Repair and regeneration of osteochondral defects in the articular joints

People suffering from pain due to osteoarthritic or rheumatoidal changes in the joints are still waiting for a better treatment. Although some studies have achieved success in repairing small cartilage defects, there is no widely accepted method for complete repair of osteochondral defects. Also joint replacements have not yet succeeded in replacing of natural cartilage without complications. Therefore, there is room for a new medical approach, which outperforms currently used methods. The aim of this study is to show potential of using a tissue engineering approach for regeneration of osteochondral defects. The critical review of currently used methods for treatment of osteochondral defects is also provided. In this study, two kinds of hybrid scaffolds developed in Hutmacher's group have been analysed. The first biphasic scaffold consists of fibrin and PCL. The fibrin serves as a cartilage phase while the porous PCL scaffold acts as the subchondral phase. The second system comprises of PCL and PCL-TCP. The scaffolds were fabricated via fused deposition modeling which is a rapid prototyping system. Bone marrow-derived mesenchymal cells were isolated from New Zealand White rabbits, cultured in vitro and seeded into the scaffolds. Bone regenerations of the subchondral phases were quantified via micro CT analysis and the results demonstrated the potential of the porous PCL and PCL-TCP scaffolds in promoting bone healing. Fibrin was found to be lacking in this aspect as it degrades rapidly. On the other hand, the porous PCL scaffold degrades slowly hence it provides an effective mechanical support. This study shows that in the field of cartilage repair or replacement, tissue engineering may have big impact in the future. In vivo bone and cartilage engineering via combining a novel composite, biphasic scaffold technology with a MSC has been shown a high potential in the knee defect regeneration in the animal models. However, the clinical application of tissue engineering requires the future research work due to several problems, such as scaffold design, cellular delivery and implantation strategies.

Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits

The objective of this study was to evaluate the feasibility and potential of a hybrid scaffold system in large- and high-load-bearing osteochondral defects repair. The implants were made of medical-grade PCL (mPCL) for the bone compartment whereas fibrin glue was used for the cartilage part. Both matrices were seeded with allogenic bone marrow-derived mesenchymal cells (BMSC) and implanted in the defect (4 mm diameter×5 mm depth) on medial femoral condyle of adult New Zealand White rabbits. Empty scaffolds were used at the control side. Cell survival was tracked via fluorescent labeling. The regeneration process was evaluated by several techniques at 3 and 6 months post-implantation. Mature trabecular bone regularly formed in the mPCL scaffold at both 3 and 6 months post-operation. Micro-Computed Tomography showed progression of mineralization from the host–tissue interface towards the inner region of the grafts. At 3 months time point, the specimens showed good cartilage repair. In contrast, the majority of 6 months specimens revealed poor remodeling and fissured integration with host cartilage while other samples could maintain good cartilage appearance. In vivo viability of the transplanted cells was demonstrated for the duration of 5 weeks. The results demonstrated that mPCL scaffold is a potential matrix for osteochondral bone regeneration and that fibrin glue does not inherit the physical properties to allow for cartilage regeneration in a large and high-load-bearing defect site. Keywords: Osteochondral tissue engineering; Scaffold; Bone marrow-derived precursor cells; Fibrin glue

