Alternative and complementary therapies in osteoarthritis and cartilage repair.

Osteoarthritis (OA) is the most common joint condition and, with a burgeoning ageing population, is due to increase in prevalence. Beyond conventional medical and surgical interventions, there are an increasing number of 'alternative' therapies. These alternative therapies may have a limited evidence base and, for this reason, are often only afforded brief reference (or completely excluded) from current OA guidelines. Thus, the aim of this review was to synthesize the current evidence regarding autologous chondrocyte implantation (ACI), mesenchymal stem cell (MSC) therapy, platelet-rich plasma (PRP), vitamin D and other alternative therapies. The majority of studies were in knee OA or chondral defects. Matrix-assisted ACI has demonstrated exceedingly limited, symptomatic improvements in the treatment of cartilage defects of the knee and is not supported for the treatment of knee OA. There is some evidence to suggest symptomatic improvement with MSC injection in knee OA, with the suggestion of minimal structural improvement demonstrated on MRI and there are positive signals that PRP may also lead to symptomatic improvement, though variation in preparation makes inter-study comparison difficult. There is variability in findings with vitamin D supplementation in OA, and the only recommendation which can be made, at this time, is for replacement when vitamin D is deplete. Other alternative therapies reviewed have some evidence (though from small, poor-quality studies) to support improvement in symptoms and again there is often a wide variation in dosage and regimens. For all these therapeutic modalities, although controlled studies have been undertaken to evaluate effectiveness in OA, these have often been of small size, limited statistical power, uncertain blindness and using various methodologies. These deficiencies must leave the question as to whether they have been validated as effective therapies in OA (or chondral defects). The conclusions of this review are that all alternative interventions definitely require clinical trials with robust methodology, to assess their efficacy and safety in the treatment of OA beyond contextual and placebo effects.

Demethylation of an NF-κB enhancer element orchestrates iNOS induction in osteoarthritis and is associated with altered chondrocyte cell cycle.

OBJECTIVE: To examine the methylation profile of the nuclear factor (NF)-κB enhancer region at -5.8 kb of inducible nitric oxide synthase (iNOS) and the subsequent role in the induction of osteoarthritis (OA) via cell cycle regulation. METHODS: Percentage methylation was determined by pyrosequencing, gene expression by qRT-PCR and cell proliferation was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Transient transfections were induced to determine the effect of the NF-κB enhancer region on cell proliferation and the influence of DNA methylation. RESULTS: In vitro de-methylation with 5-aza-dC showed decreased levels of DNA methylation at CpG sites localised at -5.8 kb, which correlated with higher levels of iNOS expression. In vitro methylation of the NF-κB enhancer region at -5.8 kb increased the percentage of cells at G0/G1 cell cycle phase. Loss of methylation within this region correlated with, enhanced proliferation and increased number of cells at G2/M phase. OA chondrocytes demonstrated up-regulation of the G0/G1 cell cycle progression markers Cyclin D1 and CDK6 in contrast to control cells. We demonstrate the loss of methylation that occurs at specific CpG sites localised at the -5.8 kb NF-κB enhancer region of the iNOS gene in OA chondrocytes permits the binding of this transcription factor activating the expression of iNOS. This results in subsequent altered cell cycle regulation, altered proliferative phenotype and transmission of the pathogenic phenotype to daughter cells. CONCLUSIONS: This study indicates that inhibition of cell cycle progression by iNOS enhancer hypermethylation is capable of reducing pro-inflammatory responses via down-regulation of NF-κB with important therapeutic implications in OA.

Epigenetic regulation of interleukin-8, an inflammatory chemokine, in osteoarthritis.

