Revascularization and tissue regeneration of an empty root canal space is enhanced by a direct blood supply and stem cells

Background Regenerative endodontics is an innovative treatment concept aiming to regenerate pulp, dentin and root structures. In the diseased or necrotic tooth, the limitation in vascular supply renders successful tissue regeneration/generation in a whole tooth challenging. The aim of this study is to evaluate the ability of vascularized tissue to develop within a pulpless tooth using tissue engineering techniques. Materials and methods A pulpless tooth chamber, filled with collagen I gel containing isolated rat dental pulp cells (DPC) and angiogenic growth factors, was placed into a hole created in the femoral cortex or into its own tooth socket, respectively. The gross, histological and biochemical characteristics of the de novo tissue were evaluated at 4 and 8weeks post-transplantation. Results Tooth revascularization and tissue generation was observed only in the femur group, confirming the important role of vascular supply in tissue regeneration. The addition of cells and growth factors significantly promoted connective tissue production in the tooth chamber. Conclusion Successful revascularization and tissue regeneration in this model demonstrate the importance of a direct vascular supply and the advantages of a stem cell approach. © 2012 John Wiley & Sons A/S.

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.

Morphometric modelling of ageing in the human pubic symphysis : sexual dimorphism in an Australian population

Despite the prominent use of the pubic symphysis for age estimation in forensic anthropology, little has been documented regarding the quantitative morphological and micro-architectural changes of this surface. Specifically, utilising post-mortem computed tomography data from a large, contemporary Australian adult population, this study aimed to evaluate sexual dimorphism in the morphology and bone composition of the symphyseal surface; and temporal characterisation of the pubic symphysis in individuals of advancing age. The sample consisted of multi-slice computed tomography (MSCT) scans of the pubic symphysis(slice thickness: 0.5 mm, overlap: 0.1 mm) of 200 individuals of Caucasian ancestry aged 15–70 years, obtained in 2011. Surface rendering reconstruction of the symphyseal surface was conducted in OsiriX1 (v.4.1) and quantitative analyses in Rapidform XOSTM and OsteomeasureTM. Morphometric variables including inter-pubic distance, surface area, circumference, maximum height and width of the symphyseal surface and micro-architectural assessment of cortical and trabecular bone compositions were quantified using novel automated engineering software capabilities. The major results of this study are correlated with the macroscopic ossification and degeneration pattern of the symphyseal surface, demonstrating significant age-related changes in the morphometric and bone tissue variables between 15 and 70 years. Regardless of sex, the overall dimensions of the symphyseal surface increased with age, coupled with a decrease in bone mass in the trabecular and cortical bone compartments. Significant differences between the ventral, dorsal and medial cortical surfaces were observed, which may be correlated to bone formation activity dependent on muscle activity and ligamentous attachments. Our study demonstrates significant sexual dimorphism at this site, with males exhibiting greater surface dimensions than females. These baseline results provide a detailed insight into the changes in the structure of the pubic symphysis with ageing and sexually dimorphic features associated with the cortical and trabecular bone profiles.

Graphene-oxide-modified β-tricalcium phosphate bioceramics stimulate in vitro and in vivo osteogenesis

Graphene oxide (GO) has attracted much interest for applications in bone tissue engineering; however, until now the interaction between GO and stem cells, and the in vivo bone-forming ability of GO has not been explored. The aim of this study was to produce a GO-modified β-tricalcium phosphate (β-TCP-GRA) biceramics and then explore the material’s osteogenic capacity in vitro and in vivo, as well as unravel some of the molecular mechanisms behind this. β-TCP-GRA disks and scaffolds were successfully prepared by a simple GO/water suspension soaking method in combination with heat treatment. These scaffolds were found to significantly enhance the proliferation, alkaline phosphatase activity and osteogenic gene expression of human bone marrow stromal cells (hBMSCs), when compared to β-TCP without GO modification (controls). Activation of the Wnt/β-catenin signaling pathway in hBMSCs appears to be the mechanism behind this osteogenic induction by β-TCP-GRA. β-TCP-GRA scaffolds led to an increased rate of in vivo new bone formation compared to β-TCP controls, indicative of the stimulatory effect of GO on in vivo osteogenesis, making GO modification of β-TCP a very promising method for applications in bone tissue engineering, in particular for the regeneration of large bone defects.

