Diagnosing acute bone and joint infection in children.

Acute bone and joint infections in children are serious, and misdiagnosis can threaten limb and life. Most young children who present acutely with pain, limping, and/or loss of function have transient synovitis, which will resolve spontaneously within a few days. A minority will have a bone or joint infection. Clinicians are faced with a diagnostic challenge: children with transient synovitis can safely be sent home, but children with bone and joint infection require urgent treatment to avoid complications. Clinicians often respond to this challenge by using a series of rudimentary decision support tools, based on clinical, haematological, and biochemical parameters, to differentiate childhood osteoarticular infection from other diagnoses. However, these tools were developed without methodological expertise in diagnostic accuracy and do not consider the importance of imaging (ultrasound scan and MRI). There is wide variation in clinical practice with regard to the indications, choice, sequence, and timing of imaging. This variation is most likely due to the lack of evidence concerning the role of imaging in acute bone and joint infection in children. We describe the first steps of a large UK multicentre study, funded by the National Institute for Health Research, which seeks to integrate definitively the role of imaging into a decision support tool, developed with the assistance of individuals with expertise in the development of clinical prediction tools.

Bio-inspired design of a magnetically active trilayered scaffold for cartilage tissue engineering.

An important topic in cartilage tissue engineering is the development of biomimetic scaffolds which mimic the depth-dependent material properties of the native tissue. We describe an advanced trilayered nanocomposite hydrogel (ferrogel) with a gradient in compressive modulus from the top to the bottom layers (p < 0.05) of the construct. Further, the scaffold was able to respond to remote external stimulation, exhibiting an elastic, depth-dependent strain gradient. When bovine chondrocytes were seeded into the ferrogels and cultured for up to 14 days, there was good cell viability and a biochemical gradient was measured with sulphated glycosaminoglycan increasing with depth from the surface. This novel construct provides tremendous scope for tailoring location-specific cartilage replacement tissue; by varying the density of magnetic nanoparticles, concentration of base hydrogel and number of cells, physiologically relevant depth-dependent gradients may be attained. © 2015 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Development of Composite Poly(Lactide-co-Glycolide)- Nanodiamond Scaffolds for Bone Cell Growth.

There are relatively few nanotechnologies that can produce nanocomposite scaffolds for cell growth. Electrospinning has emerged as the foremost method of producing nanofibrous biomimetic scaffolds for tissue engineering applications. In this study diamond nanoparticles were integrated into a polymer solution to develop a nanocomposite scaffold containing poly(lactide-co-glycolide) (PLGA) loaded with diamond nanoparticles. To investigate the effect of adding diamond nanoparticles to PLGA scaffolds, primary human mesenchymal stem cells (hMSCs) were seeded on the scaffolds. The cytocompatibility results showed that addition of diamond nanoparticles did not impinge upon cell proliferation, nor was there a cytotoxic cellular response after 9 days in culture. Scanning electron microscopy, transmission electron microscopy, atomic force microscopy and confocal microscopy enabled qualitative characterization of the fibres and revealed cell morphology and number. Furthermore, surface roughness was measured to evaluate diamond nanoparticle modifications, and no significant difference was found between the diamond nanocomposite and pure polymer scaffolds. On the other hand, bright spots on phase images performed by atomic force microscopy suggested a higher hardness at certain points on fibers of the PLGA-nanodiamond composites, which was supported by nanoindentation measurements. This study shows that PLGA nanofibers can be reinforced with nanodiamond without adversely affecting cell behaviour, and thus it sets the foundation for future application of these scaffolds in bone tissue engineering.

The application of multiple biophysical cues to engineer functional neocartilage for treatment of osteoarthritis. Part II: signal transduction.