OBJECTIVE: To determine whether altered IL8 methylation status is associated with increased expression of IL8 in human osteoarthritic (OA) chondrocytes. METHODS: IL8 expression levels and the percentage CpG methylation in human chondrocytes were quantified by qRT-PCR and pyrosequencing in OA patients and in non-OA osteoporotic controls. The effect of CpG methylation on IL8 promoter activity was determined using a CpG-free vector; co-transfections with expression vectors encoding nuclear factor-kappa B (NF-κB), AP-1 and C/EBP were subsequently undertaken to analyse for IL8 promoter activity in response to changes in methylation status. RESULTS: IL8 expression in OA patients was 37-fold higher than in osteoporotic controls. Three CpG sites in the IL8 promoter were significantly demethylated in OA patients. Multiple regression analysis revealed that the degree of methylation of the CpG site located at -116-bp was the strongest predictor of IL8 expression. In vitro DNA methylation was noted to decrease IL8 promoter basal activity. Furthermore, NF-κB, AP-1 and C/EBP strongly enhanced IL8 promoter activity whilst DNA methylation inhibited the effects of these three transcription factors. CONCLUSIONS: The present study demonstrates the key role of DNA methylation status on the expression of IL8 in human chondrocytes. We demonstrate a quantitative relationship between percentage methylation and gene expression within clinical samples. These studies provide direct evidence linking the activation of IL8, DNA demethylation and the induction of the OA process with important therapeutic implications therein for patients with this debilitating disease.

Cold water cleaning of brain proteins, biofilm and bone - harnessing an ultrasonically activated stream.

In the absence of sufficient cleaning of medical instruments, contamination and infection can result in serious consequences for the health sector and remains a significant unmet challenge. In this paper we describe a novel cleaning system reliant on cavitation action created in a free flowing fluid stream where ultrasonic transmission to a surface, through the stream, is achieved using careful design and control of the device architecture, sound field and the materials employed. Cleaning was achieved with purified water at room temperature, moderate fluid flow rates and without the need for chemical additives or the high power consumption associated with conventional strategies. This study illustrates the potential in harnessing an ultrasonically activated stream to remove biological contamination including brain tissue from surgical stainless steel substrates, S. epidermidis biofilms from glass, and fat/soft tissue matter from bone structures with considerable basic and clinical applications.

Tissue engineered bone using select growth factors: A comprehensive review of animal studies and clinical translation studies in man.

There is a growing socio-economic need for effective strategies to repair damaged bone resulting from disease, trauma and surgical intervention. Bone tissue engineering has received substantial investment over the last few decades as a result. A multitude of studies have sought to examine the efficacy of multiple growth factors, delivery systems and biomaterials within in vivo animal models for the repair of critical-sized bone defects. Defect repair requires recapitulation of in vivo signalling cascades, including osteogenesis, chondrogenesis and angiogenesis, in an orchestrated spatiotemporal manner. Strategies to drive parallel, synergistic and consecutive signalling of factors including BMP-2, BMP-7/OP-1, FGF, PDGF, PTH, PTHrP, TGF-ß3, VEGF and Wnts have demonstrated improved bone healing within animal models. Enhanced bone repair has also been demonstrated in the clinic following European Medicines Agency and Food and Drug Administration approval of BMP-2, BMP-7/OP-1, PDGF, PTH and PTHrP. The current review assesses the in vivo and clinical data surrounding the application of growth factors for bone regeneration. This review has examined data published between 1965 and 2013. All bone tissue engineering studies investigating in vivo response of the growth factors listed above, or combinations thereof, utilising animal models or human trials were included. All studies were compiled from PubMed-NCBI using search terms including 'growth factor name', 'in vivo', 'model/animal', 'human', and 'bone tissue engineering'. Focus is drawn to the in vivo success of osteoinductive growth factors incorporated within material implants both in animals and humans, and identifies the unmet challenges within the skeletal regenerative area.

The effect of oxygen tension on human articular chondrocyte matrix synthesis: integration of experimental and computational approaches.

Significant oxygen gradients occur within tissue engineered cartilaginous constructs. Although oxygen tension is an important limiting parameter in the development of new cartilage matrix, its precise role in matrix formation by chondrocytes remains controversial, primarily due to discrepancies in the experimental setup applied in different studies. In this study, the specific effects of oxygen tension on the synthesis of cartilaginous matrix by human articular chondrocytes were studied using a combined experimental-computational approach in a "scaffold-free" 3D pellet culture model. Key parameters including cellular oxygen uptake rate were determined experimentally and used in conjunction with a mathematical model to estimate oxygen tension profiles in 21-day cartilaginous pellets. A threshold oxygen tension (pO2 ≈ 8% atmospheric pressure) for human articular chondrocytes was estimated from these inferred oxygen profiles and histological analysis of pellet sections. Human articular chondrocytes that experienced oxygen tension below this threshold demonstrated enhanced proteoglycan deposition. Conversely, oxygen tension higher than the threshold favored collagen synthesis. This study has demonstrated a close relationship between oxygen tension and matrix synthesis by human articular chondrocytes in a "scaffold-free" 3D pellet culture model, providing valuable insight into the understanding and optimization of cartilage bioengineering approaches.

