Regulation of proteasome structure and function

Proteasomes are cylindrical particles made up of a stack of four heptameric rings. In animal cells the outer rings are made up of 7 different types of alpha subunits and the inner rings are composed of 7 out of 10 possible different beta subunits. Regulatory complexes can bind to the ends of the cylinder.We have investigated aspects of the assembly, activity and subunit composition of core proteasome particles and 26S proteasomes, the localization of proteasome subpopulations, and the possible role of phosphorylation in determining proteasome localization, activities and association with regulatory components.

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the c~ and /3 subunits of the simpler proteasome isolated from Thermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that /3-type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these/3-type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulated in vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.

Isolation and inheritance of microsatellite loci for the oily bittering (Acheilognathus koreensis): applications for analysis of genetic diversity of wild populations

The oily bittering Acheilognathus koreensis is a freshwater species that is endemic to Korea and is experiencing severe declines in natural populations as a result of habitat fragmentation and water pollution. For the conservation and restoration of this species, it is necessary to assess its genetic diversity at the population level. We developed 13 polymorphic microsatellite loci that were used to analyze the genetic diversity of two populations collected from the Kum River and the Tamjin River in Korea. All loci exhibited Mendelian inheritance patterns when examined in controlled crosses. Both populations revealed high levels of variability, with the number of alleles ranging from 3 to 20 and observed and expected heterozygosities ranging from 0.500 to 0.969 and from 0.529 to 0.938, respectively. None of the loci showed significant deviation from Hardy–Weinberg equilibrium, and one pair of loci showed significant linkage disequilibrium after Bonferroni correction. Pairwise F ST and genetic distance estimation showed significant differences between two populations. These results suggest that the microsatellites developed herein can be used to study the genetic diversity, population structure and conservation measure of A. koreensis.

Quantitative and qualitative assessment of the regenerative potential of osteoblasts versus bone marrow derived mesenchymal stem cells in the reconstruction of critical sized segmental tibial bone defects in a large animal model

This thesis is about the use of different cells for bone tissue engineering. The cells were used in combination with a novel biomaterial in a large tibial bone defects in a sheep model. Furthermore this study developed a novel cell delivery procedure for bone tissue engineering. This novel procedure of cell delivery could overcome the current problems of cell-based tissue engineering and serve as a baseline for the translation of novel concepts into clinical application.

Multilineage differentiation potential of bone and cartilage cells derived from explant culture

To date, mesenchymal stem cells (MSCs) from various tissues have been reported, but the yield and differentiation potential of different tissue-derived MSCs is still not clear. This study was undertaken in an attempt to investigate the multilineage stem cell potential of bone and cartilage explant cultures in comparison with bone marrow derived mesenchymal stem cells (BMSCs). The results showed that the surface antigen expression of tissue-derived cells was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing markers related to adhesion (CD29, CD166) and stem cells (CD90, CD105). The tissue-derived cells were able to differentiate into osteoblast, chondrocyte and adipocyte lineage pathways when stimulated in the appropriate differentiating conditions. However, compared with BMSCs, tissue-derived cells showed less capacity for multilineage differentiation when the level of differentiation was assessed in monolayer culture by analysing the expression of tissue-specific genes by reverse transcription polymerase chain reaction (RT-PCR) and histology. In high density pellet cultures, tissue-derived cells were able to differentiate into chondrocytes, expressing chondrocyte markers such as proteoglycans, type II collagen and aggrecan. Taken together, these results indicate that cells derived from tissue explant cultures reserved certain degree of differentiation properties of MSCs in vitro.