Mild systemic hypoxia and photopic visual field sensitivity

Purpose: Flickering stimuli increase the metabolic demand of the retina,making it a sensitive perimetric stimulus to the early onset of retinal disease. We determine whether flickering stimuli are a sensitive indicator of vision deficits resulting from to acute, mild systemic hypoxia when compared to standard static perimetry. Methods: Static and flicker visual perimetry were performed in 14 healthy young participants while breathing 12% oxygen (hypoxia) under photopic illumination. The hypoxia visual field data were compared with the field data measured during normoxia. Absolute sensitivities (in dB) were analysed in seven concentric rings at 1°, 3°, 6°, 10°, 15°, 22° and 30° eccentricities as well as mean defect (MD) and pattern defect (PD) were calculated. Preliminary data are reported for mesopic light levels. Results: Under photopic illumination, flicker and static visual field sensitivities at all eccentricities were not significantly different between hypoxia and normoxia conditions. The mean defect and pattern defect were not significantly different for either test between the two oxygenation conditions. Conclusion: Although flicker stimulation increases cellular metabolism, flicker photopic visual field impairment is not detected during mild hypoxia. These findings contrast with electrophysiological flicker tests in young participants that show impairment at photopic illumination during the same levels of mild hypoxia. Potential mechanisms contributing to the difference between the visual fields and electrophysiological flicker tests including variability in perimetric data, neuronal adaptation and vascular autoregulation, are considered. The data have implications for the use of visual perimetry in the detection of ischaemic/hypoxic retinal disorders under photopic and mesopic light levels.

Fabrication of osteochondral scaffolds with stereolithography

The osteochondral defect is a classical model for a multiple-tissue problem[1]. Tissue engineering of either bone or cartilage imposes different demands on a scaffold concerning porosity, pore size and interconnectivity. Furthermore, local release of tissue-specific growth factors necessitates a tailored architecture. For the fabrication of an osteochondral scaffold with region specific architecture, an advanced technique is required. Stereolithography is a rapid prototyping technique that allows for the creation of such 3D polymer objects with well-defined architecture. Its working principle is the partial irradiation of a resin, causing a liquid-solid transition. By irradiating this resin by a computer-driven light source, a solid 3D object is constructed layer by layer. To make biodegradable polymers applicable in stereolithography, low-molecular weight polymers have to be functionalised with double bonds to enable photo-initiated crosslinking.

Resorbable composite scaffolds for bone tissue engineering

Bone loss associated with trauma osteo-degenerative diseases and tumors has tremendous socioeconomic impact related to personal and occupation disability and health care costs. Bone grafting is often critical to surgical therapies. Autogenous bone is presently the preferred grafting material; however, this holds several disadvantages such as donor site morbidity. In the present climate of increasing life expectancy with an ensuing increase in bone-related injuries, orthopaedic surgery is undergoing a paradigm shift from bone-grafting to bone engineering, where a scaffold is implanted to provide adequate load bearing and enhance tissue regeneration. Our group at Queensland University of Technology (QUT) have developed, characterised and tested polycaprolactone/ tricalcium phosphate (PCL/TCP) composite scaffolds for low load-bearing bone defects. These scaffolds are being further developed for application in higher load bearing sites. Our approach emphasizes the importance of the biomaterials’ structural design, the scaffold architecture and structural and nutritional requirements for cell culture. These first-generation scaffolds made from medical grade PCL (mPCL) have been studied for more than 5 years within a clinical setting 1. This paper describes the application of second-generation scaffolds in small and large animal bone defect models and the ensuing bone regeneration as shown by histology and µCT.

Differences between in vitro viability and differentiation and in vivo bone-forming efficacy of human mesenchymal stem cells cultured on PCL-TCP scaffolds

Human mesenchymal stem cells (hMSCs) possess great therapeutic potential for the treatment of bone disease and fracture non-union. Too often however, in vitro evidence alone of the interaction between hMSCs and the biomaterial of choice is used as justification for continued development of the material into the clinic. Clearly for hMSC-based regenerative medicine to be successful for the treatment of orthopaedic trauma, it is crucial to transplant hMSCs with a suitable carrier that facilitates their survival, optimal proliferation and osteogenic differentiation in vitro and in vivo. This motivated us to evaluate the use of polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds produced by fused deposition modeling for the delivery of hMSCs. When hMSCs were cultured on the PCL-TCP scaffolds and imaged by a combination of phase contrast, scanning electron and confocal laser microscopy, we observed five distinct stages of colonization over a 21-day period that were characterized by cell attachment, spreading, cellular bridging, the formation of a dense cellular mass and the accumulation of a mineralized extracellular matrix when induced with osteogenic stimulants. Having established that PCL-TCP scaffolds are able to support hMSC proliferation and osteogenic differentiation, we next tested the in vivo efficacy of hMSC-loaded PCL-TCP scaffolds in nude rat critical-sized femoral defects. We found that fluorescently labeled hMSCs survived in the defect site for up to 3 weeks post-transplantation. However, only 50% of the femoral defects treated with hMSCs responded favorably as determined by new bone volume. As such, we show that verification of hMSC viability and differentiation in vitro is not sufficient to predict the efficacy of transplanted stem cells to consistently promote bone formation in orthotopic defects in vivo.