Assessing the potential of colony morphology for dissecting the CFU-F population from human bone marrow stromal cells.

Mesenchymal stem cells (MSCs) provide an ideal cell source for bone tissue engineering strategies. However, bone marrow stromal cell (BMSC) populations that contain MSCs are highly heterogeneous expressing a wide variety of proliferative and differentiation potentials. Current MSC isolation methods employing magnetic-activated and fluorescent-activated cell sorting can be expensive and time consuming and, in the absence of specific MSC markers, fail to generate homogeneous populations. We have investigated the potential of various colony morphology descriptors to provide correlations with cell growth potential. Density-independent colony forming unit-fibroblastic (CFU-F) capacity is a MSC prerequisite and resultant colonies display an array of shapes and sizes that might be representative of cell function. Parent colonies were initially categorised according to their diameter and cell density and grouped before passage for the subsequent assessment of progeny colonies. Whereas significant morphological differences between distinct parent populations indicated a correlation with immunophenotype, enhanced CFU-F capacity was not observed when individual colonies were isolated according to these morphological parameters. Colony circularity, an alternative morphological measure, displayed a strong correlation with subsequent cell growth potential. The current study indicates the potential of morphological descriptors for predicting cell growth rate and suggests new directions for research into dissection of human BMSC CFU-F populations.

A tissue engineering strategy for the treatment of avascular necrosis of the femoral head.

BACKGROUND & PURPOSE: Skeletal stem cells (SSCs) and impaction bone grafting (IBG) can be combined to produce a mechanically stable living bone composite. This novel strategy has been translated to the treatment of avascular necrosis of the femoral head. Surgical technique, clinical follow-up and retrieval analysis data of this translational case series is presented. METHODS: SSCs and milled allograft were impacted into necrotic bone in five femoral heads of four patients. Cell viability was confirmed by parallel in vitro culture of the cell-graft constructs. Patient follow-up was by serial clinical and radiological examination. Tissue engineered bone was retrieved from two retrieved femoral heads and was analysed by histology, microcomputed tomography (µCT) and mechanical testing. RESULTS: Three patients remain asymptomatic at 22- to 44-month follow-up. One patient (both hips) required total hip replacement due to widespread residual necrosis. Retrieved tissue engineered bone demonstrated a mature trabecular micro-architecture histologically and on µCT. Bone density and axial compression strength were comparable to trabecular bone. CONCLUSIONS: Clinical follow-up shows this to be an effective new treatment for focal early stage avascular necrosis of the femoral head. Unique retrieval analysis of clinically translated tissue engineered bone has demonstrated regeneration of tissue that is both structurally and functionally analogous to normal trabecular bone.

Maternal high-fat diet: effects on offspring bone structure.

UNLABELLED: Peak bone mass is believed to partly be programmed in utero. Mouse dams and offspring were given a high-fat diet and offspring studied as adults. Female offspring from high-fat dams exhibited altered trabecular structure indicative of in utero programming. In utero nutrition has consequences in later life. INTRODUCTION: Epidemiological studies suggest that skeletal growth is programmed during intrauterine and early postnatal life. We hypothesise that development of optimal peak bone mass has, in part, a foetal origin and investigated this using a mouse model of maternal dietary fat excess. METHODS: Offspring from mouse dams fed either standard chow (C) or lifetime high-fat diet (HF) were maintained on a HF diet to adulthood. Femur samples were taken at 30 weeks of age and bone structure, adiposity and strength analysed. Sample sizes were four to six for each sex and each diet group. RESULTS: Offspring from HF-fed dams showed increased adiposity in the femur in comparison to offspring from C-fed dams. Female offspring from HF dams exhibited altered trabecular structure indicative of in utero programming. CONCLUSIONS: A maternal HF diet during pregnancy increases bone marrow adiposity and alters bone structure in their offspring.

Skeletal stem cells and bone regeneration: translational strategies from bench to clinic.

