential involvement of mitogen activated protein kinase (MAPK) signalling pathway during this process.

Altered cell interactions of subchondral bone osteoblasts and articular chondrocytes in osteoarthritis is through the mediation of ERK1/2 phosphorylation

Introduction: Osteoarthritis (OA) is the most common musculoskeletal disorder and represents a major health burden to society. In the course of the pathological development of OA, articular cartilage chondrocytes (ACCs) undergo a typical phenotype changes characterized by the expression of hypertrophic differentiation markers. Also, the adjacent subchondral bone shows signs of abnormal mineral density and enhanced production of bone turnover markers, indicative of osteoblast dysfunction. However, the mechanism(s) by which these changes occur during the OA development are not completely understood. Materials and Methods: ACCs and subchondral bone osteoblasts (SBOs) were harvested from OA and healthy patients for the cross-talk studies between normal and OA ACCs and SBOs. The involvement of mitogen activated protein kinase (MAPK) signalling pathway during the cell-cell interactions was analysed by zymography, ELISA and western blotting methods. Results: The direct and in-direct co-culture studies showed that OA (ACCs and SBOs) cells induced osteoarthritic changes of normal (ACC and SBOs) cells. This altered cell interaction induced by OA cells significantly aggravated the proteolytic activity, which resulted cartilage degeneration. The altered cell interaction appeared to significantly activate ERK 1/2 phosphorylation and inhibition of MAPK-ERK 1/2 pathway reversed the osteoarthrtitic phenotypic changes. Discussion and Conclusion: Our study has demonstrated that the altered bi-directional communication of SBOs and ACCs are critical for initiation and progression of OA related changes and that this process is mediated by MAPK signalling pathways. Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA related disorders.

Delineating breast cancer cell interactions with engineered bone microenvironments

The mechanisms leading to colonization of metastatic breast cancer cells (BCa) in the skeleton are still not fully understood. Here, we demonstrate that mineralized extracellular matrices secreted by primary human osteoblasts (hOBM) modulate cellular processes associated with BCa colonization of bone. A panel of four BCa cell lines of different bone-metastatic potential (T47D, SUM1315, MDA-MB-231, and the bone-seeking subline MDA-MB-231BO) was cultured on hOBM. After 3 days, the metastatic BCa cells had undergone morphological changes on hOBM and were aligned along the hOBM's collagen type I fibrils that were decorated with bone-specific proteins. In contrast, nonmetastatic BCa cells showed a random orientation on hOBM. Atomic force microscopy-based single-cell force spectroscopy revealed that the metastatic cell lines adhered more strongly to hOBM compared with nonmetastatic cells. Function-blocking experiments indicated that β1-integrins mediated cell adhesion to hOBM. In addition, metastatic BCa cells migrated directionally and invaded hOBM, which was accompanied by enhanced MMP-2 and -9 secretion. Furthermore, we observed gene expression changes associated with osteomimickry in BCa cultured on hOBM. As such, osteopontin mRNA levels were significantly increased in SUM1315 and MDA-MB-231BO cells in a β1-integrin-dependent manner after growing for 3 days on hOBM compared with tissue culture plastic. In conclusion, our results show that extracellular matrices derived from human osteoblasts represent a powerful experimental platform to dissect mechanisms underlying critical steps in the development of bone metastases.

Use of genetically modified muscle and fat grafts to repair defects in bone and cartilage

We report a novel technology for the rapid healing of large osseous and chondral defects, based upon the genetic modification of autologous skeletal muscle and fat grafts. These tissues were selected because they not only possess mesenchymal progenitor cells and scaffolding properties, but also can be biopsied, genetically modified and returned to the patient in a single operative session. First generation adenovirus vector carrying cDNA encoding human bone morphogenetic protein-2 (Ad.BMP-2) was used for gene transfer to biopsies of muscle and fat. To assess bone healing, the genetically modified (“gene activated”) tissues were implanted into 5mm-long critical size, mid-diaphyseal, stabilized defects in the femora of Fischer rats. Unlike control defects, those receiving gene-activated muscle underwent rapid healing, with evidence of radiologic bridging as early as 10 days after implantation and restoration of full mechanical strength by 8 weeks. Histologic analysis suggests that the grafts rapidly differentiated into cartilage, followed by efficient endochondral ossification. Fluorescence in situ hybridization detection of Y-chromosomes following the transfer of male donor muscle into female rats demonstrated that at least some of the osteoblasts of the healed bone were derived from donor muscle. Gene activated fat also healed critical sized defects, but less quickly than muscle and with more variability. Anti-adenovirus antibodies were not detected. Pilot studies in a rabbit osteochondral defect model demonstrated the promise of this technology for healing cartilage defects. Further development of these methods should provide ways to heal bone and cartilage more expeditiously, and at lower cost, than is presently possible.

Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis

As microenvironmental factors such as three-dimensionality and cell–matrix interactions are increasingly being acknowledged by cancer biologists, more complex 3D in vitro models are being developed to study tumorigenesis and cancer progression. To better understand the pathophysiology of bone metastasis, we have established and validated a 3D indirect co-culture model to investigate the paracrine interactions between prostate cancer (PCa) cells and human osteoblasts. Co-culture of the human PCa, LNCaP cells embedded within polyethylene glycol hydrogels with human osteoblasts in the form of a tissue engineered bone construct (TEB), resulted in reduced proliferation of LNCaP cells. LNCaP cells in both monoculture and co-culture were responsive to the androgen analog, R1881, as indicated by an increase in the expression (mRNA and/or protein induction) of androgen-regulated genes including prostate specific antigen and fatty acid synthase. Microarray gene expression analysis further revealed an up-regulation of bone markers and other genes associated with skeletal and vasculature development and a significant activation of transforming growth factor β1 downstream genes in LNCaP cells after co-culture with TEB. LNCaP cells co-cultured with TEB also unexpectedly showed similar changes in classical androgen-responsive genes under androgen-deprived conditions not seen in LNCaP monocultures. The molecular changes of LNCaP cells after co-culturing with TEBs suggest that osteoblasts exert a paracrine effect that may promote osteomimicry and modulate the expression of androgen-responsive genes in LNCaP cells. Taken together, we have presented a novel 3D in vitro model that allows the study of cellular and molecular changes occurring in PCa cells and osteoblasts that are relevant to metastatic colonization of bone. This unique in vitro model could also facilitate cancer biologists to dissect specific biological hypotheses via extensive genomic or proteomic assessments to further our understanding of the PCa-bone crosstalk.

A humanized tissue-engineered in vivo model to dissect interactions between human prostate cancer cells and human bone

Currently used xenograft models for prostate cancer bone metastasis lack the adequate tissue composition necessary to study the interactions between human prostate cancer cells and the human bone microenvironment. We introduce a tissue engineering approach to explore the interactions between human tumor cells and a humanized bone microenvironment. Scaffolds, seeded with human primary osteoblasts in conjunction with BMP7, were implanted into immunodeficient mice to form humanized tissue engineered bone constructs (hTEBCs) which consequently resulted in the generation of highly vascularized and viable humanized bone. At 12 weeks, PC3 and LNCaP cells were injected into the hTEBCs. Seven weeks later the mice were euthanized. Micro-CT, histology, TRAP, PTHrP and osteocalcin staining results reflected the different characteristics of the two cell lines regarding their phenotypic growth pattern within bone. Microvessel density, as assessed by vWF staining, showed that tumor vessel density was significantly higher in LNCaP injected hTEBC implants than in those injected with PC3 cells (p\0.001). Interestingly, PC3 cells showed morphological features of epithelial and mesenchymal phenotypes suggesting a cellular plasticity within this microenvironment. Taken together, a highly reproducible humanized model was established which is successful in generating LNCaP and PC3 tumors within a complex humanized bone microenvironment. This model simulates the conditions seen clinically more closely than any other model described in the literature to date and hence represents a powerful experimental platform that can be used in future work to investigate specific biological questions relevant to bone metastasis.