Atomistic numerical investigation of single-crystal copper nanowire with surface defects

Based on the embedded atom method (EAM), a molecular dynamics (MD) simulation is performed to study the single-crystal copper nanowire with surface defects through tension. The tension simulations for nanowire without defect are first carried out under different temperatures, strain rates and time steps and then surface defect effects for nanowire are investigated. The stress-strain curves obtained by the MD simulations of various strain rates show a rate below 1 x 10(9) s-1 will exert less effect on the yield strength and yield point, and the Young's modulus is independent of strain rate. a time step below 5 fs is recommend for the atomic model during the MD simulation. It is observed that high temperature leads to low Young's modulus, as well as the yield strength. The surface defects on nanowires are systematically studied in considering different defect orientations. It is found that the surface defect serves as a dislocation source, and the yield strength shows 34.20% decresse with 45 degree surface defect. Both yield strength and yield point are significantly influenced by the surface defects, except the Young's modulus.

Low frequency of CHEK2 1100delC allele in Australian multiple-case breast cancer families : functional analysis in heterozygous individuals

A protein-truncating variant of CHEK2, 1100delC, is associated with a moderate increase in breast cancer risk. We have determined the prevalence of this allele in index cases from 300 Australian multiple-case breast cancer families, 95% of which had been found to be negative for mutations in BRCA1 and BRCA2. Only two (0.6%) index cases heterozygous for the CHEK2 mutation were identified. All available relatives in these two families were genotyped, but there was no evidence of co-segregation between the CHEK2 variant and breast cancer. Lymphoblastoid cell lines established from a heterozygous carrier contained approximately 20% of the CHEK2 1100delC mRNA relative to wild-type CHEK2 transcript. However, no truncated CHK2 protein was detectable. Analyses of expression and phosphorylation of wild-type CHK2 suggest that the variant is likely to act by haploinsufficiency. Analysis of CDC25A degradation, a downstream target of CHK2, suggests that some compensation occurs to allow normal degradation of CDC25A. Such compensation of the 1100delC defect in CHEK2 might explain the rather low breast cancer risk associated with the CHEK2 variant, compared to that associated with truncating mutations in BRCA1 or BRCA2.

hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex

hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).

A polymorphism in the dysbindin gene (DTNBP1) associated with multiple psychiatric disorders including schizophrenia

Background A number of studies have found associations between dysbindin (DTNBP1) polymorphisms and schizophrenia. Recently we identified a DTNBP1 SNP (rs9370822) that is strongly associated with schizophrenia. Individuals diagnosed with schizophrenia were nearly three times as likely to carry the CC genotype compared to the AA genotype. Methods To investigate the importance of this SNP in the function of DTNBP1, a number of psychiatric conditions including addictive behaviours and anxiety disorders were analysed for association with rs9370822. Results The DTNBP1 polymorphism was significantly associated with post-traumatic stress disorder (PTSD) as well as nicotine and opiate dependence but not alcohol dependence. Individuals suffering PTSD were more than three times as likely to carry the CC genotype compared to the AA genotype. Individuals with nicotine or opiate dependence were more than twice as likely to carry the CC genotype compared to the AA genotype. Conclusions This study provides further support for the importance of DTNBP1 in psychiatric conditions and suggests that there is a common underlying molecular defect involving DTNBP1 that contributes to the development of several anxiety and addictive disorders that are generally recognised as separate clinical conditions. These disorders may actually be different expressions of a single metabolic pathway perturbation. As our participant numbers are limited our observations should be viewed with caution until they are independently replicated.

