Conceptual models of carcinogenesis typically consist of an evolutionary sequence of heritable changes in genes controlling proliferation, apoptosis, and senescence. We propose that these steps are necessary but not sufficient to produce invasive breast cancer because intraductal tumour growth is also constrained by hypoxia and acidosis that develop as cells proliferate into the lumen and away from the underlying vessels. This requires evolution of glycolytic and acid-resistant phenotypes that, we hypothesise, is critical for emergence of invasive cancer. Mathematical models demonstrate severe hypoxia and acidosis in regions of intraductal tumours more than 100 μm from the basement membrane. Subsequent evolution of glycolytic and acid-resistant phenotypes leads to invasive proliferation. Multicellular spheroids recapitulating ductal carcinoma in situ (DCIS) microenvironmental conditions demonstrate upregulated glucose transporter 1 (GLUT1) as adaptation to hypoxia followed by growth into normoxic regions in qualitative agreement with model predictions. Clinical specimens of DCIS exhibit periluminal distribution of GLUT-1 and Na⁺/H⁺ exchanger (NHE) indicating transcriptional activation by hypoxia and clusters of the same phenotype in the peripheral, presumably normoxic regions similar to the pattern predicted by the models and observed in spheroids. Upregulated GLUT-1 and NHE-1 were observed in microinvasive foci and adjacent intraductal cells. Adaptation to hypoxia and acidosis may represent key events in transition from in situ to invasive cancer.

Gelatin has been used for many years in pharmaceutical formulation, cell culture and tissue engineering on account of its excellent biocompatibility, ease of processing and availability at low cost. Over the last decade gelatin has been extensively evaluated for numerous ocular applications serving as cell-sheet carriers, bio-adhesives and bio-artificial grafts. These different applications naturally have diverse physical, chemical and biological requirements and this has prompted research into the modification of gelatin and its derivatives. The crosslinking of gelatin alone or in combination with natural or synthetic biopolymers has produced a variety of scaffolds that could be suitable for ocular applications. This review focuses on methods to crosslink gelatin-based materials and how the resulting materials have been applied in ocular tissue engineering. Critical discussion of recent innovations in tissue engineering and regenerative medicine will highlight future opportunities for gelatin-based materials in ophthalmology.

The extracellular matrix (ECM) of mammalian tissues has been isolated, decellularized and utilized as a scaffold to facilitate the repair and reconstruction of numerous tissues. Recent studies have suggested that superior function and complex tissue formation occurred when ECM scaffolds were derived from site-specific homologous tissues compared with heterologous tissues. The objectives of the present study were to apply a stringent decellularization process to demineralized bone matrix (DBM), prepared from bovine bone, and to characterize the structure and composition of the resulting ECM materials and DBM itself. Additionally, we sought to produce a soluble form of DBM and ECM which could be induced to form a hydrogel. Current clinical delivery of DBM particles for treatment of bone defects requires incorporation of the particles within a carrier liquid. Differences in osteogenic activity, inflammation and nephrotoxicity have been reported with various carrier liquids. The use of hydrogel forms of DBM or ECM may reduce the need for carrier liquids. DBM and ECM hydrogels exhibited sigmoidal gelation kinetics consistent with a nucleation and growth mechanism, with ECM hydrogels characterized by lower storage moduli than the DBM hydrogels. Enhanced proliferation of mouse primary calvarial cells was achieved on ECM hydrogels, compared with collagen type I and DBM hydrogels. These results show that DBM and ECM hydrogels have distinct structural, mechanical and biological properties and have the potential for clinical delivery without the need for carrier liquids.

BACKGROUND AND AIMS Intestinal epithelial cell derived antimicrobial peptides of the defensin family may play a major role in host defence against microorganisms. Our aims were to (i) isolate, characterise, and investigate the processing of human defensin 5 (HD-5) in normal Paneth cells and (ii) investigate expression of HD-5 in active inflammatory bowel disease (IBD). METHODS Antiserum raised against chemically synthesised putative mature HD-5 was used for immunohistochemistry and purification of HD-5 from extracts of normal terminal ileal crypts. RESULTS In normal and Crohn's disease terminal ileum, HD-5 immunoreactivity was seen in Paneth cells and in some villous epithelial cells. Normal colonic mucosa did not express HD-5 but HD-5 immunoreactivity was seen in cells in the colonic crypt region of many IBD samples.N-terminal amino acid sequence analysis of HD-5 purified from normal terminal ileal Paneth cells consistently showed the predicted sequence of the precursor form of the peptide. Following stimulation of isolated intact normal terminal ileal crypts, a truncated form of HD-5, with theN-terminal sequence GEDNQLAIS, was detected in the supernatant. CONCLUSIONS (i) HD-5 is present only in the precursor form in normal terminal ileal Paneth cells and is processed to the mature form during and/or after secretion, (ii) some villous epithelial cells express HD-5, and (iii) HD-5 is expressed by metaplastic Paneth cells in the colon in IBD.