Application of high voltage, high frequency pulsed electromagnetic field on cortical bone tissue

Over the last few decades, electric and electromagnetic fields have achieved important role as stimulator and therapeutic facility in biology and medicine. In particular, low magnitude, low frequency, pulsed electromagnetic field has shown significant positive effect on bone fracture healing and some bone diseases treatment. Nevertheless, to date, little attention has been paid to investigate the possible effect of high frequency, high magnitude pulsed electromagnetic field (pulse power) on functional behaviour and biomechanical properties of bone tissue. Bone is a dynamic, complex organ, which is made of bone materials (consisting of organic components, inorganic mineral and water) known as extracellular matrix, and bone cells (live part). The cells give the bone the capability of self-repairing by adapting itself to its mechanical environment. The specific bone material composite comprising of collagen matrix reinforced with mineral apatite provides the bone with particular biomechanical properties in an anisotropic, inhomogeneous structure. This project hypothesized to investigate the possible effect of pulse power signals on cortical bone characteristics through evaluating the fundamental mechanical properties of bone material. A positive buck-boost converter was applied to generate adjustable high voltage, high frequency pulses up to 500 V and 10 kHz. Bone shows distinctive characteristics in different loading mode. Thus, functional behaviour of bone in response to pulse power excitation were elucidated by using three different conventional mechanical tests applying three-point bending load in elastic region, tensile and compressive loading until failure. Flexural stiffness, tensile and compressive strength, hysteresis and total fracture energy were determined as measure of main bone characteristics. To assess bone structure variation due to pulse power excitation in deeper aspect, a supplementary fractographic study was also conducted using scanning electron micrograph from tensile fracture surfaces. Furthermore, a non-destructive ultrasonic technique was applied for determination and comparison of bone elasticity before and after pulse power stimulation. This method provided the ability to evaluate the stiffness of millimetre-sized bone samples in three orthogonal directions. According to the results of non-destructive bending test, the flexural elasticity of cortical bone samples appeared to remain unchanged due to pulse power excitation. Similar results were observed in the bone stiffness for all three orthogonal directions obtained from ultrasonic technique and in the bone stiffness from the compression test. From tensile tests, no significant changes were found in tensile strength and total strain energy absorption of the bone samples exposed to pulse power compared with those of the control samples. Also, the apparent microstructure of the fracture surfaces of PP-exposed samples (including porosity and microcracks diffusion) showed no significant variation due to pulse power stimulation. Nevertheless, the compressive strength and toughness of millimetre-sized samples appeared to increase when the samples were exposed to 66 hours high power pulsed electromagnetic field through screws with small contact cross-section (increasing the pulsed electric field intensity) compare to the control samples. This can show the different load-bearing characteristics of cortical bone tissue in response to pulse power excitation and effectiveness of this type of stimulation on smaller-sized samples. These overall results may address that although, the pulse power stimulation can influence the arrangement or the quality of the collagen network causing the bone strength and toughness augmentation, it apparently did not affect the mineral phase of the cortical bone material. The results also confirmed that the indirect application of high power pulsed electromagnetic field at 500 V and 10 kHz through capacitive coupling method, was athermal and did not damage the bone tissue construction.

Studies of bone cell adhesion and proliferation on ion implanted titanium discs

Argon ions were implanted on titanium discs to study its effect on bone cell adhesion and proli feration. Polished titanium discs were prepared and implanted with argon ions with different doses. Afterwards the samples were sterilized using UV light, inocu lated with human bone cells and incubated. Once fixed and rinsed, image analysis has been used to quantify the number of cells attached to the titanium discs. Cell proliferation tests were also conducted after a period of 120 hours. Cell adhesion was seen to be higher with ion im planted surface. SEM analysis has shown that the cells attached spread more on ion implanted surface. The numbers of cells attached were seen to be higher on implanted surfaces; they tend to occupy wider areas with healthier cells.

