Perfusion model system to generate engineered grafts with controlled cellular distributions

Engineered tissue grafts, which mimic the spatial variations of cell density and extracellular matrix present in native tissues, could facilitate more efficient tissue regeneration and integration. We previously demonstrated that cells could be uniformly seeded throughout a 3D scaffold having a random pore architecture using a perfusion bioreactor2. In this work, we aimed to generate 3D constructs with defined cell distributions based on rapid prototyped scaffolds manufactured with a controlled gradient in porosity. Computational models were developed to assess the influence of fluid flow, associated with pore architecture and perfusion regime, on the resulting cell distribution.

Nuclear localization of NPR1 is required for activation of PR gene expression

Systemic acquired resistance (SAR) is a broad-spectrum resistance in plants that involves the upregulation of a battery of pathogenesis-related (PR) genes. NPR1 is a key regulator in the signal transduction pathway that leads to SAR. Mutations in NPR1 result in a failure to induce PR genes in systemic tissues and a heightened susceptibility to pathogen infection, whereas overexpression of the NPR1 protein leads to increased induction of the PR genes and enhanced disease resistance. We analyzed the subcellular localization of NPR1 to gain insight into the mechanism by which this protein regulates SAR. An NPR1–green fluorescent protein fusion protein, which functions the same as the endogenous NPR1 protein, was shown to accumulate in the nucleus in response to activators of SAR. To control the nuclear transport of NPR1, we made a fusion of NPR1 with the glucocorticoid receptor hormone binding domain. Using this steroid-inducible system, we clearly demonstrate that nuclear localization of NPR1 is essential for its activity in inducing PR genes.

A myosin from a higher plant has structural similarities to class V myosins

In plant cells, myosin is believed to be the molecular motor responsible for actin-based motility processes such as cytoplasmic streaming and directed vesicle transport. In an effort to characterize plant myosin, a cDNA encoding a myosin heavy chain was isolated from Arabidopsis thaliana. The predicted product of the MYA1 gene is 173 kDa and is structurally similar to the class V myosins. It is composed of the highly-conserved NH2-terminal "head" domain, a putative calmodulin-binding "neck" domain an alpha-helical coiled-coil domain, and a COOH-terminal domain. Northern blot analysis shows that the Arabidopsis MYA1 gene is expressed in all the major plant tissues (flower, leaf, root, and stem). We suggest that the MYA1 myosin may be involved in a general intracellular transport process in plant cells.

Formalin fixation affects equilibrium partitioning of an ionic contrast agent-microcomputed tomography (EPIC-[mu]CT) imaging of osteochondral samples

Equilibrium Partitioning of an Ionic Contrast agent with microcomputed tomography (EPIC-[mu]CT) is a non-invasive technique to quantify and visualize the three-dimensional distribution of glycosaminoglycans (GAGs) in fresh cartilage tissue. However, it is unclear whether this technique is applicable to already fixed tissues. Therefore, this study aimed at investigating whether formalin fixation of bovine cartilage affects X-ray attenuation, and thus the interpretation of EPIC-[mu]CT data.Design Osteochondral samples (n = 24) were incubated with ioxaglate, an ionic contrast agent, for 22 h prior to [mu]CT scanning. The samples were scanned in both formalin-fixed and fresh conditions. GAG content was measured using a biochemical assay and normalized to wet weight, dry weight, and water content to determine potential reasons for differences in X-ray attenuation.Results The expected zonal distribution of contrast agent/GAGs was observed for both fixed and fresh cartilage specimens. However, despite no significant differences in GAG concentrations or physical properties between fixed and fresh samples, the average attenuation levels of formalin-fixed cartilage were 14.3% lower than in fresh samples.Conclusions EPIC-[mu]CT is useful for three-dimensional visualization of GAGs in formalin-fixed cartilage. However, a significant reduction in X-ray attenuation for fixed (compared to fresh) cartilage must be taken into account and adjusted for accordingly when quantifying GAG concentrations using EPIC-[mu]CT.