Development of an online condition monitoring system for slow speed machinery

One of the main challenges of slow speed machinery condition monitoring is that the energy generated from an incipient defect is too weak to be detected by traditional vibration measurements due to its low impact energy. Acoustic emission (AE) measurement is an alternative for this as it has the ability to detect crack initiations or rubbing between moving surfaces. However, AE measurement requires high sampling frequency and consequently huge amount of data are obtained to be processed. It also requires expensive hardware to capture those data, storage and involves signal processing techniques to retrieve valuable information on the state of the machine. AE signal has been utilised for early detection of defects in bearings and gears. This paper presents an online condition monitoring (CM) system for slow speed machinery, which attempts to overcome those challenges. The system incorporates relevant signal processing techniques for slow speed CM which include noise removal techniques to enhance the signal-to-noise and peak-holding down sampling to reduce the burden of massive data handling. The analysis software works under Labview environment, which enables online remote control of data acquisition, real-time analysis, offline analysis and diagnostic trending. The system has been fully implemented on a site machine and contributing significantly to improve the maintenance efficiency and provide a safer and reliable operation.

Mesoporous bioglass/silk composite scaffolds for bone tissue engineering

In the past 20 years, mesoporous materials have been attracted great attention due to their significant feature of large surface area, ordered mesoporous structure, tunable pore size and volume, and well-defined surface property. They have many potential applications, such as catalysis, adsorption/separation, biomedicine, etc. [1]. Recently, the studies of the applications of mesoporous materials have been expanded into the field of biomaterials science. A new class of bioactive glass, referred to as mesoporous bioactive glass (MBG), was first developed in 2004. This material has a highly ordered mesopore channel structure with a pore size ranging from 5–20 nm [1]. Compared to non-mesopore bioactive glass (BG), MBG possesses a more optimal surface area, pore volume and improved in vitro apatite mineralization in simulated body fluids [1,2]. Vallet-Regí et al. has systematically investigated the in vitro apatite formation of different types of mesoporous materials, and they demonstrated that an apatite-like layer can be formed on the surfaces of Mobil Composition of Matters (MCM)-48, hexagonal mesoporous silica (SBA-15), phosphorous-doped MCM-41, bioglass-containing MCM-41 and ordered mesoporous MBG, allowing their use in biomedical engineering for tissue regeneration [2-4]. Chang et al. has found that MBG particles can be used for a bioactive drug-delivery system [5,6]. Our study has shown that MBG powders, when incorporated into a poly (lactide-co-glycolide) (PLGA) film, significantly enhance the apatite-mineralization ability and cell response of PLGA films. compared to BG [7]. These studies suggest that MBG is a very promising bioactive material with respect to bone regeneration. It is known that for bone defect repair, tissue engineering represents an optional method by creating three-dimensional (3D) porous scaffolds which will have more advantages than powders or granules as 3D scaffolds will provide an interconnected macroporous network to allow cell migration, nutrient delivery, bone ingrowth, and eventually vascularization [8]. For this reason, we try to apply MBG for bone tissue engineering by developing MBG scaffolds. However, one of the main disadvantages of MBG scaffolds is their low mechanical strength and high brittleness; the other issue is that they have very quick degradation, which leads to an unstable surface for bone cell growth limiting their applications. Silk fibroin, as a new family of native biomaterials, has been widely studied for bone and cartilage repair applications in the form of pure silk or its composite scaffolds [9-14]. Compared to traditional synthetic polymer materials, such as PLGA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the chief advantage of silk fibroin is its water-soluble nature, which eliminates the need for organic solvents, that tend to be highly cytotoxic in the process of scaffold preparation [15]. Other advantages of silk scaffolds are their excellent mechanical properties, controllable biodegradability and cytocompatibility [15-17]. However, for the purposes of bone tissue engineering, the osteoconductivity of pure silk scaffolds is suboptimal. It is expected that combining MBG with silk to produce MBG/silk composite scaffolds would greatly improve their physiochemical and osteogenic properties for bone tissue engineering application. Therefore, in this chapter, we will introduce the research development of MBG/silk scaffolds for bone tissue engineering.