The effect of osteoimmunomodulation on the osteogenic effects of cobalt incorporated β-tricalcium phosphate

Osteoblast lineage cells are direct effectors of osteogenesis and are, therefore, commonly used to evaluate the in vitro osteogenic capacity of bone substitute materials. This method has served its purposes when testing novel bone biomaterials; however, inconsistent results between in vitro and in vivo studies suggest the mechanisms that govern a material's capacity to mediate osteogenesis are not well understood. The emerging field of osteoimmunology and immunomodulation has informed a paradigm shift in our view of bone biomaterials–from one of an inert to an osteoimmunomodulatory material–highlighting the importance of immune cells in materials-mediated osteogenesis. Neglecting the importance of the immune response during this process is a major shortcoming of the current evaluation protocol. In this study we evaluated a potential angiogenic bone substitute material cobalt incorporated with β-tricalcium phosphate (CCP), comparing the traditional “one cell type” approach with a “multiple cell types” approach to assess osteogenesis, the latter including the use of immune cells. We found that CCP extract by itself was sufficient to enhance osteogenic differentiation of bone marrow stem cells (BMSCs), whereas this effect was cancelled out when macrophages were involved. In response to CCP, the macrophage phenotype switched to the M1 extreme, releasing pro-inflammatory cytokines and bone catabolic factors. When the CCP materials were implanted into a rat femur condyle defect model, there was a significant increase of inflammatory markers and bone destruction, coupled with fibrous encapsulation rather than new bone formation. These findings demonstrated that the inclusion of immune cells (macrophages) in the in vitro assessment matched the in vivo tissue response, and that this method provides a more accurate indication of the essential role of immune cells when assessing materials-stimulated osteogenesis in vitro.

Understanding the relationship between interfragmentary movement and callus growth in fracture healing: A novel computational approach

Introduction & Aims Optimising fracture treatments requires a sound understanding of relationships between stability, callus development and healing outcomes. This has been the goal of computational modelling, but discrepancies remain between simulations and experimental results. We compared healing patterns vs fixation stiffness between a novel computational callus growth model and corresponding experimental data. Hypothesis We hypothesised that callus growth is stimulated by diffusible signals, whose production is in turn regulated by mechanical conditions at the fracture site. We proposed that introducing this scheme into computational models would better replicate the observed tissue patterns and the inverse relationship between callus size and fixation stiffness. Method Finite element models of bone healing under stiff and flexible fixation were constructed, based on the parameters of a parallel rat femoral osteotomy study. An iterative procedure was implemented, to simulate the development of callus and its mechanical regulation. Tissue changes were regulated according to published mechano-biological criteria. Predictions of healing patterns were compared between standard models, with a pre-defined domain for callus development, and a novel approach, in which periosteal callus growth is driven by a diffusible signal. Production of this signal was driven by local mechanical conditions. Finally, each model’s predictions were compared to the corresponding histological data. Results Models in which healing progressed within a prescribed callus domain predicted that greater interfragmentary movements would displace early periosteal bone formation further from the fracture. This results from artificially large distortional strains predicted near the fracture edge. While experiments showed increased hard callus size under flexible fixation, this was not reflected in the standard models. Allowing the callus to grow from a thin soft tissue layer, in response to a mechanically stimulated diffusible signal, results in a callus shape and tissue distribution closer to those observed histologically. Importantly, the callus volume increased with increasing interfragmentary movement. Conclusions A novel method to incorporate callus growth into computational models of fracture healing allowed us to successfully capture the relationship between callus size and fixation stability observed in our rat experiments. This approach expands our toolkit for understanding the influence of different fixation strategies on healing outcomes.