Engineering an Ecosystem : Taking Cues from Nature’s Paradigm to Build Tissue in the Lab and the Body

This manuscript took a 'top down' approach to understanding survival of inhabitant cells in the ecosystem bone, working from higher to lower length and time scales through the hierarchical ecosystem of bone. Our working hypothesis is that nature “engineered” the skeleton using a 'bottom up' approach,where mechanical properties of cells emerge from their adaptation to their local me-chanical milieu. Cell aggregation and formation of higher order anisotropic struc- ture results in emergent architectures through cell differentiation and extracellular matrix secretion. These emergent properties, including mechanical properties and architecture, result in mechanical adaptation at length scales and longer time scales which are most relevant for the survival of the vertebrate organism [Knothe Tate and von Recum 2009]. We are currently using insights from this approach to har-ness nature’s regeneration potential and to engineer novel mechanoactive materials [Knothe Tate et al. 2007, Knothe Tate et al. 2009]. In addition to potential applications of these exciting insights, these studies may provide important clues to evolution and development of vertebrate animals. For instance, one might ask why mesenchymal stem cells condense at all? There is a putative advantage to self-assembly and cooperation, but this advantage is somewhat outweighed by the need for infrastructural complexity (e.g., circulatory systems comprised of specific differentiated cell types which in turn form conduits and pumps to overcome limitations of mass transport via diffusion, for example; dif-fusion is untenable for multicellular organisms larger than 250 microns in diameter. A better question might be: Why do cells build skeletal tissue? Once cooperatingcells in tissues begin to deplete local sources of food in their aquatic environment, those that have evolved a means to locomote likely have an evolutionary advantage. Once the environment becomes less aquarian and more terrestrial, self-assembled organisms with the ability to move on land might have conferred evolutionary ad-vantages as well. So did the cytoskeleton evolve several length scales, enabling the emergence of skeletal architecture for vertebrate animals? Did the evolutionary advantage of motility over noncompliant terrestrial substrates (walking on land) favor adaptations including emergence of intracellular architecture (changes in the cytoskeleton and upregulation of structural protein manufacture), inter-cellular con- densation, mineralization of tissues, and emergence of higher order architectures?How far does evolutionary Darwinism extend and how can we exploit this knowl- edge to engineer smart materials and architectures on Earth and new, exploratory environments?[Knothe Tate et al. 2008]. We are limited only by our ability to imagine. Ultimately, we aim to understand nature, mimic nature, guide nature and/or exploit nature’s engineering paradigms without engineer-ing ourselves out of existence.

Purification and Properties of Closteroviruslike Particles Associated with Grapevine Corky Bark Disease

Closteroviruslike particles, designated as grapevine corky bark-associated virus (GCBaV), were purified from mature leaves and stem phloem tissue of a corky bark-affected grapevine that had indexed negative for other grapevine viruses. Electron microscopy of purified preparations revealed the presence of flexuous rod-shaped viruslike particles that were about 13 nm in diameter and between 1,400 and 2,000 nm long, with a helical pitch of 3.4 nm. In purified preparations, the GCBaV particles degraded within a few weeks, unlike grapevine leafroll associated virus (GLRaV), which was stable for more than 1 mo under the same storage condition. The molecular weight of the coat protein of GCBaV was 24,000. A large dsRNA molecule (about 15.3 kbp), along with lower molecular weight species, was detected in tissues of corky bark-diseased grapevines, but not in healthy grapevines. Polyclonal antisera were produced in rabbits against purified or partially purified virus preparations. In direct enzyme-linked immunosorbent assay (ELISA), antisera to GCBaV did not react to the serologically distinct types (II and III) of the long closteroviruses associated with grapevine leafroll disease and grapevine virus A (GVA), and vice versa. This antiserum also reacted in ELISA with other corky bark-affected grapevines. Our data suggest that closteroviruslike particles, designated as GCBaV, may be the causal agent of corky bark disease. However, definitive proof is still lacking. The inclusion of GCBaV in the group of closteroviruses with citrus tristeza virus is proposed.

Chlamydial infection of immune cells : altered function and implications for disease

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects the genital and ocular mucosa of humans, causing infections that can lead to pelvic inflammatory disease, infertility, and blinding trachoma. C. pneumoniae is a respiratory pathogen that is the cause of 12–15% of community-acquired pneumonia. Both chlamydial species were believed to be restricted to the epithelia of the genital, ocular, and respiratory mucosa; however, increasing evidence suggests that both these pathogens can be isolated from peripheral blood of both healthy individuals and patients with inflammatory conditions such as coronary artery disease and asthma. Chlamydia can also be isolated from brain tissues of patients with degenerative neurological disorders such as Alzheimer’s disease and multiple sclerosis, and also from certain lymphomas. An increasing number of in vitro studies suggest that some chlamydial species can infect immune cells, at least at low levels. These infections may alter immune cell function in a way that promotes chlamydial persistence in the host and contributes to the progression of several chronic inflammatory diseases. In this paper, we review the evidence for the growth of Chlamydia in immune cells, particularly monocytes/macrophages and dendritic cells, and describe how infection may affect the function of these cells.

