The microRNA expression signature on modified titanium implant surfaces influences genetic mechanisms leading to osteogenic differentiation

Topographically and chemically modified titanium implants are recognized to have improved osteogenic properties; however, the molecular regulation of this process remains unknown. This study aimed to determine the microRNA profile and the potential regulation of osteogenic differentiation following early exposure of osteoprogenitor cells to sand-blasted, large-grit acid-etched (SLA) and hydrophilic SLA (modSLA) surfaces. Firstly, the osteogenic characteristics of the primary osteoprogenitor cells were confirmed using ALP activity and Alizarin Red S staining. The effect of smooth (SMO), SLA and modSLA surfaces on the TGF-β/BMP (BMP2, BMP6, ACVR1) and non-canonical WNT/Ca2+ (WNT5A, FZD6) pathways, as well as the integrins ITGB1 and ITGA2, was determined. It was revealed that the modified titanium surfaces could induce the activation of TGF-β/BMP and non-canonical WNT/Ca2+ signaling genes. The expression pattern of microRNAs (miRNAs) related to cell differentiation was evaluated. Statistical analysis of the differentially regulated miRNAs indicated that 35 and 32 miRNAs were down-regulated on the modSLA and SLA surfaces respectively, when compared with the smooth surface (SMO). Thirty-one miRNAs that were down-regulated were common to both modSLA and SLA. There were 10 miRNAs up-regulated on modSLA and nine on SLA surfaces, amongst which eight were the same as observed on modSLA. TargetScan predictions for the down-regulated miRNAs revealed genes of the TGF-β/BMP and non-canonical Ca2+ pathways as targets. This study demonstrated that modified titanium implant surfaces induce differential regulation of miRNAs, which potentially regulate the TGF-β/BMP and WNT/Ca2+ pathways during osteogenic differentiation on modified titanium implant surfaces.

Development of a 3D culture system to study the skeletal metastasis of prostate cancer

In the cancer research field, most in vitro studies still rely on two-dimensional (2D) cultures. However, the trend is rapidly shifting towards using a three-dimensional (3D) culture system. This is because 3D models better recapitulate the microenvironment of cells, and therefore, yield cellular and molecular responses that more accurately describe the pathophysiology of cancer. By adopting technology platforms established by the tissue engineering discipline, it is now possible to grow cancer cells in extracellular matrix (ECM)-like environments and dictate the biophysical and biochemical properties of the matrix. In addition, 3D models can be modified to recapitulate different stages of cancer progression for instance from the initial development of tumor to metastasis. Inevitably, to recapitulate a heterotypic condition, comprising more than one cell type, it requires a more complex 3D model. To date, 3D models that are available for studying the prostate cancer (CaP)-bone interactions are still lacking. Therefore, the aim of this study is to establish a co-culture model that allows investigation of direct and indirect CaP-bone interactions. Prior to that, 3D polyethylene glycol (PEG)-based hydrogel cultures for CaP cells were first developed and growth conditions were optimised. Characterization of the 3D hydrogel cultures show that LNCaP cells form a multicellular mass that resembles avascular tumor. In comparison to 2D cultures, besides the difference in cell morphology, the response of LNCaP cells to the androgen analogue (R1881) stimulation is different compared to the cells in 2D cultures. This discrepancy between 2D and 3D cultures is likely associated with the cell-cell contact, density and ligand-receptor interactions. Following the 3D monoculture study, a 3D direct co-culture model of CaP cells and the human tissue engineered bone (hTEBC) construct was developed. Interactions between the CaP cells and human osteoblasts (hOBs) resulted in elevation of Matrix Metalloproteinase 9 (MMP9) for PC-3 cells and Prostate Specific Antigen (PSA) for LNCaP cells. To further investigate the paracrine interaction of CaP cells and (hOBs), a 3D indirect co-culture model was developed, where LNCaP cells embedded within PEG hydrogels were co-cultured with hTEBC. It was found that the cellular changes observed reflect the early event of CaP colonizing the bone site. In the absence of androgens, interestingly, up-regulation of PSA and other kallikreins is also detected in the co-culture compared to the LNCaP monoculture. This non androgenic stimulation could be triggered by the soluble factors secreted by the hOB such as Interleukin-6. There are also decrease in alkaline phosphatase (ALP) activity and down-regulation of genes of the hOB when co-cultured with LNCaP cells that have not been previously described. These genes include transforming growth factor β1 (TGFβ1), osteocalcin and Vimentin. However, no changes to epithelial markers (e.g E-cadherin, Cytokeratin 8) were observed in both cell types from the co-culture. Some of these intriguing changes observed in the co-cultures that had not been previously described have enriched the basic knowledge of the CaP cell-bone interaction. From this study, we have shown evidence of the feasibility and versatility of our established 3D models. These models can be adapted to test various hypotheses for studies pertaining to underlying mechanisms of bone metastasis and could provide a vehicle for anticancer drug screening purposes in the future.