Clinoenstatite coatings have high bonding strength, bioactive ion release, and osteoimmunomodulatory effects that enhance in vivo osseointegration

A number of coating materials have been developed over past two decades seeking to improve the osseointegration of orthopedic metal implants. Despite the many candidate materials trialed, their low rate of translation into clinical applications suggests there is room for improving the current strategies for their development. We therefore propose that the ideal coating material(s) should possess the following three properties: (i) high bonding strength, (ii) release of functional ions, and (iii) favourable osteoimmunomodulatory effects. To test this proposal, we developed clinoenstatite (CLT, MgSiO3), which as a coating material has high bonding strength, cytocompability and immunomodulatory effects that are favourable for in vivo osteogenesis. The bonding strength of CLT coatings was 50.1 ± 3.2 MPa, more than twice that of hydroxyapatite (HA) coatings, at 23.5 ± 3.5 MPa. CLT coatings released Mg and Si ions, and compared to HA coatings, induced an immunomodulation more conducive for osseointegration, demonstrated by downregurelation of pro-inflammatory cytokines, enhancement of osteogenesis, and inhibition of osteoclastogenesis. In vivo studies demonstrated that CLT coatings improved osseointegration with host bone, as shown by the enhanced biomechanical strength and increased de novo bone formation, when compared with HA coatings. These results support the notion that coating materials with the proposed properties can induce an in vivo environment better suited for osseointegration. These properties could, therefore, be fundamental when developing high-performance coating materials.

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.

Advanced tissue engineering scaffold design for regeneration of the complex hierarchical periodontal structure

AIM: This study investigated the ability of an osteoconductive biphasic scaffold to simultaneously regenerate alveolar bone, periodontal ligament and cementum. MATERIALS AND METHODS: A biphasic scaffold was built by attaching a fused deposition modelled bone compartment to a melt electrospun periodontal compartment. The bone compartment was coated with a calcium phosphate (CaP) layer for increasing osteoconductivity, seeded with osteoblasts and cultured in vitro for 6 weeks. The resulting constructs were then complemented with the placement of PDL cell sheets on the periodontal compartment, attached to a dentin block and subcutaneously implanted into athymic rats for 8 weeks. Scanning electron microscopy, X-ray diffraction, alkaline phosphatase and DNA content quantification, confocal laser microscopy, micro computerized tomography and histological analysis were employed to evaluate the scaffold's performance. RESULTS: The in vitro study showed that alkaline phosphatase activity was significantly increased in the CaP-coated samples and they also displayed enhanced mineralization. In the in vivo study, significantly more bone formation was observed in the coated scaffolds. Histological analysis revealed that the large pore size of the periodontal compartment permitted vascularization of the cell sheets, and periodontal attachment was achieved at the dentin interface. CONCLUSIONS: This work demonstrates that the combination of cell sheet technology together with an osteoconductive biphasic scaffold could be utilized to address the limitations of current periodontal regeneration techniques.

Magnetic and morphological properties of ferrofluid-impregnated hydroxyapatite/collagen scaffolds

In this article we present the morphological and magnetic characterization of ferrofluid-impregnated biomimetic scaffolds made of hydroxyapatite and collagen used for bone reconstruction. We describe an innovative and simple impregnation process by which the ferrofluid is firmly adsorbed onto the hydroxyapatite/collagen scaffolds. The process confers sufficient magnetization to attract potential magnetic carriers, which may be used to transport bioactive agents that favour bone regeneration. The crystalline structure of the magnetite contained in the ferrofluid is preserved and its quantity, estimated from the weight gain due to the impregnation process, is consistent with that obtained from energy dispersive X-ray spectroscopy. The magnetization, measured with a superconducting quantum interference device, is uniform throughout the scaffolds, demonstrating the efficiency of the impregnation process. The field emission gun scanning electron microscopy characterization demonstrates that the process does not alter the morphology of the hydroxyapatite/collagen scaffolds, which is essential for the preservation of their bioactivity and consequently for their effectiveness in promoting bone formation.

An N-ethyl-n-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, maybe due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh -120/+ mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh-120/+ mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh -120/+ mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh-120/+ mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.