Formation of one-dimensional capped ZnO nanoparticle assemblies at the air/water interface

The self-assembling behavior and microscopic structure of zinc oxide nanoparticle Langmuir-Blodgett monolayer films were investigated for the case of zinc oxide nanoparticles coated with a hydrophobic layer of dodecanethiol. Evolution of nanoparticle film structure as a function of surface pressure (π) at the air-water interface was monitored in situ using Brewster’s angle microscopy, where it was determined that π=16 mN/m produced near-defect-free monolayer films. Transmission electron micrographs of drop-cast and Langmuir-Schaefer deposited films of the dodecanethiol-coated zinc oxide nanoparticles revealed that the nanoparticle preparation method yielded a microscopic structure that consisted of one-dimensional rodlike assemblies of nanoparticles with typical dimensions of 25 x 400 nm, encased in the organic dodecanethiol layer. These nanoparticle-containing rodlike micelles were aligned into ordered arrangements of parallel rods using the Langmuir-Blodgett technique.

Enhancing vascularized bone formation using tissue engineered periosteum

Objectives: The periosteum plays an indispensable role in both bone formation and bone defect healing. The aim of this project is to produce tissue engineered periosteum for bone defect treatment. Methods: In this study we constructed an artificial in vitro periosteum by incorporating osteogenic differentiated bone marrow stromal cells (BMSCs) and cobalt chloride (CoCl2)-treated BMSCs. The engineered periostea were implanted both subcutaneously and into skull bone defects in SCID mice to investigate ectopic and orthotopic osteogenesis and vascularisation. After two weeks in subcutaneous and four weeks in bone defect areas, the implanted constructs were assessed for ectopic and orthotopic osteogenesis and vascularisation by micro-CT, histomorphometrical and immunohistochemical methods. Results: The results showed that CoCl2 pre-treated BMSCs induced higher degree of vascularisation and enhanced osteogenesis within the implants in both ectopic and orthotopic areas. Conclusion: This study provided a novel approach using BMSCs sourced from the same patient for both osteogenic and pro-angiogenic purposes in constructing tissue engineered periosteum to enhance vascularized osteogenesis.

Limitations and challenges in MSC delivery for bone regeneration

Objective: Regeneration of osseous defects by tissue-engineering or cell delivery approach provides a novel means of treatment utilizing cell biology, materials sciences, and molecular biology. The concept of in vitro explanted mesenchymal stem cells (MSCs) with an ability to induce new bone formation has been demonstrated in some small animal models. However, contradictory results have been reported regarding the regenerative capacity of MSCs after ex vivo expansion due to the lack of the understanding of microenvironment for MSC differentiation in vivo. ----- ----- Methods: In our laboratory tissue-derived and bone marrow-derived MSCs have been investigated in their osteogenesis. Cell morphology and proliferation were studied by microscopy, confocal microscopy, FACS and cell counting. Cell differentiation and matrix formation were analysed by matrix staining, quantitative PCR, and immunohistochemistry. A SCID skull defect model was used for cell transplantation studies.----- ----- Results: It was noted that tissue-derived and bone marrow-derived MSCs showed similar characteristics in cell surface marker expression, mesenchymal lineage differentiation potential, and cell population doubling. MSCs from both sources could initiate new bone formation in bone defects after delivery into a critical size defects. The bone forming cells were from both transplanted cells and endogenous cells from the host. Interestingly, the majority of in vitro osteogenic differentiated cells did not form new bone directly even though mineralized matrix was synthesized in vitro by MSCs. Furthermore, no new bone formation was detected when MSCs were transplanted subcutaneously.----- ----- Conclusion: This study unveiled the limitations of MSC delivery in bone regeneration and proposed that in vivo microenvironment needs to be optimized for MSC delivery in osteogenesis.