The evaluation of a biphasic osteochondral implant coupled with an electrospun membrane in a large animal model

Conventional clinical therapies are unable to resolve osteochondral defects adequately, hence tissue engineering solutions are sought to address the challenge. A biphasic implant which was seeded with Mesenchymal Stem Cells (MSC) and coupled with an electrospun membrane was evaluated as an alternative. This dual phase construct comprised of a Polycaprolactone (PCL) cartilage scaffold and a Polycaprolactone - Tri Calcium Phosphate (PCL - TCP) osseous matrix. Autologous MSC was seeded into the entire implant via fibrin and the construct was inserted into critically sized osteochondral defects located at the medial condyle and patellar groove of pigs. The defect was resurfaced with a PCL - collagen electrospun mesh that served as a substitute for periosteal flap in preventing cell leakage. Controls either without implanted MSC or resurfacing membrane were included. After 6 months, cartilaginous repair was observed with a low occurrence of fibrocartilage at the medial condyle. Osteochondral repair was promoted and host cartilage degeneration was arrested as shown by the superior Glycosaminoglycan (GAG) maintenance. This positive morphological outcome was supported by a higher relative Young's modulus which indicated functional cartilage restoration. Bone in growth and remodeling occurred in all groups with a higher degree of mineralization in the experimental group. Tissue repair was compromised in the absence of the implanted cells or the resurfacing membrane. Moreover healing was inferior at the patellar groove as compared to the medial condyle and this was attributed to the native biomechanical features.

Investigating the effects of preinduction on human adipose-derived precursor cells in an athymic rat model

The osteogenic potential of human adipose-derived precursor cells seeded on medical-grade polycaprolactone-tricalcium phosphate scaffolds was investigated in this in vivo study. Three study groups were investigated: (1) induced—stimulated with osteogenic factors only after seeding into scaffold; (2) preinduced—induced for 2 weeks before seeding into scaffolds; and (3) uninduced—cells without any introduced induction. For all groups, scaffolds were implanted subcutaneously into the dorsum of athymic rats. The scaffold/cell constructs were harvested at the end of 6 or 12 weeks and analyzed for osteogenesis. Gross morphological examination using scanning electron microscopy indicated good integration of host tissue with scaffold/cell constructs and extensive tissue infiltration into the scaffold interior. Alizarin Red histology and immunostaining showed a heightened level of mineralization and an increase in osteonectin, osteopontin, and collagen type I protein expression in both the induced and preinduced groups compared with the uninduced groups. However, no significant differences were observed in these indicators when compared between the induced and preinduced groups.

Ovine bone and marrow derived progenitor cells : isolation, characterization, and osteogenic potential

Recently, research has focused on bone marrow derived multipotent mesenchymal precursor cells (MPC) for their potential clinical use in bone engineering. Prior to clinical application, MPC-based treatment concepts need to be evaluated in preclinical, immunocompetent, large animal models. Sheep in particular are considered a valid model for orthopaedic and trauma related research. However, ovine MPC and their osteogenic potential remain poorly characterized. In the present study, ex vivo expanded MPC isolated from ovine bone marrow proliferated at a higher rate than osteoblasts (OB) derived from tibial compact bone as assessed using standard 2D culture. MPC expressed the respective phenotypic profile typical for different mesenchymal cell populations (CD14-/CD31-/CD45- /CD29+/CD44+/CD166+) and showed a multilineage differentiation potential. When compared to OB, MPC had a higher mineralization potential under standard osteogenic culture conditions and expressed typical markers such as osteocalcin, osteonectin and type I collagen at the mRNA and protein level. After 4 weeks in 3D culture, MPC constructs demonstrated higher cell density and mineralization, whilst cell viability on the scaffolds was assessed >90%. Cells displayed a spindle-like morphology and formed an interconnected network. Implanted subcutaneously into NOD/SCID mice on type I collagen coated polycaprolactone-tricalciumphosphate (mPCL-TCP) scaffolds, MPC presented a higher developmental potential than osteoblasts. In summary, this study provides a detailed in vitro characterisation of ovine MPC from a bone engineering perspective and suggests that MPC provide promising means for future bone disease related treatment applications.