Clinical imperatives for new bone to replace or restore the function of traumatized or bone lost as a consequence of age or disease has led to the need for therapies or procedures to generate bone for skeletal applications. Tissue regeneration promises to deliver specifiable replacement tissues and the prospect of efficacious alternative therapies for orthopaedic applications such as non-union fractures, healing of critical sized segmental defects and regeneration of articular cartilage in degenerative joint diseases. In this paper we review the current understanding of the continuum of cell development from skeletal stem cells, osteoprogenitors through to mature osteoblasts and the role of the matrix microenvironment, vasculature and factors that control their fate and plasticity in skeletal regeneration. Critically, this review addresses in vitro and in vivo models to investigate laboratory and clinical based strategies for the development of new technologies for skeletal repair and the key translational points to clinical success. The application of developmental paradigms of musculoskeletal tissue formation specifically, understanding developmental biology of bone formation particularly in the adult context of injury and disease will, we propose, offer new insights into skeletal cell biology and tissue regeneration allowing for the critical integration of stem cell science, tissue engineering and clinical applications. Such interdisciplinary, iterative approaches will be critical in taking patient aspirations to clinical reality.

Characterisation and evaluation of the regenerative capacity of Stro-4+ enriched bone marrow mesenchymal stromal cells using bovine extracellular matrix hydrogel and a novel biocompatible melt electro-written medical-grade polycaprolactone scaffold.

Many skeletal tissue regenerative strategies centre around the multifunctional properties of bone marrow derived stromal cells (BMSC) or mesenchymal stem/stromal cells (MSC)/bone marrow derived skeletal stem cells (SSC). Specific identification of these particular stem cells has been inconclusive. However, enriching these heterogeneous bone marrow cell populations with characterised skeletal progenitor markers has been a contributing factor in successful skeletal bone regeneration and repair strategies. In the current studies we have isolated, characterised and enriched ovine bone marrow mesenchymal stromal cells (oBMSCs) using a specific antibody, Stro-4, examined their multipotential differentiation capacity and, in translational studies combined Stro-4+ oBMSCs with a bovine extracellular matrix (bECM) hydrogel and a biocompatible melt electro-written medical-grade polycaprolactone scaffold, and tested their bone regenerative capacity in a small in vivo, highly vascularised, chick chorioallantoic membrane (CAM) model and a preclinical, critical-sized ovine segmental tibial defect model. Proliferation rates and CFU-F formation were similar between unselected and Stro-4+ oBMSCs. Col1A1, Col2A1, mSOX-9, PPARG gene expression were upregulated in respective osteogenic, chondrogenic and adipogenic culture conditions compared to basal conditions with no significant difference between Stro-4+ and unselected oBMSCs. In contrast, proteoglycan expression, alkaline phosphatase activity and adipogenesis were significantly upregulated in the Stro-4+ cells. Furthermore, with extended cultures, the oBMSCs had a predisposition to maintain a strong chondrogenic phenotype. In the CAM model Stro-4+ oBMSCs/bECM hydrogel was able to induce bone formation at a femur fracture site compared to bECM hydrogel and control blank defect alone. Translational studies in a critical-sized ovine tibial defect showed autograft samples contained significantly more bone, (4250.63 mm3, SD = 1485.57) than blank (1045.29 mm3, SD = 219.68) ECM-hydrogel (1152.58 mm3, SD = 191.95) and Stro-4+/ECM-hydrogel (1127.95 mm3, SD = 166.44) groups. Stro-4+ oBMSCs demonstrated a potential to aid bone repair in vitro and in a small in vivo bone defect model using select scaffolds. However, critically, translation to a large related preclinical model demonstrated the complexities of bringing small scale reported stem-cell material therapies to a clinically relevant model and thus facilitate progression to the clinic.

The cell in the ink: Improving biofabrication by printing stem cells for skeletal regenerative medicine.

Recent advances in regenerative medicine have confirmed the potential to manufacture viable and effective tissue engineering 3D constructs comprising living cells for tissue repair and augmentation. Cell printing has shown promising potential in cell patterning in a number of studies enabling stem cells to be precisely deposited as a blueprint for tissue regeneration guidance. Such manufacturing techniques, however, face a number of challenges including; (i) post-printing cell damage, (ii) proliferation impairment and, (iii) poor or excessive final cell density deposition. The use of hydrogels offers one approach to address these issues given the ability to tune these biomaterials and subsequent application as vectors capable of delivering cell populations and as extrusion pastes. While stem cell-laden hydrogel 3D constructs have been widely established in vitro, clinical relevance, evidenced by in vivo long-term efficacy and clinical application, remains to be demonstrated. This review explores the central features of cell printing, cell-hydrogel properties and cell-biomaterial interactions together with the current advances and challenges in stem cell printing. A key focus is the translational hurdles to clinical application and how in vivo research can reshape and inform cell printing applications for an ageing population.

