Narrow Results

In this chapter we discuss how to engineer 3D pulmonary tissue constructs in vitro using primary isolates of foetal mouse distal lung cells. When cultured in hydrogel-based 3D constructs, the mixed cell population, comprised epithelial, mesenchymal and endothelial cells, organised into alveolar forming unit (AFU)-like sacculated structures, which, in terms of morphology and cytodifferentiation, were reminiscent of native distal lung. By using a unique, serum-free medium supplemented with a cocktail of tissue-specific growth factors, we were able to induce concomitant alveolisation and neovascularisation when culturing the cells in the hydrogels, but not in scaffolds composed of synthetic polymers. Our data suggest that our in vitro model is capable of recapitulating the parallel morphogenesis of epithelial and endothelial pulmonary tissue components, which may occur through dynamic paracrine interactions. These results also stress the importance of the complex input from co-cultures, tissue-specific growth factors and integrin signalling for successful tissue engineering in vitro. In a mouse model in vivo, incorporation of the primary lung cell isolates into Matrigel plugs, implanted either subcutaneously (s.c.), or under the kidney capsule, leads to the formation of sacculated AFUs in close proximity to patent capillaries. Effective functional vascularisation, however, was only observed upon addition of angiogenic growth factors to the scaffolds and their controlled release over time. Use of a fluorescent cell tracker confirmed that the neovessels in the constructs comprised endothelial cells from both the host and the grafts. These data demonstrate that it is feasible to generate vascularised pulmonary tissue constructs in vivo with proper epithelial differentiation, and that the degree of vascularisation may be manipulated by incorporating the release of an angiogenic factor within the construct.

Food spoilage is mainly caused by microorganisms, such as bacteria. In this study, we measure the autofluorescence in meat samples longitudinally over a week in an attempt to develop a method to rapidly detect meat spoilage using fluorescence spectroscopy. Meat food is a biological tissue, which contains intrinsic fluorophores, such as tryptophan, collagen, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) etc. As meat spoils, it undergoes various morphological and chemical changes. The concentrations of the native fluorophores present in a sample may change. In particular, the changes in NADH and FAD are associated with microbial metabolism, which is the most important process of the bacteria in food spoilage. Such changes may be revealed by fluorescence spectroscopy and used to indicate the status of meat spoilage. Therefore, such native fluorophores may be unique, reliable and non-subjective indicators for detection of spoiled meat. The results of the study show that the relative concentrations of all above fluorophores change as the meat samples kept in room temperature (~19℃) spoil. The changes become more rapidly after about two days. For the meat samples kept in a freezer (~ -12℃), the changes are much less or even unnoticeable over a-week-long storage.

Tumor invasion, the process by which tumor cells break away from their primary tumor and gain access to vascular systems, is an important step in cancer metastasis. Most current 3D tumor invasion assays consisted of a single tumor cell embedded within an extracellular matrix (ECM). These assays taught us much of what we know today on how key biophysical (e.g., ECM stiffiness) and biochemical (e.g., cytokine gradients) parameters within the tumor microenvironment guided and regulated tumor invasion. One limitation of the single tumor cell invasion assays was that it did not account for cell–cell adhesion within the tumor. In this chapter, we developed a micrometer scale 3D co-culture spheroid invasion assay that recapitulated physiologically realistic tumor microenvironment and was compatible with microscopic imaging. Micrometer scale co-culture spheroids (1:1 ratio of metastatic breast cancer MDA-MB-231 and non-tumorigenic epithelial MCF-10A cells) were made using an array of microwells, and then were embedded within a collagen matrix in a microfluidic platform. Real time imaging of tumor spheroid invasion revealed that the spatial distribution of the two cell types within the tumor spheroid critically regulated tumor invasion. This work linked tumor architecture with tumor invasion and highlighted the importance of the biophysical cues within the bulk of the tumor in tumor invasion.

In the treatment of wound, a good understanding of the principles of wound healing is essential. A moist wound healing environment is needed. A range of new generation dressings has emerged in addition to traditional dressings such as gauze and tulle gras. These include low-adhesive dressings, transparent dressings, hydrocolloids, hydrogels, alginates, foams, hydrofibres, anti-microbial dressings, de-odouriser dressings and collagen dressings. The choice of dressing depends on a proper assessment of the wound (presence of ischaemia, infection, etc.) and matching the properties of the various dressings available to best meet the individual needs of that particular wound.

The intervertebral disc is organized with a concentrated proteoglycan solution, the central nucleus pulposus, held within the strong collagen network of the outer annulus fibrosus. The disc exhibits a viscoelastic response when subjected to loads and deformations. Disc degeneration, and/or spondylotic changes that are generally considered to be associated with aging, result in a spinal segment with decreased stiffness. However, in the cervical spine of cerebral palsy patients suffering from athetotic movements of the neck, there is a very early onset of disc degeneration and spondylotic change. Acceleration of disc degeneration has been shown to take place in the spines of animals subjected to excessive extension-flexion of the head and neck. Repetitive torsion of the disc has led to structural regression in in vitro studies using animal spines. Delaminated lamellae and/or disruption of the annulus fibrosus are always recognized in the early stages of the destructive process of the intervertebral disc structure. Disruption of the collagen network may be a result of fatigue failure by repetitive loading, which in turn causes the high tensile stresses in the annulus fibrosus from the development of large hydrostatic pressures within the nucleus pulposus. Loosening of the collagen network may be a key factor leading to the loss of proteoglycans and water, finally inducing the development of disc degeneration. A “degenerated disc” can be induced through pure mechanical fatigue failure of the tissue, as an age-independent degradation of the cartilaginous tissue.

The etiology of most of the degenerative changes in the spine continues to remain obscure. However, several lines of evidence suggest that genetic factors may play an important role in the onset of degenerative changes, in addition to various environmental factors. We have generated transgenic mice expressing mutant αl(IX) collagen in the cartilage matrix. They developed progressive intervertebral disc degeneration with age as well as joint degeneration. Both radiologic and histologic studies indicated that cervical and lumbar disc degeneration was more advanced in the transgenic mice than in control littermates. The initial degenerative changes included shrinkage and replacement of the nucleus pulposus with consolidated fibrous tissue, that resulted in a loss of nuclear-anular demarcation. Partial disruption in the lamellar structure of the anulus fibrosus also occurred at this stage. With age, the disc degeneration progressively advanced and sometimes caused herniation of disc material and mild osteophyte formation. These findings imply that genetic abnormalities of cartilage matrix components, such as type IX collagen, may be responsible for certain degenerative diseases in the spine.

Self-organized nanocomposites of hydroxyapatite (HAp) and collagen were prepared by controlling temperature and pH on the basis of a biomimetic process. Transmission electron microscopic observations indicated that the composites prepared at pH 8-9 and 40°C had the bone-like structure in which the c-axes of HAp nanocrystals aligned along collagen fibers forming bundles of about 20μm in length and 1μm in diameter. The mechanical strength of the composites obtained was dependent on the degree of self-organization: the maximum 3-point bending strength was 39.5±0.88MPa and Young’s modulus 2.50±0.38GPa. When implanted in Wister rats’ craniums and beagles’ bilateral radii, osteoblasts and osteoclasts were induced near the composites after two weeks, and the composites were covered with a newly formed bone after 12 weeks.