Solving navigational uncertainty using grid cells on robots

To successfully navigate their habitats, many mammals use a combination of two mechanisms, path integration and calibration using landmarks, which together enable them to estimate their location and orientation, or pose. In large natural environments, both these mechanisms are characterized by uncertainty: the path integration process is subject to the accumulation of error, while landmark calibration is limited by perceptual ambiguity. It remains unclear how animals form coherent spatial representations in the presence of such uncertainty. Navigation research using robots has determined that uncertainty can be effectively addressed by maintaining multiple probabilistic estimates of a robot's pose. Here we show how conjunctive grid cells in dorsocaudal medial entorhinal cortex (dMEC) may maintain multiple estimates of pose using a brain-based robot navigation system known as RatSLAM. Based both on rodent spatially-responsive cells and functional engineering principles, the cells at the core of the RatSLAM computational model have similar characteristics to rodent grid cells, which we demonstrate by replicating the seminal Moser experiments. We apply the RatSLAM model to a new experimental paradigm designed to examine the responses of a robot or animal in the presence of perceptual ambiguity. Our computational approach enables us to observe short-term population coding of multiple location hypotheses, a phenomenon which would not be easily observable in rodent recordings. We present behavioral and neural evidence demonstrating that the conjunctive grid cells maintain and propagate multiple estimates of pose, enabling the correct pose estimate to be resolved over time even without uniquely identifying cues. While recent research has focused on the grid-like firing characteristics, accuracy and representational capacity of grid cells, our results identify a possible critical and unique role for conjunctive grid cells in filtering sensory uncertainty. We anticipate our study to be a starting point for animal experiments that test navigation in perceptually ambiguous environments.

Preclinical animal models for segmental bone defect research and tissue engineering

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, however they are limited in access and availability and associated with donor site morbidity, haemorrhage, risk of infection, insufficient transplant integration, graft devitalisation, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. Analysing the tissue engineering literature it can be concluded that bone regeneration has become a focus area in the field. Hence, a considerable number of research groups and commercial entities work on the development of tissue engineered constructs for bone regeneration. However, bench to bedside translations are still infrequent as the process towards approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. In translational orthopaedic research, the utilisation of large preclinical animal models is a conditio sine qua non. Consequently, to allow comparison between different studies and their outcomes, it is essential that animal models, fixation devices, surgical procedures and methods of taking measurements are well standardized to produce reliable data pools as a base for further research directions. The following chapter reviews animal models of the weight-bearing lower extremity utilized in the field which include representations of fracture-healing, segmental bone defects, and fracture non-unions.

Periodontal regeneration and mesenchymal stem cells

The ultimate goal of periodontal therapy is to regenerate periodontal supporting tissues, but this is hard to achieve as the results of periodontal techniques for regeneration are clinically unpredictable. Stem cells owing to their plasticity and proliferation potential provides a new paradigm for periodontal regeneration. Stem cells from mesenchyme can self renew and generate new dental tissues (including dentin and cementum), alveolar bone and periodontal ligament, and thus they have great potential in periodontal regeneration. This chapter presents an insight into mesenchymal stem cells and their potential use in periodontal regeneration. In this chapter the cellular and molecular biology in periodontal regeneration will be introduced, followed by a range of conventional surgical procedures for periodontal regeneration will be discussed. Mesenchymal stem cells applied in regenerated periodontal tissue and their biological characterizations in vitro will be also introduced. Lastly, the use of mesenchymal stem cell to repair periodontal tissues in large animal models will be also reviewed.

Inhibition of p38 pathway leads to OA-like changes in a rat animal model

Objectives The p38 mitogen-activated protein kinase (MAPK) signal transduction pathway is involved in a variety of inflammatory responses, including cytokine generation, cell differentiation proliferation and apoptosis. Here, we examined the effects of systemic p38 MAPK inhibition on cartilage cells and osteoarthritis (OA) disease progression by both in vitro and in vivo approaches. Methods p38 kinase activity was evaluated in normal and OA cartilage cells by measuring the amount of phosphorylated protein. To examine the function of p38 signaling pathway in vitro, normal chondrocytes were isolated and differentiated in the presence or absence of p38 inhibitor; SB203580 and analysed for chondrogenic phenotype. Effect of systemic p38 MAPK inhibition in normal and OA (induced by menisectomy) rats were analysed by treating animals with vehicle alone (DMS0) or p38 inhibitor (SB203580). Damage to the femur and tibial plateau was evaluated by modified Mankin score, histology and immunohistochemistry. Results Our in vitro studies have revealed that a down-regulation of chondrogenic and increase of hypertrophic gene expression occurs in the normal chondrocytes, when p38 is neutralized by a pharmacological inhibitor. We further observed that the basal levels of p38 phosphorylation were decreased in OA chondrocytes compared with normal chondrocytes. These findings together indicate the importance of this pathway in the regulation of cartilage physiology and its relevance to OA pathogenesis. At in vivo level, systematic administration of a specific p38 MAPK inhibitor, SB203580, continuously for over a month led to a significant loss of proteoglycan; aggrecan and cartilage thickness. On the other hand, SB203580 treated normal rats showed a significant increase in TUNEL positive cells, cartilage hypertrophy markers such as Type 10 collagen, Runt-related transcription factor and Matrix metalloproteinase-13 and substantially induced OA like phenotypic changes in the normal rats. In addition, menisectomy induced OA rat models that were treated with p38 inhibitor showed aggravation of cartilage damage. Conclusions In summary, this study has provided evidence that the component of the p38 MAPK pathway is important to maintain the cartilage health and its inhibition can lead to severe cartilage degenerative changes. The observations in this study highlight the possibility of using activators of the p38 pathway as an alternative approach in the treatment of OA.