Printing bone in a gel: using nanocomposite bioink to print functionalised bone scaffolds.

Free-form printing offers a novel biofabrication approach to generate complex shapes by depositing hydrogel materials within a temporary supportive environment. However, printed hydrogels typically lack the requisite mechanical properties and functionality of the desired tissue, limiting application and, more importantly, safety and efficacy of the implant. The study authors have developed an innovative nanoclay-based bioink to print high shape fidelity functional constructs for potential skeletal application. Laponite® (LAP) nanoclay was combined with gellan gum (GG) to generate a printable hydrogel that was highly stable in vitro, displayed limited swelling ability compared with the silicate-free control and remained stable over time. An agarose fluid gel was found to provide the requisite support for the deposition of the material ink and preservation of the printed structure before crosslinking. Printed C2C12 myoblasts remained viable and displayed extensive proliferation over 21 days in culture. Cell-laden scaffolds demonstrated functionality within 1 day of culture in vitro and that was preserved over 3 weeks. Analysis of absorption and release mechanisms from LAP-GG using model proteins (lysozyme and bovine serum albumin) demonstrated the retention capability of the clay-based materials for compound localisation and absence of burst release. Vascular endothelial growth factor â€‹was loaded within the agarose fluid gel and absorbed by the material ink via absorption during deposition. The 3D-printed constructs were implanted on the chorioallantoic membrane of a 10-day-old developing chick. Extensive and preferential vasculature infiltration was observed in LAP-GG-loaded vascular endothelial growth factor constructs compared with controls (p<0.01 and p<0.0001) after only 7 days of incubation. The current studies demonstrate, for the first time, the application of innovative LAP-GG 3D constructs in the generation of growth factor-loaded 3D constructs for potential application in skeletal tissue repair.

Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs.

Due to an increasing aging population the need for innovative approaches to aid skeletal repair and reconstruction is a significant socio-economic increasing problem. The emerging discipline of tissue engineering has sort to augment the growth and repair of bone loss particularly in areas of trauma, degeneration and revision surgery. However, the initiation and development of a fully functional vascular network are critical for bioengineered bone to repair large osseous defects, whether the material is osteosynthetic (poly (d,l)-lactic acid, PLA) or natural bone allograft. Quantification and three-dimensional visualization of new vessel networks remain a problem in bone tissue engineering constructs. A novel technique utilising a radio-opaque dye and micro-computed tomography (muCT) has been developed and applied to study angiogenesis in an impaction bone graft model. Tissue-engineered constructs combining human bone marrow stromal cells (HBMSC) with natural allograft and synthetic grafts (PLA) were impacted and implanted into the subcutis of MF-1 nu/nu mice for a period of 28 days. Microfil consisting of radio-opaque polymer was perfused through the mice and scanned using a Bench Top CT system for micro-computed tomography. Analysis of three-dimensional muCT reconstructions demonstrated an increase in vessel volume and vessel number in the impacted scaffolds/HBMC compared to scaffolds alone. Vessel volume: allograft/HBMSC=0.57 mm(3)+/-0.19; allograft=0.04 mm(3)+/-0.04; PLA/HBMSC=1.19 mm(3)+/-0.31; and PLA=0.12 mm(3)+/-0.01. Penetrating vessel number: allograft/HBMSC=22.33+/-3.21; allograft=3.67+/-1.153; PLA/HBMSC=32.67+/-8.33; and PLA=7.67+/-3.06. Type 1 collagen and von Willebrand factor immunohistochemistry in scaffold/HBMSC constructs indicated the osteogenic cell phenotype, and new blood vessel formation respectively. Contrast-enhanced 3D reconstructions facilitated the visualization and quantification of neovascularisation. This novel technique has been used to demonstrate neovascularisation in impacted tissue engineered constructs providing a facile approach with wide experimental application.

Osteogenesis and angiogenesis: the potential for engineering bone.