The unique mechanoelectrochemical environment of cartilage has motivated researchers to investigate the effect of multiple biophysical cues, including mechanical, magnetic, and electrical stimulation, on chondrocyte biology. It is well established that biophysical stimuli promote chondrocyte proliferation, differentiation, and maturation within "biological windows" of defined dose parameters, including mode, frequency, magnitude, and duration of stimuli (see companion review Part I: Cellular Response). However, the underlying molecular mechanisms and signal transduction pathways activated in response to multiple biophysical stimuli remain to be elucidated. Understanding the mechanisms of biophysical signal transduction will deepen knowledge of tissue organogenesis, remodeling, and regeneration and aiding in the treatment of pathologies such as osteoarthritis. Further, this knowledge will provide the tissue engineer with a potent toolset to manipulate and control cell fate and subsequently develop functional replacement cartilage. The aim of this article is to review chondrocyte signal transduction pathways in response to mechanical, magnetic, and electrical cues. Signal transduction does not occur along a single pathway; rather a number of parallel pathways appear to be activated, with calcium signaling apparently common to all three types of stimuli, though there are different modes of activation. Current tissue engineering strategies, such as the development of "smart" functionalized biomaterials that enable the delivery of growth factors or integration of conjugated nanoparticles, may further benefit from targeting known signal transduction pathways in combination with external biophysical cues.

The application of multiple biophysical cues to engineer functional neocartilage for treatment of osteoarthritis. Part I: cellular response.

Osteoarthritis (OA) is a complex disease of the joint for which current treatments are unsatisfactory, thus motivating development of tissue engineering (TE)-based therapies. To date, TE strategies have had some success, developing replacement tissue constructs with biochemical properties approaching that of native cartilage. However, poor biomechanical properties and limited postimplantation integration with surrounding tissue are major shortcomings that need to be addressed. Functional tissue engineering strategies that apply physiologically relevant biophysical cues provide a platform to improve TE constructs before implantation. In the previous decade, new experimental and theoretical findings in cartilage biomechanics and electromechanics have emerged, resulting in an increased understanding of the complex interplay of multiple biophysical cues in the extracellular matrix of the tissue. The effect of biophysical stimulation on cartilage, and the resulting chondrocyte-mediated biosynthesis, remodeling, degradation, and repair, has, therefore, been extensively explored by the TE community. This article compares and contrasts the cellular response of chondrocytes to multiple biophysical stimuli, and may be read in conjunction with its companion paper that compares and contrasts the subsequent intracellular signal transduction cascades. Mechanical, magnetic, and electrical stimuli promote proliferation, differentiation, and maturation of chondrocytes within established dose parameters or "biological windows." This knowledge will provide a framework for ongoing studies incorporating multiple biophysical cues in TE functional neocartilage for treatment of OA.

The design and development of a high-throughput magneto-mechanostimulation device for cartilage tissue engineering.

To recapitulate the in vivo environment and create neo-organoids that replace lost or damaged tissue requires the engineering of devices, which provide appropriate biophysical cues. To date, bioreactors for cartilage tissue engineering have focused primarily on biomechanical stimulation. There is a significant need for improved devices for articular cartilage tissue engineering capable of simultaneously applying multiple biophysical (electrokinetic and mechanical) stimuli. We have developed a novel high-throughput magneto-mechanostimulation bioreactor, capable of applying static and time-varying magnetic fields, as well as multiple and independently adjustable mechanical loading regimens. The device consists of an array of 18 individual stations, each of which uses contactless magnetic actuation and has an integrated Hall Effect sensing system, enabling the real-time measurements of applied field, force, and construct thickness, and hence, the indirect measurement of construct mechanical properties. Validation tests showed precise measurements of thickness, within 14 µm of gold standard calliper measurements; further, applied force was measured to be within 0.04 N of desired force over a half hour dynamic loading, which was repeatable over a 3-week test period. Finally, construct material properties measured using the bioreactor were not significantly different (p=0.97) from those measured using a standard materials testing machine. We present a new method for articular cartilage-specific bioreactor design, integrating combinatorial magneto-mechanostimulation, which is very attractive from functional and cost viewpoints.

Nanofibrous poly(lactide-co-glycolide) membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering.