Mesenchymal stem cells and nano-structured surfaces

Mesenchymal stem cells (MSCs) represent multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells). Their multi-potency provides a great promise as a cell source for tissue engineering and cell-based therapy for many diseases, particularly bone diseases and bone formation. To be able to direct and modulate the differentiation of MSCs into the desired cell types in situ in the tissue, nanotechnology is introduced and used to facilitate or promote cell growth and differentiation. These nano-materials can provide a fine structure and tuneable surface in nanoscales to help the cell adhesion and promote the cell growth and differentiation of MSCs. This could be a dominant direction in future for stem cells based therapy or tissue engineering for various diseases. Therefore, the isolation, manipulation, and differentiation of MSCs are very important steps to make meaningful use of MSCs for disease treatments. In this chapter, we have described a method of isolating MSC from human bone marrow, and how to culture and differentiate them in vitro. We have also provided research methods on how to use MSCs in an in vitro model and how to observe MSC biological response on the surface of nano-scaled materials.

Epithelial-mesenchymal transition and mesenchymal-epithelial transition are essential for the acquisition of stem cell properties in hTERT-immortalised oral epithelial cells

BACKGROUND INFORMATION: Evidence has shown that mesenchymal-epithelial transition (MET) and epithelial-mesenchymal transition (EMT) are linked to stem cell properties. We currently lack a model showing how the occurrence of MET and EMT in immortalised cells influences the maintenance of stem cell properties. Thus, we established a project aiming to investigate the roles of EMT and MET in the acquisition of stem cell properties in immortalised oral epithelial cells. RESULTS: In this study, a retroviral transfection vector (pLXSN-hTERT) was used to immortalise oral epithelial cells by insertion of the hTERT gene (hTERT(+)-oral mucosal epithelial cell line [OME]). The protein and RNA expression of EMT transcriptional factors (Snail, Slug and Twist), their downstream markers (E-cadherin and N-cadherin) and embryonic stem cell markers (OCT4, Nanog and Sox2) were studied by reverse transcription PCR and Western blots in these cells. Some EMT markers were detected at both mRNA and protein levels. Adipocytes and bone cells were noted in the multi-differentiation assay, showing that the immortal cells underwent EMT. The differentiation assay for hTERT(+)-OME cells revealed the recovery of epithelial phenotypes, implicating the presence of MET. The stem cell properties were confirmed by the detection of appropriate markers. Altered expression of alpha-tubulin and gamma-tubulin in both two-dimensional-cultured (without serum) and three-dimensional-cultured hTERT(+)-OME spheroids indicated the re-programming of cytoskeleton proteins which is attributed to MET processes in hTERT(+)-OME cells. CONCLUSIONS: EMT and MET are essential for hTERT-immortalised cells to maintain their epithelial stem cell properties.

A 3D in vitro model reflecting the androgen deprivation state in prostate cancer bone metastasis

In castrate-resistant prostate cancer (CRPC), the prevailing organ for metastasis is bone, where the survival of cancer cells is regulated by the permissive metastatic niche offered by the bone marrow. The tumour microenvironment and cellular interactions with the matrix and bone cells enable metastasis and lead to cancer cells becoming androgen resistant. Hence, 3D models that mimic CRPC in terms of an androgen deprivation state (ADS) are needed to identify the mechanisms for CPRC growth in bone and further develop therapeutic strategies.

Kallikrein-related peptidase 4 activation of protease-activated receptor family members and association with prostate cancer

