Collagen is the most frequently used protein in the fields of biomaterials and regenerative medicine. Within the skin, collagen type I and III are the most abundant, while collagen type VII is associated with pathologies of the dermal–epidermal junction. The focus of this review is mainly collagens I and III, with a brief overview of collagen VII. Currently, the majority of collagen is extracted from animal sources; however, animal-derived collagen has a number of shortcomings, including immunogenicity, batch-to-batch variation, and pathogenic contamination. Recombinant collagen is a potential solution to the aforementioned issues, although production of correctly post-translationally modified recombinant human collagen has not yet been performed at industrial scale. This review provides an overview of current collagen sources, associated shortcomings, and potential resolutions. Recombinant expression systems are discussed, as well as the issues associated with each method of expression.

Cellular distribution of cystic fibrosis transmembrane conductance regulator (CFTR) immunofluorescence was detected by monoclonal antibody directed to the C terminus of killifish CFTR (kfCFTR) in chloride cells of fresh water (FW) adapted fish and animals transferred to sea water (SW) for 24h, 48h and 14+ days. Confocal microscopy allowed localization within mitochondria-rich (MR) cells to be determined as superficial (i.e. in the apical membrane) or deeper within the cytoplasm of the cells. In FW, 90 % of MR cells had diffuse kfCFTR immunofluorescence in the central part of the cytosol, with only 8.1 % having apical kfCFTR, which was 6.6±0.54 μm below the microridges of surrounding pavement cells. Curiously, FW but not SW pavement cells also had positive immunofluorescence to kfCFTR. After 24h in SW, a time when kfCFTR expression is elevated, a condensed punctate immunofluorescence appeared among 18.8 % of MR cells, 13.4±0.66 μm(mean ± S.E.M.) below the surface of the cells. By 48h, a majority(76.3 %) of MR cells had punctate kfCFTR distribution and the distance from the surface was less (7.8±0.2 μm), a distribution approaching the SW-acclimated condition (i.e. all MR cells showing kfCFTR immunofluorescence,6.1±0.04 μm below the surface). In contrast, NKCC immunofluorescence was condensed and localized in lateral parts of MR cell complexes in FW animals and then redistributed to the whole basal cytoplasm after acclimation to SW. CFTR, the anion channel responsible for Cl^- secretion in marine teleosts, redistributes in MR cells during SW acclimation by condensation of a diffuse distribution below the apical crypt, followed by translocation and insertion in the apical membrane. NKCC, the cotransporter that translocates Cl^- across the basolateral membrane, moves from an eccentric cytosolic location in FW to a diffuse basolateral localization in SW chloride cells.

SUMMARY: Freshwater-adapted killifish (Fundulus heteroclitus) opercular epithelia were dissected and subjected to blood-side hypertonic bathing solution in Ussing-style chambers to simulate the increase in blood osmolality during migration to sea water. Conversely, seawater-acclimated killifish opercular epithelia were subjected to hypotonic bathing solutions to simulate the initial stages of migration to fresh water. Freshwater-acclimation (hypertonic stress) induced a rapid (approximately 30min) increase in membrane conductance (Gt) from 3.10±0.56 to 7.52±1.15mScm−2 (P<0.01, N=27), whereas seawater-acclimation (hypotonic stress) induced a rapid decrease in Gt from 8.22±1.15 to 4.41±1.00mScm−2 (P<0.01, N=27; means ± s.e.m.). Control seawater-acclimated membranes had a density of apical crypts (where chloride cells are exposed to the environment; detected by scanning electron microscopy) of 1133±96.4cryptsmm−2 (N=12), whereas the hypotonically shocked specimens had a lower crypt density of 870±36.7cryptsmm−2 (P<0.01 N=10; means ± s.e.m.). Hypertonic shock of freshwater membranes increased crypt density from 383.3±73.9 (N=12) to 630±102.9cryptsmm−2 (P<0.05; N=11; means ± s.e.m.). There was no change in density of chloride cells, as detected by fluorescence microscopy; hence, osmotic stress changes the degree of exposure, not the number of chloride cells. Cytochalasin D (5.0μmoll−1) completely blocked the conductance response to hypotonic shock and the reduction in apical crypt density measured by scanning electron microscopy, while phalloidin (33μmoll−1), colchicine (3×10−4moll−1) and griseofulvin (1.0μmoll−1) were ineffective. Actin imaging by phalloidin staining and confocal microscopy revealed extensive actin cords in pavement cell microridges and a ring of actin at the apex of chloride cells. We conclude that the actin cytoskeleton of chloride cells is required to maintain crypt opening and that osmotic shock causes chloride cells to adjust their apical crypt size.

Euryhaline teleost fish adapt rapidly to salinity change and reduce their rate of ion secretion on entry to fresh water. Killifish (Fundulus heteroclitus) transferred from full-strength sea water to fresh water showed large reductions in plasma [Na+] and osmolality at 6 h which were corrected by 24 h. To mimic this in vitro, a hypotonic shock of 20–70 mosmol kg−1 was applied on the basolateral side of opercular epithelia. This hypotonic shock reversibly reduced the short-circuit current (Isc, equivalent to the rate of secretion of Cl−) in a dose-dependent fashion, with a 40 mosmol kg−1 hypotonic shock reducing Isc by 58±4.6 % in 40 min. Similar reductions in [NaCl], but with added mannitol to maintain osmolality, were without effect, indicating that the effect was purely osmotic. Hypotonic inhibition of Isc was accompanied by reductions in epithelial conductance (Gt) but no significant change in transepithelial potential (Vt). The hypotonic inhibition was apparently not Ca2+-mediated because Ca2+-depleted salines, thapsigargin and ionomycin all failed to block the reduction in Isc produced by hypotonic shock. The inhibition was not mediated via a reduction in intracellular cyclic AMP level because cyclic AMP levels, measured by radioimmunoassay, were unchanged by hypotonic shock and by 1.0 μmol l−1 clonidine (which inhibits Isc by changing intracellular [Ca2+]) but were increased markedly by 1.0 μmol l−1 isoproterenol, a positive control. The protein tyrosine kinase inhibitor genistein (100 μmol l−1), but not its inactive analogue daidzein, inhibited Isc in normal osmolality but produced a stimulation of Isc after hypotonic shock (and after clonidine treatment). The inhibitory effects of genistein and hypotonicity were not additive, suggesting that the same portion of the Isc was inhibited by both treatments. These data are consistent with a model for Cl− transport regulation involving tyrosine phosphorylation in cell-swelling-induced inhibition of Cl− secretion when euryhaline teleosts adapt to fresh water.