Investigating the links between muscle strength, sun exposure, dietary vitamin D intake and the vitamin D status of ambulatory older adults in South East Queensland

Vitamin D deficiency and insufficiency are now seen as a contemporary health problem in Australia with possible widespread health effects not limited to bone health1. Despite this, the Vitamin D status (measured as serum 25-hydroxyvitamin D (25(OH)D)) of ambulatory adults has been overlooked in this country. Serum 25(OH)D status is especially important among this group as studies have shown a link between Vitamin D and fall risk in older adults2. Limited data also exists on the contributions of sun exposure via ultraviolet radiation and dietary intake to serum 25(OH)D status in this population. The aims of this project were to assess the serum 25(OH)D status of a group of older ambulatory adults in South East Queensland, to assess the association between their serum 25(OH)D status and functional measures as possible indicators of fall risk, obtain data on the sources of Vitamin D in this population and assess whether this intake was related to serum 25(OH)D status and describe sun protection and exposure behaviors in this group and investigate whether a relationship existed between these and serum 25(OH)D status. The collection of this data assists in addressing key gaps identified in the literature with regard to this population group and their Vitamin D status in Australia. A representative convenience sample of participants (N=47) over 55 years of age was recruited for this cross-sectional, exploratory study which was undertaken in December 2007 in south-east Queensland (Brisbane and Sunshine coast). Participants were required to complete a sun exposure questionnaire in addition to a Calcium and Vitamin D food frequency questionnaire. Timed up and go and handgrip dynamometry tests were used to examine functional capacity. Serum 25(OH)D status and blood measures of Calcium, Phosphorus and Albumin were determined through blood tests. The Mean and Median serum 25-Hydroxyvitamin D (25(OH)D) for all participants in this study was 85.8nmol/L (Standard Deviation 29.7nmol/L) and 81.0nmol/L (Range 22-158nmol/L), respectively. Analysis at the bivariate level revealed a statistically significant relationship between serum 25(OH)D status and location, with participants living on the Sunshine Coast having a mean serum 25(OH)D status 21.3nmol/L higher than participants living in Brisbane (p=0.014). While at the descriptive level there was an apparent trend towards higher outdoor exposure and increasing levels of serum 25(OH)D, no statistically significant associations between the sun measures of outdoor exposure, sun protection behaviors and phenotypic characteristics and serum 25(OH)D status were observed. Intake of both Calcium and Vitamin D was low in this sample with sixty-eight (68%) of participants not meeting the Estimated Average Requirements (EAR) for Calcium (Median=771.0mg; Range=218.0-2616.0mg), while eighty-seven (87%) did not meet the Adequate Intake for Vitamin D (Median=4.46ug; Range=0.13-30.0ug). This raises the question of how realistic meeting the new Adequate Intakes for Vitamin D is, when there is such a low level of Vitamin D fortification in this country. However, participants meeting the Adequate Intake (AI) for Vitamin D were observed to have a significantly higher serum 25(OH)D status compared to those not meeting the AI for Vitamin D (p=0.036), showing that meeting the AI for Vitamin D may play a significant role in determining Vitamin D status in this population. By stratifying our data by categories of outdoor exposure time, a trend was observed between increased importance of Vitamin D dietary intake as a possible determinant of serum 25(OH)D status in participants with lower outdoor exposures. While a trend towards higher Timed Up and Go scores in participants with higher 25(OH) D status was seen, this was only significant for females (p=0.014). Handgrip strength showed statistically significant association with serum 25(OH)D status. The high serum 25(OH)D status in our sample almost certainly explains the limited relationship between functional measures and serum 25(OH)D. However, the observation of an association between slower Time Up and Go speeds, and lower serum 25(OH)D levels, even with a small sample size, is significant as slower Timed Up and Go speeds have been associated with increased fall risk in older adults3. Multivariable regression analysis revealed Location as the only significant determinant of serum 25(OH)D status at p=0.014, with trends (p=>0.1) for higher serum 25(OH)D being shown for participants that met the AI for Vitamin D and rated themselves as having a higher health status. The results of this exploratory study show that 93.6% of participants had adequate 25(OH)D status-possibly due to measurement being taken in the summer season and the convenience nature of the sample. However, many participants do not meet their dietary Calcium and Vitamin D requirements, which may indicate inadequate intake of these nutrients in older Australians and a higher risk of osteoporosis. The relationship between serum 25(OH)D and functional measures in this population also requires further study, especially in older adults displaying Vitamin D insufficiency or deficiency.