Two areas of particular importance in prostate cancer progression are primary tumour development and metastasis. These processes involve a number of physiological events, the mediators of which are still being discovered and characterised. Serine proteases have been shown to play a major role in cancer invasion and metastasis. The recently discovered phenomenon of their activation of a receptor family known as the protease activated receptors (PARs) has extended their physiological role to that of signaling molecule. Several serine proteases are expressed by malignant prostate cancer cells, including members of the kallikreinrelated peptidase (KLK) serine protease family, and increasingly these are being shown to be associated with prostate cancer progression. KLK4 is highly expressed in the prostate and expression levels increase during prostate cancer progression. Critically, recent studies have implicated KLK4 in processes associated with cancer. For example, the ectopic over-expression of KLK4 in prostate cancer cell lines results in an increased ability of these cells to form colonies, proliferate and migrate. In addition, it has been demonstrated that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone forming cells). The ability of KLK4 to influence cellular behaviour is believed to be through the selective cleavage of specific substrates. Identification of relevant in vivo substrates of KLK4 is critical to understanding the pathophysiological roles of this enzyme. Significantly, recent reports have demonstrated that several members of the KLK family are able to activate PARs. The PARs are relatively new members of the seven transmembrane domain containing G protein coupled receptor (GPCR) family. PARs are activated through proteolytic cleavage of their N-terminus by serine proteases, the resulting nascent N-terminal binds intramolecularly to initiate receptor activation. PARs are involved in a number of patho-physiological processes, including vascular repair and inflammation, and a growing body of evidence suggests roles in cancer. While expression of PAR family members has been documented in several types of cancers, including prostate, the role of these GPCRs in prostate cancer development and progression is yet to be examined. Interestingly, several studies have suggested potential roles in cellular invasion through the induction of cytoskeletal reorganisation and expression of basement membrane-degrading enzymes. Accordingly, this program of research focussed on the activation of the PARs by the prostate cancer associated enzyme KLK4, cellular processing of activated PARs and the expression pattern of receptor and agonist in prostate cancer. For these studies KLK4 was purified from the conditioned media of stably transfected Sf9 insect cells expressing a construct containing the complete human KLK4 coding sequence in frame with a V5 epitope and poly-histidine encoding sequences. The first aspect of this study was the further characterisation of this recombinant zymogen form of KLK4. The recombinant KLK4 zymogen was demonstrated to be activatable by the metalloendopeptidase thermolysin and amino terminal sequencing indicated that thermolysin activated KLK4 had the predicted N-terminus of mature active KLK4 (31IINED). Critically, removal of the pro-region successfully generated a catalytically active enzyme, with comparable activity to a previously published recombinant KLK4 produced from S2 insect cells. The second aspect of this study was the activation of the PARs by KLK4 and the initiation of signal transduction. This study demonstrated that KLK4 can activate PAR-1 and PAR-2 to mobilise intracellular Ca2+, but failed to activate PAR-4. Further, KLK4 activated PAR-1 and PAR-2 over distinct concentration ranges, with KLK4 activation and mobilisation of Ca2+ demonstrating higher efficacy through PAR-2. Thus, the remainder of this study focussed on PAR-2. KLK4 was demonstrated to directly cleave a synthetic peptide that mimicked the PAR-2 Nterminal activation sequence. Further, KLK4 mediated Ca2+ mobilisation through PAR-2 was accompanied by the initiation of the extra-cellular regulated kinase (ERK) cascade. The specificity of intracellular signaling mediated through PAR-2 by KLK4 activation was demonstrated by siRNA mediated protein depletion, with a reduction in PAR-2 protein levels correlating to a reduction in KLK4 mediated Ca2+mobilisation and ERK phosphorylation. The third aspect of this study examined cellular processing of KLK4 activated PAR- 2 in a prostate cancer cell line. PAR-2 was demonstrated to be expressed by five prostate derived cell lines including the prostate cancer cell line PC-3. It was also demonstrated by flow cytometry and confocal microscopy analyses that activation of PC-3 cell surface PAR-2 by KLK4 leads to internalisation of this receptor in a time dependent manner. Critically, in vivo relevance of the interaction between KLK4 and PAR-2 was established by the observation of the co-expression of receptor and agonist in primary prostate cancer and prostate cancer bone lesion samples by immunohistochemical analysis. Based on the results of this study a number of exciting future studies have been proposed, including, delineating differences in KLK4 cellular signaling via PAR-1 and PAR-2 and the role of PAR-1 and PAR-2 activation by KLK4 in prostate cancer cells and bone cells in prostate cancer progression.