Biological responses of human bone marrow mesenchymal stem cells to Sr-M-Si (M = Zn, Mg) silicate bioceramics

Strontium (Sr), Zinc (Zn), magnesium (Mg), and silicon (Si) are reported to be essential trace elements for the growth and mineralization of bone. We speculated that the combination of these bioactive elements in bioceramics may be effective to regulate the osteogenic property of boneforming cells. In this study, two Sr-containing silicate bioceramics, Sr2ZnSi2O7 (SZS) and Sr2MgSi2O7 (SMS), were prepared. The biological response of human bone marrow mesenchymal stem cells (BMSCs) to the two bioceramics (in the forms of powders and dense ceramic bulks) was systematically studied. In powder form, the effect of powder extracts on the viability and alkaline phosphatase (ALP) activity of BMSCs was investigated. In ceramic disc form, both direct and indirect coculture of BMSCs with ceramic discs were used to investigate their biological response, including attachment, proliferation, ALP activity, and bone-related genes expression. Beta-tricalcium phosphate (b-TCP) and akermanite (Ca2MgSi2O7, CMS) were used as control materials. The results showed that the Sr, Zn, and Si (or Sr, Mg, and Si)-containing ionic products from SZS and SMS powders enhanced ALP activity of BMSCs, compared to those from b-TCP. Both SZS and SMS ceramic discs supported the growth of BMSCs, and most importantly, significantly enhanced the ALP activity and bone-related genes expression of BMSCs as compared to b-TCP. The results suggest that the specific combination of bioactive ions (Sr, Zn, Si, e.g.) in bioceramics is a viable way to improve the biological performance of biomaterials, and the form of materials and surface properties were nonnegligible factors to influence cell response.

Is flapless implant surgery a viable option in posterior maxilla? : a review

This article reviews the literature on the outcome of flapless surgery for dental implants in the posterior maxilla. The literature search was carried out in using the keywords: flapless, dental implants and maxilla. A hand search and Medline search were carried out on studies published between 1971 and 2011. The authors included research involving a minimum of 15 dental implants with a followup period of 1 year, an outcome measurement of implant survival, but excluded studies involving multiple simultaneous interventions, and studies with missing data. The Cochrane approach for cohort studies and Oxford Centre for Evidence- Based Medicine were applied. Of the 56 published papers selected, 14 papers on the flapless technique showed high overall implant survival rates. The prospective studies yielded 97.01% (95% CI: 90.72–99.0) while retrospective studies or case series illustrated 95.08% (95% CI: 91.0–97.93) survival. The average of intraoperative complications was 6.55% using the flapless procedure. The limited data obtained showed that flapless surgery in posterior maxilla areas could be a viable and predictable treatment method for implant placement. Flapless surgery tends to be more applicable in this area of the mouth. Further long-term clinical controlled studies are needed.

A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex

This study describes the design of a biphasic scaffold composed of a Fused Deposition Modeling scaffold (bone compartment) and an electrospun membrane (periodontal compartment) for periodontal regeneration. In order to achieve simultaneous alveolar bone and periodontal ligament regeneration a cell-based strategy was carried out by combining osteoblast culture in the bone compartment and placement of multiple periodontal ligament (PDL) cell sheets on the electrospun membrane. In vitro data showed that the osteoblasts formed mineralized matrix in the bone compartment after 21 days in culture and that the PDL cell sheet harvesting did not induce significant cell death. The cell-seeded biphasic scaffolds were placed onto a dentin block and implanted for 8 weeks in an athymic rat subcutaneous model. The scaffolds were analyzed by μCT, immunohistochemistry and histology. In the bone compartment, a more intense ALP staining was obtained following seeding with osteoblasts, confirming the μCT results which showed higher mineralization density for these scaffolds. A thin mineralized cementum-like tissue was deposited on the dentin surface for the scaffolds incorporating the multiple PDL cell sheets, as observed by H&E and Azan staining. These scaffolds also demonstrated better attachment onto the dentin surface compared to no attachment when no cell sheets were used. In addition, immunohistochemistry revealed the presence of CEMP1 protein at the interface with the dentine. These results demonstrated that the combination of multiple PDL cell sheets and a biphasic scaffold allows the simultaneous delivery of the cells necessary for in vivo regeneration of alveolar bone, periodontal ligament and cementum. © 2012 Elsevier Ltd.