Implication of 3D culture system for study of prostate cancer (CaP) mediated bone metastasis

Introduction : For the past decade, three dimensional (3D) culture has served as a foundation for regenerative medicine study. With an increasing awareness of the importance of cell-cell and cell-extracellular matrix interactions which are lacking in 2D culture system, 3D culture system has been employed for many other applications namely cancer research. Through development of various biomaterials and utilization of tissue engineering technology, many in vivo physiological responses are now better understood. The cellular and molecular communication of cancer cells and their microenvironment, for instance can be studied in vitro in 3D culture system without relying on animal models alone. Predilection of prostate cancer (CaP) to bone remains obscure due to the complexity of the mechanisms and lack of proper model for the studies. In this study, we aim to investigate the interaction between CaP cells and osteoblasts simulating the natural bone metastasis. We also further investigate the invasiveness of CaP cells and response of androgen sensitve CaP cells, LNCaP to synthetic androgen.----- Method : Human osteoblast (hOB) scaffolds were prepared by seeding hOB on medical grade polycaprolactone-tricalcium phosphate (mPLC-TCP) scaffolds and induced to produce bone matrix. CaP cell lines namely wild type PC3 (PC3-N), overexpressed prostate specific antigen PC3 (PC3k3s5) and LNCaP were seeded on hOB scaffolds as co-cultures. Morphology of cells was examined by Phalloidin-DAPI and SEM imaging. Gelatin zymography was performed on the 48 hours conditioned media (CM) from co-cultures to determine matrix metalloproteinase (MMP) activity. Gene expression of hOB/LNCaP co-cultures which were treated for 48 hours with 1nM synthetic androgen R1881 were analysed by quantitative real time PCR (qRT-PCR).----- Results : Co-culture of PCC/hOB revealed that the morphology of PCCs on the tissue engineered bone matrix varied from homogenous to heterogenous clusters. Enzymatically inactive pro-MMP2 was detected in CM from hOBs and PCCs cultured on scaffolds. Elevation in MMP9 activity was found only in hOB/PC3N co-culture. hOB/LNCaP co-culture showed increase in expression of key enzymes associated with steroid production which also corresponded to an increase in prostate specific antigen (PSA) and MMP9.----- Conclusions : Upregulation of MMP9 indicates involvement of ECM degradation during cancer invasion and bone metastases. Expression of enzymes involved in CaP progression, PSA, which is not expressed in osteoblasts, demonstrates that crosstalk between PCCs and osteoblasts may play a part in the aggressiveness of CaP. The presence of steroidogenic enzymes, particularly, RDH5, in osteoblasts and stimulated expression in co-culture, may indicate osteoblast production of potent androgens, fuelling cancer cell proliferation. Based on these results, this practical 3D culture system may provide greater understanding into CaP mediated bone metastasis. This allows the role of the CaP/hOB interaction with regards to invasive property and steroidogenesis to be further explored.

Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair

Healing large bone defects and non-unions remains a significant clinical problem. Current treatments, consisting of auto and allografts, are limited by donor supply and morbidity, insufficient bioactivity and risk of infection. Biotherapeutics, including cells, genes and proteins, represent promising alternative therapies, but these strategies are limited by technical roadblocks to biotherapeutic delivery, cell sourcing, high cost, and regulatory hurdles. In the present study, the collagen-mimetic peptide, GFOGER, was used to coat synthetic PCL scaffolds to promote bone formation in critically-sized segmental defects in rats. GFOGER is a synthetic triple helical peptide that binds to the [alpha]2[beta]1 integrin receptor involved in osteogenesis. GFOGER coatings passively adsorbed onto polymeric scaffolds, in the absence of exogenous cells or growth factors, significantly accelerated and increased bone formation in non-healing femoral defects compared to uncoated scaffolds and empty defects. Despite differences in bone volume, no differences in torsional strength were detected after 12 weeks, indicating that bone mass but not bone quality was improved in this model. This work demonstrates a simple, cell/growth factor-free strategy to promote bone formation in challenging, non-healing bone defects. This biomaterial coating strategy represents a cost-effective and facile approach, translatable into a robust clinical therapy for musculoskeletal applications.