Mesenchymal stem cells

Cell based therapies as they apply to tissue engineering and regenerative medicine, require cells capable of self renewal and differentiation, and a prerequisite is to be able to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies therefore figures as an integral part of tissue engineering. Stem cells serve as a reserve for biological repair, having the potential to differentiate into a number of specialised cell types within the body; they therefore represent the most useful candidates for cell based therapies. The primary goal of stem cell research is to produce cells that are both patient specific, as well as having properties suitable for the specific conditions for which they are intended to remedy. From a purely scientific perspective, stem cells allow scientists to gain a deeper understanding of developmental biology and regenerative therapies. Stem cells have acquired a number of uses for applications in regenerative medicine, immunotherapy, gene therapy, but it is in the area of tissue engineering that they generate most excitement, primarily as a result of their capacity for self-renewal and pluripotency. A unique feature of stem cells is their ability to maintain an uncommitted quiescent state in vivo and then, once triggered by conditions such as disease, injury or natural wear or tear, serve as a reservoir and natural support system to replenish lost cells. Although these cells retain the plasticity to differentiate into various tissues, being able to control this differentiation process is still one of the biggest challenges facing stem cell research. In an effort to harness the potential of these cells a number of studies have been conducted using both embryonic/foetal and adult stem cells. The use of embryonic stem cells (ESC) have been hampered by strong ethical and political concerns, this despite their perceived versatility due to their pluripotency. Ethical issues aside, other concerns raised with ESCs relates to the possibility of tumorigenesis, immune rejection and complications with immunosuppressive therapies, all of which adds layers of complications to the application ESC in research and which has led to the search for alternative sources for stem cells. The adult tissues in higher organisms harbours cells, termed adult stem cells, and these cells are reminiscent of unprogrammed stem cells. A number of sources of adult stem cells have been described. Bone marrow is by far the most accessible source of two potent populations of adult stem cells, namely haematopoietic stem cells (HSCs) and bone marrow mesenchymal stem cells (BMSCs). Autologously harvested adult stem cells can, in contrast to embryonic stem cells, readily be used in autografts, since immune rejection is not an issue; and their use in scientific research has not attracted the ethical concerns which have been the case with embryonic stem cells. The major limitation to their use, however, is the fact that adult stem cells are exceedingly rare in most tissues. This fact makes identifying and isolating these cells problematic; bone marrow being perhaps the only notable exception. Unlike the case of HSCs, there are as yet no rigorous criteria for characterizing MSCs. Changing acuity about the pluripotency of MSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to MSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their study in vitro. Also, when MSCs are cultured in vitro, there is a loss of the in vivo microenvironment, resulting in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage numbers in culture, characterized by the onset of senescence related changes. As a consequence, it is necessary to establish protocols for generating large numbers of MSCs but without affecting their differentiation potential. MSCs are capable of differentiating into mesenchymal tissue lineages, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Recent findings indicate that adult bone marrow may also contain cells that can differentiate into the mature, nonhematopoietic cells of a number of tissues, including cells of the liver, kidney, lung, skin, gastrointestinal tract, and myocytes of heart and skeletal muscle. MSCs can readily be expanded in vitro and can be genetically modified by viral vectors and be induced to differentiate into specific cell lineages by changing the microenvironment–properties which makes these cells ideal vehicles for cellular gene therapy. MSCs can also exert profound immunosuppressive effects via modulation of both cellular and innate immune pathways, and this property allows them to overcome the issue of immune rejection. Despite the many attractive features associated with MSCs, there are still many hurdles to overcome before these cells are readily available for use in clinical applications. The main concern relates to in vivo characterization and identification of MSCs. The lack of a universal biomarker, sparse in vivo distribution, and a steady age related decline in their numbers, makes it an obvious need to decipher the reprogramming pathways and critical molecular players which govern the characteristics unique to MSCs. This book presents a comprehensive insight into the biology of adult stem cells and their utility in current regeneration therapies. The adult stem cell populations reviewed in this book include bone marrow derived MSCs, adipose derived stem cells (ASCs), umbilical cord blood stem cells, and placental stem cells. The features such as MSC circulation and trafficking, neuroprotective properties, and the nurturing roles and differentiation potential of multiple lineages have been discussed in details. In terms of therapeutic applications, the strengths of MSCs have been presented and their roles in disease treatments such as osteoarthritis, Huntington’s disease, periodontal regeneration, and pancreatic islet transplantation have been discussed. An analysis comparing osteoblast differentiation of umbilical cord blood stem cells and MSCs has been reviewed, as has a comparison of human placental stem cells and ASCs, in terms of isolation, identification and therapeutic applications of ASC in bone, cartilage regeneration, as well as myocardial regeneration. It is my sincere hope that this book will update the reader as to the research progress of MSC biology and potential use of these cells in clinical applications. It will be the best reward to all contributors of this book, if their efforts herein may in some way help the readers in any part of their study, research, and career development.