Mechanical considerations in fracture fixation

Bone’s capacity to repair following trauma is both unique and astounding. However, fractures sometimes fail to heal. Hence, the goal of fracture treatment is the restoration of bone’s structure, composition and function. Fracture fixation devices should provide a favourable mechanical and biological environment for healing to occur. The use of internal fixation is increasing as these devices may be applied with less invasive techniques. Recent studies suggest however that, internal fixation devices may be overly stiff and suppresses callus formation. The degree of mechanical stability influences the healing outcome. This is determined by the stiffness of the fixation device and the degree of limb loading. This project aims to characterise the fixation stability of an internal plate fixation device and the influence of modifications to its configuration on implant stability. As there are no standardised methods for the determination of fixation stiffness, the first part of this project aims to compares different methodologies and determines the most appropriate method to characterise the stiffness of internal plate fixators. The stiffness of a fixation device also influences the physiological loads experienced by the healing bone. Since bone adapts to this applied load by undergoing changes through a remodelling process, undesirable changes could occur during the period of treatment with an implant. The second part of this project aims to develop a methodology to quantify remodelling changes. This quantification is expected to aid our understanding of the changes in pattern due to implant related remodelling and on the factors driving the remodelling process. Knowledge gained in this project is useful to understand how the configuration of internal fixation devices can promote timely healing and prevent undesirable bone loss.

Fast fourier analysis of structural organization in decellularized cartilage-on-bone laminates

Denaturation of tissues can provide a unique biological environment for regenerative medicine application only if minimal disruption of their microarchitecture is achieved during the decellularization process. The goal is to keep the structural integrity of such a construct as functional as the tissues from which they were derived. In this work, cartilage-on-bone laminates were decellularized through enzymatic, non-ionic and ionic protocols. This work investigated the effects of decellularization process on the microarchitecture of cartiligous extracellular matrix; determining the extent of how each process deteriorated the structural organization of the network. High resolution microscopy was used to capture cross-sectional images of samples prior to and after treatment. The variation of the microarchitecture was then analysed using a well defined fast Fourier image processing algorithm. Statistical analysis of the results revealed how significant the alternations among aforementioned protocols were (p < 0.05). Ranking the treatments by their effectiveness in disrupting the ECM integrity, they were ordered as: Trypsin> SDS> Triton X-100.

Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration.

There is a growing need for successful bone tissue engineering strategies and advanced biomaterials that mimic the structure and function of native tissues carry great promise. Successful bone repair approaches may include an osteoconductive scaffold, osteoinductive growth factors, cells with an osteogenic potential and capacity for graft vascularisation. To increase osteoinductivity of biomaterials, the local combination and delivery of growth factors has been developed. In the present study we investigated the osteogenic effects of calcium phosphate (CaP)-coated nanofiber mesh tube-mediated delivery of BMP-7 from a PRP matrix for the regeneration of critical sized segmental bone defects in a small animal model. Bilateral full-thickness diaphyseal segmental defects were created in twelve male Lewis rats and nanofiber mesh tubes were placed around the defect. Defects received either treatment with a CaP-coated nanofiber mesh tube (n = 6), an un-coated nanofiber mesh tube (n=6) a CaP-coated nanofiber mesh tube with PRP (n=6) or a CaP-coated nanofiber mesh tube in combination with 5 μg BMP-7 and PRP (n = 6). After 12 weeks, bone volume and biomechanical properties were evaluated using radiography, microCT, biomechanical testing and histology. The results demonstrated significantly higher biomechanical properties and bone volume for the BMP group compared to the control groups. These results were supported by the histological evaluations, where BMP group showed the highest rate of bone regeneration within the defect. In conclusion, BMP-7 delivery via PRP enhanced functional bone defect regeneration, and together these data support the use of BMP-7 in the treatment of critical sized defects.