The ancient gene C12orf29 : an exploration of its role in the chordate body plan

The sheep (Ovis aries) is commonly used as a large animal model in skeletal research. Although the sheep genome has been sequenced there are still only a limited number of annotated mRNA sequences in public databases. A complementary DNA (cDNA) library was constructed to provide a generic resource for further exploration of genes that are actively expressed in bone cells in sheep. It was anticipated that the cDNA library would provide molecular tools for further research into the process of fracture repair and bone homeostasis, and add to the existing body of knowledge. One of the hallmarks of cDNA libraries has been the identification of novel genes and in this library the full open reading frame of the gene C12orf29 was cloned and characterised. This gene codes for a protein of unknown function with a molecular weight of 37 kDa. A literature search showed that no previous studies had been conducted into the biological role of C12orf29, except for some bioinformatics studies that suggested a possible link with cancer. Phylogenetic analyses revealed that C12orf29 had an ancient pedigree with a homologous gene found in some bacterial taxa. This implied that the gene was present in the last common eukaryotic ancestor, thought to have existed more than 2 billion years ago. This notion was further supported by the fact that the gene is found in taxa belonging to the two major eukaryotic branches, bikonts and unikonts. In the bikont supergroup a C12orf29-like gene was found in the single celled protist Naegleria gruberi, whereas in the unikont supergroup, encompassing the metazoa, the gene is universal to all chordate and, therefore, vertebrate species. It appears to have been lost to the majority of cnidaria and protostomes taxa; however, C12orf29-like genes have been found in the cnidarian freshwater hydra and the protostome Pacific oyster. The experimental data indicate that C12orf29 has a structural role in skeletal development and tissue homeostasis, whereas in silico analysis of the human C12orf29 promoter region suggests that its expression is potentially under the control of the NOTCH, WNT and TGF- developmental pathways, as well SOX9 and BAPX1; pathways that are all heavily involved in skeletogenesis. Taken together, this investigation provides strong evidence that C12orf29 has a very important role in the chordate body plan, in early skeletal development, cartilage homeostasis, and also a possible link with spina bifida in humans.

Role of microRNAs in improved osteogenicity of topographically modified titanium implant surfaces

This project aimed at understanding the molecular mechanisms involved in the superior integration of micro-roughened titanium implant surfaces with the surrounding bone, when compared with their smooth surfaces. It involved studying the role of microRNAs and cell signaling pathways in the molecular regulation of bone cells on topographically modified titanium dental implants. The findings suggest a highly regulated microRNA-mediated control of molecular mechanisms during the process of bone formation that may be responsible for the superior osseointegration properties on micro-roughened titanium implant surfaces and indicate the possibility of using microRNA modulators to enhance osseointegration in clinically demanding circumstances.

Comparison of ovine mesenchymal cell and osteoblast osteogeic capacity in 2D and 3D

This thesis explored the different bone-forming potential of specific bone cells with differing embryological origin, on conventional culture platforms compared to 3D biocompatible scaffolds in vitro. Bone mesenchymal stem cells, mandibular osteoblasts and long bone osteoblasts from adult and juvenile sheep were compared in the study, as the embryological origin of the osteoblasts from the craniofacial and appendicular skeleton differs. The study demonstrated differing characteristics of the various cell types when cultured on the two different platforms compared and this may have an impact on future research into cell seeded tissue scaffolds to aid in vivo tissue regeneration.

Environments for zonal cartilage tissue engineering

Articular cartilage is a highly organized tissue with cellular and matrix properties that vary with depth zones. Regenerating this zonal organization has proven difficult in tissue-engineered cartilage to treat damaged cartilage. In this thesis, we evaluated the effects of culture environments that mimic aspects of the native cartilage environment on chondrocyte subpopulations. We found that decellularized cartilage matrix can improve zonal tissue-engineered cartilage. Also, chondrocytes respond to signals from bone cells and compressive stimulation in a zone-dependent manner. These results highlight the importance of a zone-specific environment to improve tissue-engineered cartilage in vitro.