The biological application of cobalt

This review collects and summarises the biological applications of the element cobalt. Small amounts of the ferromagnetic metal can be found in rock, soil, plants and animals, but is mainly obtained as a by-product of nickel and copper mining, and is separated from the ores (mainly cobaltite, erythrite, glaucodot and skutterudite) using a variety of methods. Compounds of cobalt include several oxides, including: green cobalt(II) (CoO), blue cobalt(II,III) (Co3O4), and black cobalt(III) (Co2O3); four halides including pink cobalt(II) fluoride (CoF2), blue cobalt(II) chloride (CoCl2), green cobalt(II) bromide (CoBr2), and blue-black cobalt(II) iodide (CoI2). The main application of cobalt is in its metal form in cobalt-based super alloys, though other uses include lithium cobalt oxide batteries, chemical reaction catalyst, pigments and colouring, and radioisotopes in medicine. It is known to mimic hypoxia on the cellular level by stabilizing the α subunit of hypoxia inducing factor (HIF), when chemically applied as cobalt chloride (CoCl2). This is seen in many biological research applications, where it has shown to promote angiogenesis, erythropoiesis and anaerobic metabolism through the transcriptional activation of genes such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO), contributing significantly to the pathophysiology of major categories of disease, such as myocardial, renal and cerebral ischaemia, high altitude related maladies and bone defects. As a necessary constituent for the formation of vitamin B12, it is essential to all animals, including humans, however excessive exposure can lead to tissue and cellular toxicity. Cobalt has been shown to provide promising potential in clinical applications, however further studies are necessary to clarify its role in hypoxia-responsive genes and the applications of cobalt-chloride treated tissues.

Instability prolongs the chondral phase during bone healing in sheep

In this sheep study, we investigated the influence of fixation stability on the temporal and spatial distribution of tissues in the fracture callus. As the initial mechanical conditions have been cited as being especially important for the healing outcome, it was hypothesized that differences in the path of healing would be seen as early as the initial phase of healing. ----- ----- Sixty-four sheep underwent a mid-shaft tibial osteotomy that was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture calluses were analyzed using radiological, biomechanical and histological techniques. Statistical comparison between the groups was performed using the Mann–Whitney U test for unpaired non-parametric data. ----- ----- In the callus of the tibia treated with semi-rigid fixation, remnants of the fracture haematoma remained present for longer, although new periosteal bone formation during early healing was similar in both groups. The mechanical competence of the healing callus at 6 weeks was inferior compared to tibiae treated with rigid fixation. Semi-rigid fixation resulted in a larger cartilage component of the callus, which persisted longer. Remodeling processes were initiated earlier in the rigid group, while new bone formation continued throughout the entire investigated period in the semi-rigid group. ----- ----- In this study, evidence is provided that less rigid fixation increased the time required for healing. The process of intramembranous ossification appeared during the initial stages of healing to be independent of mechanical stability. However, the delay in healing was related to a prolonged chondral phase.

Temporal tissue patterns in bone healing of sheep

Secondary fracture healing in long bones leads to the successive formation of intricate patterns of tissues in the newly formed callus. The main aim of this work was to quantitatively describe the topology of these tissue patterns at different stages of the healing process and to generate averaged images of tissue distribution. This averaging procedure was based on stained histological sections (2, 3, 6, and 9 weeks post-operatively) of 64 sheep with a 3 mm tibial mid-shaft osteotomy, stabilized either with a rigid or a semi-rigid external fixator. Before averaging, histological images were sorted for topology according to six identified tissue patterns. The averaged images were obtained for both fixation types and the lateral and medial side separately. For each case, the result of the averaging procedure was a collection of six images characterizing quantitatively the progression of the healing process. In addition, quantified descriptions of the newly formed cartilage and the bone area fractions (BA/TA) of the bony callus are presented. For all cases, a linear increase in the BA/TA of the bony callus was observed. The slope was greatest in the case of the most rigid stabilization and lowest in the case of the least stiff. This topological description of the progression of bone healing will allow quantitative validation (or falsification) of current mechano-biological theories.