Tissue engineering bone - reconstruction of critical sized segmental bone defects in a large animal model

Currently, well established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, their application, however, is associated with disadvantages. These include limited access and availability, donor site morbidity and haemorrhage, increased risk of infection, and insufficient transplant integration. As a result, recent research focuses on the development of complementary therapeutic concepts. The field of tissue engineering has emerged as an important alternative approach to bone regeneration. Tissue engineering unites aspects of cellular biology, biomechanical engineering, biomaterial sciences and trauma and orthopaedic surgery. To obtain approval by regulatory bodies for these novel therapeutic concepts the level of therapeutic benefit must be demonstrated rigorously in well characterized, clinically relevant animal models. Therefore, in this PhD project, a reproducible and clinically relevant, ovine, critically sized, high load bearing, tibial defect model was established and characterized as a prerequisite to assess the regenerative potential of a novel treatment concept in vivo involving a medical grade polycaprolactone and tricalciumphosphate based composite scaffold and recombinant human bone morphogenetic proteins.

Leucocytes, cytokines and satellite cells : what role do they play in muscle damage and regeneration following eccentric exercise?

Exercise-induced muscle damage is an important topic in exercise physiology. However several aspects of our understanding of how muscles respond to highly stressful exercise remain unclear In the first section of this review we address the evidence that exercise can cause muscle damage and inflammation in otherwise healthy human skeletal muscles. We approach this concept by comparing changes in muscle function (i.e., the force-generating capacity) with the degree of leucocyte accumulation in muscle following exercise. In the second section, we explore the cytokine response to 'muscle-damaging exercise', primarily eccentric exercise. We review the evidence for the notion that the degree of muscle damage is related to the magnitude of the cytokine response. In the third and final section, we look at the satellite cell response to a single bout of eccentric exercise, as well as the role of the cyclooxygenase enzymes (COX1 and 2). In summary, we propose that muscle damage as evaluated by changes in muscle function is related to leucocyte accumulation in the exercised muscles. 'Extreme' exercise protocols, encompassing unaccustomed maximal eccentric exercise across a large range of motion, generally inflict severe muscle damage, inflammation and prolonged recovery (> 1 week). By contrast, exercise resembling regular athletic training (resistance exercise and downhill running) typically causes mild muscle damage (myofibrillar disruptions) and full recovery normally occurs within a few days. Large variation in individual responses to a given exercise should, however be expected. The link between cytokine and satellite cell responses and exercise-induced muscle damage is not so clear The systemic cytokine response may be linked more closely to the metabolic demands of exercise rather than muscle damage. With the exception of IL-6, the sources of systemic cytokines following exercise remain unclear The satellite cell response to severe muscle damage is related to regeneration, whereas the biological significance of satellite cell proliferation after mild damage or non-damaging exercise remains uncertain. The COX enzymes regulate satellite cell activity, as demonstrated in animal models; however the roles of the COX enzymes in human skeletal muscle need further investigation. We suggest using the term 'muscle damage' with care. Comparisons between studies and individuals must consider changes in and recovery of muscle force-generating capacity.