The repair of large bone defects remains a major clinical orthopaedic challenge. Bone is a highly vascularised tissue reliant on the close spatial and temporal connection between blood vessels and bone cells to maintain skeletal integrity. Angiogenesis thus plays a pivotal role in skeletal development and bone fracture repair. Current procedures to repair bone defects and to provide structural and mechanical support include the use of grafts (autologous, allogeneic) or implants (polymeric or metallic). These approaches face significant limitations due to insufficient supply, potential disease transmission, rejection, cost and the inability to integrate with the surrounding host tissue. The engineering of bone tissue offers new therapeutic strategies to aid musculoskeletal healing. Various scaffold constructs have been employed in the development of tissue-engineered bone; however, an active blood vessel network is an essential pre-requisite for these to survive and integrate with existing host tissue. Combination therapies of stem cells and polymeric growth factor release scaffolds tailored to promote angiogenesis and osteogenesis are under evaluation and development actively to stimulate bone regeneration. An understanding of the cellular and molecular interactions of blood vessels and bone cells will enhance and aid the successful development of future vascularised bone scaffold constructs, enabling survival and integration of bioengineered bone with the host tissue. The role of angiogenic and osteogenic factors in the adaptive response and interaction of osteoblasts and endothelial cells during the multi step process of bone development and repair will be highlighted in this review, with consideration of how some of these key mechanisms can be combined with new developments in tissue engineering to enable repair and growth of skeletal fractures. Elucidation of the processes of angiogenesis, osteogenesis and tissue engineering strategies offer exciting future therapeutic opportunities for skeletal repair and regeneration in orthopaedics.

Adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330nm deep microgrooves.

The surface microtexture of an orthopaedic device can regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved to include the field of surface modification; in particular, nanotechnology has allowed for the development of experimental nanoscale substrates for investigation into cell nanofeature interactions. Here primary human osteoblasts (HOBs) were cultured on ordered nanoscale groove/ridge arrays fabricated by photolithography. Grooves were 330nm deep and either 10, 25 or 100microm in width. Adhesion subtypes in HOBs were quantified by immunofluorescent microscopy and cell-substrate interactions were investigated via immunocytochemistry with scanning electron microscopy. To further investigate the effects of these substrates on cellular function, 1.7K gene microarray analysis was used to establish gene regulation profiles of mesenchymal stem cells cultured on these nanotopographies. Nanotopographies significantly affected the formation of focal complexes (FXs), focal adhesions (FAs) and supermature adhesions (SMAs). Planar control substrates induced widespread adhesion formation; 100microm wide groove/ridge arrays did not significantly affect adhesion formation yet induced upregulation of genes involved in skeletal development and increased osteospecific function; 25microm wide groove/ridge arrays were associated with a reduction in SMA and an increase in FX formation; and 10microm wide groove/ridge arrays significantly reduced osteoblast adhesion and induced an interplay of up- and downregulation of gene expression. This study indicates that groove/ridge topographies are important modulators of both cellular adhesion and osteospecific function and, critically, that groove/ridge width is important in determining cellular response.

The influence of a high fat diet on bone and soft tissue formation in Matrix Gla Protein knockout mice.

Studies suggest bone growth and development are influenced by maternal nutrition, during intrauterine and early postnatal life. This study assessed the role of MGP and a maternal high fat diet on vitamin K-dependent proteins' gene expression and their impact on bone formation. Knockout (KO) offspring were smaller than wild type (WT) littermates, yet possessed the same volume of intrascapular brown adipose tissue. The total proportion of body fat was reduced, but only in animals on a control diet. Lung air volume was observed to be comparable in both KO and WT animals on the same diet. The degree of aortic calcification was reduced in KO animals maintained on a HF diet. KO females on the high fat diet showed reduced cortical bone volume and thickness in the femur and tibia. Gene expression levels of GGCX and VKOR were reduced in control fed KO animals suggesting a potential link between gene expression levels of MGP, GGCX, and VKOR and total volumes of bone, calcified soft tissue, and iBAT; with implications for modulation of body length and mass. Our results confirm the important role for vitamin K in bone and calcified soft tissue, but now extend this role to include iBAT.

Human fibroblast and human bone marrow cell response to lithographically nanopatterned adhesive domains on protein rejecting substrates.