Size and habit of mineral particles in bone and mineralized callus during bone healing in sheep

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small-angle X-ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus.

Spatial and temporal variations of mechanical properties and mineral content of the external callus during bone healing

After bone fracture, various cellular activities lead to the formation of different tissue types, which form the basis for the process of secondary bone healing. Although these tissues have been quantified by histology, their material properties are not well understood. Thus, the aim of this study is to correlate the spatial and temporal variations in the mineral content and the nanoindentation modulus of the callus formed via intramembranous ossification over the course of bone healing. Midshaft tibial samples from a sheep osteotomy model at time points of 2, 3, 6 and 9 weeks were employed. PMMA embedded blocks were used for quantitative back scattered electron imaging and nanoindentation of the newly formed periosteal callus near the cortex. The resulting indentation modulus maps show the heterogeneity in the modulus in the selected regions of the callus. The indentation modulus of the embedded callus is about 6 GPa at the early stage. At later stages of mineralization, the average indentation modulus reaches 14 GPa. There is a slight decrease in average indentation modulus in regions distant to the cortex, probably due to remodelling of the peripheral callus. The spatial and temporal distribution of mineral content in the callus tissue also illustrates the ongoing remodelling process observed from histological analysis. Most interestingly the average indentation modulus, even at 9 weeks, remains as low as 13 GPa, which is roughly 60% of that for cortical sheep bone. The decreased indentation modulus in the callus compared to cortex is due to the lower average mineral content and may be perhaps also due to the properties of the organic matrix which might be different from normal bone.

Pressure, oxygen tension and temperature in the periosteal callus during bone healing - An in vivo study in sheep

Adequate blood supply and sufficient mechanical stability are necessary for timely fracture healing. Damage to vessels impairs blood supply; hindering the transport of oxygen which is an essential metabolite for cells involved in repair. The degree of mechanical stability determines the mechanical conditions in the healing tissues. The mechanical conditions can influence tissue differentiation and may also inhibit revascularization. Knowledge of the actual conditions in a healing fracture in vivo is extremely limited. This study aimed to quantify the pressure, oxygen tension and temperature in the external callus during the early phase of bone healing. Six Merino-mix sheep underwent a tibial osteotomy. The tibia was stabilized with a standard mono-lateral external fixator. A multi-parameter catheter was placed adjacent to the osteotomy gap on the medial aspect of the tibia. Measurements of oxygen tension and temperature were performed for ten days post-op. Measurements of pressure were performed during gait on days three and seven. The ground reaction force and the interfragmentary movements were measured simultaneously. The maximum pressure during gait increased (p=0.028) from three (41.3 [29.2-44.1] mm Hg) to seven days (71.8 [61.8-84.8] mm Hg). During the same interval, there was no change (p=0.92) in the peak ground reaction force or in the interfragmentary movement (compression: p=0.59 and axial rotation: p=0.11). Oxygen tension in the haematoma (74.1 mm Hg [68.6-78.5]) was initially high post-op and decreased steadily over the first five days. The temperature increased over the first four days before reaching a plateau at approximately 38.5 degrees C on day four. This study is the first to report pressure, oxygen tension and temperature in the early callus tissues. The magnitude of pressure increased even though weight bearing and IFM remained unchanged. Oxygen tensions were initially high in the haematoma and fell gradually with a low oxygen environment first established after four to five days. This study illustrates that in bone healing the local environment for cells may not be considered constant with regard to oxygen tension, pressure and temperature.

Novel synthetic bio-mimic polymers for potential cell delivery therapy

Cell-based therapy is one of the major potential therapeutic strategies for cardiovascular, neuronal and degenerative diseases in recent years. Synthetic biodegradable polymers have been utilized increasingly in pharmaceutical, medical and biomedical engineering. Control of the interaction of living cells and biomaterials surfaces is one of the major goals in the design and development of new polymeric biomaterials in tissue engineering. The aims of this study is to develop a novel bio-mimic polymeric materials which will facilitate the delivery cells, control cell bioactivities and enhance the focal integration of graft cells with host tissues.