BACKGROUND: Nanofibrous scaffolds loaded with bioactive nanoparticles are promising materials for bone tissue engineering. METHODS: In this study, composite nanofibrous membranes containing a copolymer of L-lactide and glycolide (PLGA) and diamond nanoparticles were fabricated by an electrospinning technique. PLGA was dissolved in a mixture of methylene chloride and dimethyl formamide (2:3) at a concentration of 2.3 wt%, and nanodiamond (ND) powder was added at a concentration of 0.7 wt% (about 23 wt% in dry PLGA). RESULTS: In the composite scaffolds, the ND particles were either arranged like beads in the central part of the fibers or formed clusters protruding from the fibers. In the PLGA-ND membranes, the fibers were thicker (diameter 270 ± 9 nm) than in pure PLGA meshes (diameter 218 ± 4 nm), but the areas of pores among these fibers were smaller than in pure PLGA samples (0.46 ± 0.02 µm(2) versus 1.28 ± 0.09 µm(2) in pure PLGA samples). The PLGA-ND membranes showed higher mechanical resistance, as demonstrated by rupture tests of load and deflection of rupture probe at failure. Both types of membranes enabled the attachment, spreading, and subsequent proliferation of human osteoblast-like MG-63 cells to a similar extent, although these values were usually lower than on polystyrene dishes. Nevertheless, the cells on both types of membranes were polygonal or spindle-like in shape, and were distributed homogeneously on the samples. From days 1-7 after seeding, their number rose continuously, and at the end of the experiment, these cells were able to create a confluent layer. At the same time, the cell viability, evaluated by a LIVE/DEAD viability/cytotoxicity kit, ranged from 92% to 97% on both types of membranes. In addition, on PLGA-ND membranes, the cells formed well developed talin-containing focal adhesion plaques. As estimated by the determination of tumor necrosis factor-alpha levels in the culture medium and concentration of intercellular adhesion molecule-1, MG-63 cells, and RAW 264.7 macrophages on these membranes did not show considerable inflammatory activity. CONCLUSION: This study shows that nanofibrous PLGA membranes loaded with diamond nanoparticles have interesting potential for use in bone tissue engineering.

The primordium of a biological joint replacement: Coupling of two stem cell pathways in biphasic ultrarapid compressed gel niches.

The impaired temporomandibular joint might be the first to benefit from applied tissue engineering techniques because it is small and tissue growth in larger amounts is challenging. Bone and cartilage require different competing environmental conditions to be cultivated in vitro. But coupling both the osteogenic and cartilaginous pathways of mesenchymal stem cell differentiation in homeostasis will be a key essential to grow osteochondral constructs or even the first biological joint replacement in the future. The aim of this study was to test a single source biomaterial and a single source cell type to engineer a biphasic osteochondral construct in vitro for future in vivo implantation. Ultrarapid tissue engineering techniques were used to create the biphasic matrix and primary human mesenchymal stem cells (MSCs) preconditioned in osteogenic and chondrogenic media were then seeded in opposite portions of the hyper-hydrated collagen gel in order to further substantiate the respective bone-like and cartilage-like layers thus potentially customising the collagen scaffold according to patient needs in regards to future biological joint replacements. After incubation for 7 days to allow cell growth and differentiation, mineralization of the bone-like layer was demonstrated using von Kossa staining and biochemical bone markers. The cartilage-like layer was demonstrated using alcian blue staining and biochemical cartilage markers. Integration of the bone-like and cartilage-like layers to simulate a tidemark layer was achieved through partial setting of the gels. Cell tracking was used to further confirm the establishment of distinct cartilage-like and bone-like areas within the single construct. This is the first report of one homogeneous human MSC population differentiating into dissimilar "bone-like" and "cartilage-like" zones hosted in a biphasic ultrarapid compressed gel phase niche and mimicking a primordial joint-like structure.

Synergy between myogenic and non-myogenic cells in a 3D tissue-engineered craniofacial skeletal muscle construct.

In vitro skeletal muscle engineering involves the culture of isolated primary myogenic cells in an environment conducive to the formation of a three-dimensional (3D) tissue construct capable of generating force. Isolated human myogenic cells have been used to study cell-cell interactions, permitting identification of functions intrinsic to skeletal muscle in two dimensions (2D). However, the independent contribution of human myogenic and non-myogenic cell types that comprise skeletal muscle to myogenic cell differentiation, force generation and matrix remodelling has yet to be established in 3D. The objective of this study was to use isolated human myogenic and non-myogenic muscle-derived cells (MDC) seeded in 3D collagen constructs to engineer a biomimetic craniofacial skeletal construct. The aim was to purify the two subpopulations of myogenic and non-myogenic cells from human masseter muscle and quantitate myogenic cell differentiation, force generation and matrix remodelling of the 3D collagen construct. The results showed that both the heterogeneous mixture of cells and the purified myogenic cell population expressed myogenin, indicative of myogenic cell differentiation. Further, there was a synergistic effect as the heterogeneous co-culture of myogenic and non-myogenic cells generated the highest peak force and expressed the most MMP-2 mRNA compared to isolated individual cell populations.