The separate influence of topographical and chemical cues on cell attachment and spreading are well documented; however, that of duel-cue substrates is less so. In this study graft copolymers that sterically stabilize biological surfaces were employed alongside nanotopographical features fabricated by colloidal lithography. This resulted in the production of a range of substrates whereby the effect of chemistry and or topography on both on human fibroblast and bone marrow cell adhesion and spreading could be observed. The current studies indicate an enhancement of cell response as a consequence of modifications in material topography, whereas the current selected chemical cues inhibited cell function. Critically, in combination, topography modulated the effects of chemical environment.

Vibration-assisted bone-graft compaction in impaction bone grafting of the femur.

The complications of impaction bone grafting in revision hip replacement includes fracture of the femur and subsidence of the prosthesis. In this in vitro study we aimed to investigate whether the use of vibration, combined with a perforated tamp during the compaction of morsellised allograft would reduce peak loads and hoop strains in the femur as a surrogate marker of the risk of fracture and whether it would also improve graft compaction and prosthetic stability. We found that the peak loads and hoop strains transmitted to the femoral cortex during graft compaction and subsidence of the stem in subsequent mechanical testing were reduced. This innovative technique has the potential to reduce the risk of intra-operative fracture and to improve graft compaction and therefore prosthetic stability.

The inferomedial femoral neck is compromised by age but not disease: Fracture toughness and the multifactorial mechanisms comprising reference point microindentation.

The influence of ageing on the fracture mechanics of cortical bone tissue is well documented, though little is known about if and how related material properties are further affected in two of the most prominent musculoskeletal diseases, osteoporosis and osteoarthritis (OA). The femoral neck, in close proximity to the most pertinent osteoporotic fracture site and near the hip joint affected by osteoarthritis, is a site of particular interest for investigation. We have recently shown that Reference Point micro-Indentation (RPI) detects differences between cortical bone from the femoral neck of healthy, osteoporotic fractured and osteoarthritic hip replacement patients. RPI is a new technique with potential for in vivo bone quality assessment. However, interpretation of RPI results is limited because the specific changes in bone properties with pathology are not well understood and, further, because it is not conclusive what properties are being assessed by RPI. Here, we investigate whether the differences previously detected between healthy and diseased cortical bone from the femoral neck might reflect changes in fracture toughness. Together with this, we investigate which additional properties are reflected in RPI measures. RPI (using the Biodent device) and fracture toughness tests were conducted on samples from the inferomedial neck of bone resected from donors with: OA (41 samples from 15 donors), osteoporosis (48 samples from 14 donors) and non age-matched cadaveric controls (37 samples from 10 donoros) with no history of bone disease. Further, a subset of indented samples were imaged using micro-computed tomography (3 osteoporotic and 4 control samples each from different donors) as well as fluorescence microscopy in combination with serial sectioning after basic fuchsin staining (7 osteoporotic and 5 control samples from 5 osteoporotic and 5 control donors). In this study, the bulk indentation and fracture resistance properties of the inferomedial femoral neck in osteoporotic fracture, severe OA and control bone were comparable (p > 0.05 for fracture properties and <10% difference for indentation) but fracture toughness reduced with advancing age (7.0% per decade, r = -0.36, p = 0.029). Further, RPI properties (in particular, the indentation distance increase, IDI) showed partial correlation with fracture toughness (r = -0.40, p = 0.023) or derived elastic modulus (r = -0.40, p = 0.023). Multimodal indent imaging revealed evidence of toughening mechanisms (i.e. crack deflection, bridging and microcracking), elastoplastic response (in terms of the non-conical imprint shape and presence of pile-up) and correlation of RPI with damage extent (up to r = 0.79, p = 0.034) and indent size (up to r = 0.82, p < 0.001). Therefore, crack resistance, deformation resistance and, additionally, micro-structure (porosity: r = 0.93, p = 0.002 as well as pore proximity: r = -0.55, p = 0.027 for correlation with IDI) are all contributory to RPI. Consequently, it becomes clear that RPI measures represent a multitude of properties, various aspects of bone quality, but are not necessarily strongly correlated to a single mechanical property. In addition, osteoporosis or osteoarthritis do not seem to further influence fracture toughness of the inferomedial femoral neck beyond natural ageing. Since bone is highly heterogeneous, whether this finding can be extended to the whole femoral neck or whether it also holds true for other femoral neck quadrants or other material properties remains to be shown.