Shaping development through mechanical strain: The transcriptional basis of diet-induced phenotypic plasticity in a cichlid fish

Adaptive phenotypic plasticity, the ability of an organism to change its phenotype to match local environments, is increasingly recognized for its contribution to evolution. However, few empirical studies have explored the molecular basis of plastic traits. The East African cichlid fish Astatoreochromis alluaudi displays adaptive phenotypic plasticity in its pharyngeal jaw apparatus, a structure that is widely seen as an evolutionary key innovation that has contributed to the remarkable diversity of cichlid fishes. It has previously been shown that in response to different diets, the pharyngeal jaws change their size, shape and dentition: hard diets induce an adaptive robust molariform tooth phenotype with short jaws and strong internal bone structures, while soft diets induce a gracile papilliform tooth phenotype with elongated jaws and slender internal bone structures. To gain insight into the molecular underpinnings of these adaptations and enable future investigations of the role that phenotypic plasticity plays during the formation of adaptive radiations, the transcriptomes of the two divergent jaw phenotypes were examined. Our study identified a total of 187 genes whose expression differs in response to hard and soft diets, including immediate early genes, extracellular matrix genes and inflammatory factors. Transcriptome results are interpreted in light of expression of candidate genesmarkers for tooth size and shape, bone cells and mechanically sensitive pathways. This study opens up new avenues of research at new levels of biological organization into the roles of phenotypic plasticity during speciation and radiation of cichlid fishes.

The effects of osteoporosis on osseointegration in the rat maxilla

The study investigated the effects of oestrogen deficiency on dental implant in a rat model. An osteoporosis rat model was successfully established for dental implant research and it was noted that bone cells functioned differently in osteoporotic condition during the healing of dental implant. The study further demonstrated that implant surface roughness could stimulate bone formation, therefore, improve the bone healing in osteoporotic condition.

Multilineage differentiation potential of bone and cartilage cells derived from explant culture

To date, mesenchymal stem cells (MSCs) from various tissues have been reported, but the yield and differentiation potential of different tissue-derived MSCs is still not clear. This study was undertaken in an attempt to investigate the multilineage stem cell potential of bone and cartilage explant cultures in comparison with bone marrow derived mesenchymal stem cells (BMSCs). The results showed that the surface antigen expression of tissue-derived cells was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing markers related to adhesion (CD29, CD166) and stem cells (CD90, CD105). The tissue-derived cells were able to differentiate into osteoblast, chondrocyte and adipocyte lineage pathways when stimulated in the appropriate differentiating conditions. However, compared with BMSCs, tissue-derived cells showed less capacity for multilineage differentiation when the level of differentiation was assessed in monolayer culture by analysing the expression of tissue-specific genes by reverse transcription polymerase chain reaction (RT-PCR) and histology. In high density pellet cultures, tissue-derived cells were able to differentiate into chondrocytes, expressing chondrocyte markers such as proteoglycans, type II collagen and aggrecan. Taken together, these results indicate that cells derived from tissue explant cultures reserved certain degree of differentiation properties of MSCs in vitro.

Cellular senescence and longevity of osteophyte-derived mesenchymal stem cells compared to patient-matched bone marrow stromal cells

This study aimed to determine the cellular aging of osteophyte-derived mesenchymal cells (oMSCs) in comparison to patient-matched bone marrow stromal cells (bMSCs). Extensive expansion of the cell cultures was performed and early and late passage cells (passages 4 and 9, respectively) were used to study signs of cellular aging, telomere length, telomerase activity, and cell-cycle-related gene expression. Our results showed that cellular aging was more prominent in bMSCs than in oMSCs, and that oMSCs had longer telomere length in late passages compared with bMSCs, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSCs and not in bMSCs. In osteophyte tissues telomerase-positive cells were found to be located perivascularly and were Stro-1 positive. Fifteen cell-cycle regulator genes were investigated and only three genes (APC, CCND2, and BMP2) were differentially expressed between bMSC and oMSC. Our results indicate that oMSCs retain a level of telomerase activity in vitro, which may account for the relatively greater longevity of these cells, compared with bMSCs, by preventing replicative senescence. J. Cell. Biochem. 108: 839-850, 2009. (c) 2009 Wiley-Liss, Inc.