Genomics of ankylosing spondylitis

Ankylosing spondylitis (AS) is the prototypic and most prevalent and debilitating spondyloarthropathy, a group of arthritides where the spine and pelvis are specifically targeted. Unlike many other forms of arthritis in which joint damage is mediated through tissue destruction, in AS uncontrolled bone formation occurs, frequently resulting in joint fusion and consequently significant disability. It is estimated that there are 2.4 million spondyloarthritis sufferers in the U.S., twice as many as rheumatoid arthritis. The pathogenesis of AS is very poorly understood and both genetics and gene expression profiling approaches have been utilized to elucidate the underlying mechanisms and pathways that drive the disease. Using powerful genome-wide association study approaches a number of candidate genes have been found to be associated with AS. However, although such approaches can identify genes that can contribute to the disease process, they do not inform us of the actual changes in gene/cell activity at any point in the disease process. Expression profiling allows us to take a "snapshot" of cellular activity and what gene activity changes are underlying those changes. A number of expression profiling studies have been undertaken in AS, looking at both circulating cells and tissues from affected joints. The results to date have been somewhat disappointing with little consensus on gene activity changes due to the low power of the studies undertaken. Some more recent better powered studies have identified diagnostic expression profiles that do point to a possible role for expression profiling in early AS diagnosis. Future studies will require collaborative approaches to target specific disease stages and sites with larger numbers of samples.

Genetic disorders of the LRP5-Wnt signalling pathway affecting the skeleton

Osteoporosis is a common, increasingly prevalent and potentially debilitating condition of men and women. Genetic factors are major determinants of bone mass and the risk of fracture, but few genes have been definitively demonstrated to be involved. The identification of these factors will provide novel insights into the processes of bone formation and loss and thus the pathogenesis of osteoporosis, enabling the rational development of novel therapies. In this article, we present the extensive genetic and functional data indicating that the LRP5 gene and the Wnt signalling pathway are key players in bone formation and the risk of osteoporosis, and that LRP5 signalling is essential for normal morphology, developmental processes and bone health.

Groucho homologue Grg5 interacts with the transcription factor Runx2-Cbfa1 and modulates its activity during postnatal growth in mice

Runx2-Cbfal, a Runt transcription factor, plays important roles during skeletal development. It is required for differentiation and function of osteoblasts. In its absence, chondrocyte hypertrophy is severely impaired and there is no vascularization of cartilage templates during skeletal development. These tissue-specific functions of Runx2 are likely to be dependent on its interaction with other proteins. We have therefore searched for proteins that may modulate the activity of Runx2. The yeast two-hybrid system was used to identify a groucho homologue, Grg5, as a Runx2-interacting protein. Grg5 enhances Runx2 activity in a cell culture-based assay and by analyses of postnatal growth in mice we demonstrate that Grg5 and Runx2 interact genetically. We also show that Runx2 haploinsufficiency in the absence of Grg5 results in a more severe delay in ossification of cranial sutures and fontanels than occurs with Runx2 haploinsufficiency on a wild-type background. Finally, we find shortening of the proliferative and hypertrophic zones, and expansion of the resting zone in the growth plates of Runx2(+/-)Grg5(-/-) mice that are associated with reduced Ihh expression and Indian hedgehog (Ihh) signaling. We therefore conclude that Grg5 enhances Runx2 activity in vivo.

Growth defect in Grg5 null mice is associated with reduced Ihh signaling in growth plates

Gene-targeted disruption of Grg5, a mouse homologue of Drosophila groucho (gro), results in postnatal growth retardation in mice. The growth defect, most striking in approximately half of the Grg5 null mice, occurs during the first 4-5 weeks of age, but most mice recover retarded growth later. We used the nonlinear mixed-effects model to fit the growth data of wild-type, heterozygous, and Grg5 null mice. On the basis of preliminary evidence suggesting an interaction between Grg5 and the transcription factor Cbfa1/Runx2, critical for skeletal development, we further investigated the skeleton in the mice. A long bone growth plate defect was identified, which included shorter zones of proliferative and hypertrophic chondrocytes and decreased trabecular bone formation. This decreased trabecular bone formation is likely caused by a reduced recruitment of osteoblasts into the growth plate region of Grg5 null mice. Like the growth defect, the growth plate and trabecular bone abnormality improved as the mice grew older. The growth plate defect was associated with reduced Indian hedgehog expression and signaling. We suggest that Grg5, a transcriptional coregulator, modulates the activities of transcription factors, such as Cbfa1/Runx2 in vivo to affect Ihh expression and the function of long bone growth plates.