Influence of plate bending stiffness on mechanical properties of healing fracture, mouse femora

Mechanically well-defined stabilization systems have only recently become available, providing standardized conditions for studying the role of the mechanical environment on mouse bone fracture healing. The aim of this study was to characterize the time course of strength recovery and callus development of mouse femoral osteotomies stabilized with either low or high flexibility (in bending and torsion) internal fixation plates. Animals were euthanized and femora excised at 14, 21, and 28 days post-osteotomy for microCT analysis and torsional strength testing. While a larger mineralized callus was observed in osteotomies under more flexible conditions at all time points, the earlier bridging of the mineralized callus under less flexible conditions by 1 week resulted in an earlier recovery of torsional strength in mice stabilized with low flexibility fixation. Ultimate torque values for these bones were significantly higher at 14 and 21 days post-osteotomy compared to bones with the more flexible stabilization. Our study confirms the high reproducibility of the results that are achieved with this new implant system, therefore making it ideal for studying the influence of the mechanical environment on murine fracture healing under highly standardized conditions.

Evolutionary design of bone scaffolds with reference to material selection

The favourable scaffold for bone tissue engineering should have desired characteristic features, such as adequate mechanical strength and three-dimensional open porosity, which guarantee a suitable environment for tissue regeneration. In fact, the design of such complex structures like bone scaffolds is a challenge for investigators. One of the aims is to achieve the best possible mechanical strength-degradation rate ratio. In this paper we attempt to use numerical modelling to evaluate material properties for designing bone tissue engineering scaffold fabricated via the fused deposition modelling technique. For our studies the standard genetic algorithm was used, which is an efficient method of discrete optimization. For the fused deposition modelling scaffold, each individual strut is scrutinized for its role in the architecture and structural support it provides for the scaffold, and its contribution to the overall scaffold was studied. The goal of the study was to create a numerical tool that could help to acquire the desired behaviour of tissue engineered scaffolds and our results showed that this could be achieved efficiently by using different materials for individual struts. To represent a great number of ways in which scaffold mechanical function loss could proceed, the exemplary set of different desirable scaffold stiffness loss function was chosen. © 2012 John Wiley & Sons, Ltd.

Water–rock interactions : an investigation of the relationships between mineralogy and groundwater composition and flow in a subtropical basalt aquifer

A holistic study of the composition of the basalt groundwaters of the Atherton Tablelands region in Queensland, Australia was undertaken to elucidate possible mechanisms for the evolution of these very low salinity, silica- and bicarbonate-rich groundwaters. It is proposed that aluminosilicate mineral weathering is the major contributing process to the overall composition of the basalt groundwaters. The groundwaters approach equilibrium with respect to the primary minerals with increasing pH and are mostly in equilibrium with the major secondary minerals (kaolinite and smectite), and other secondary phases such as goethite, hematite, and gibbsite, which are common accessory minerals in the Atherton basalts. The mineralogy of the basalt rocks, which has been examined using X-ray diffraction and whole rock geochemistry methods, supports the proposed model for the hydrogeochemical evolution of these groundwaters: precipitation + CO 2 (atmospheric + soil) + pyroxene + feldspars + olivine yields H 4SiO 4, HCO 3 -, Mg 2+, Na +, Ca 2+ + kaolinite and smectite clays + amorphous or crystalline silica + accessory minerals (hematite, goethite, gibbsite, carbonates, zeolites, and pyrite). The variations in the mineralogical content of these basalts also provide insights into the controls on groundwater storage and movement in this aquifer system. The fresh and weathered vesicular basalts are considered to be important in terms of zones of groundwater occurrence, while the fractures in the massive basalt are important pathways for groundwater movement.

An improved model of tumour-immune system interactions

The immune system plays an important role in defending the body against tumours and other threats. Currently, mechanisms involved in immune system interactions with tumour cells are not fully understood. Here we develop a mathematical tool that can be used in aiding to address this shortfall in understanding. This paper de- scribes a hybrid cellular automata model of the interaction between a growing tumour and cells of the innate and specific immune system including the effects of chemokines that builds on previous models of tumour-immune system interactions. In particular, the model is focused on the response of immune cells to tumour cells and how the dynamics of the tumour cells change due to the immune system of the host. We present results and predictions of in silico experiments including simulations of Kaplan-Meier survival-like curves.