The influence of crosslinking agents and diamines on the pore size, morphology and the biological stability of collagen sponges and their effect on cell penetration through the sponge matrix.

Artificial skin substitutes based on autologous keratinocytes cultured on collagen substrata are being developed for treating patients with severe burns. The properties of the collagen substrate can be manipulated, for example, by crosslinking, to optimize desirable properties such as cell growth and penetration into the substrate, biological stability and mechanical strength. Collagen sponges crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and the diamine, diaminohexane, were used to determine the effect of crosslinking on pore size and morphology, on the stability of the crosslinked sponges both in cell culture media and during incubation with collagenase, and on the penetration of keratinocytes and fibroblasts through the sponge matrix. Crosslinking of the sponges reduced the pore size, particularly at the surface, and altered sponge morphology. After crosslinking the collagen fibers were thinner, and appeared lacy and delicate. Crosslinking also influenced sponge stability. In keratinocyte serum-free medium the pore size of plain collagen sponges increased with increasing incubation time, and crosslinking appeared to prevent this, and may have stabilized sponge structure. Incubation in serum-containing Dulbecco's minimum essential medium caused a marked reduction in pore size in both plain collagen and crosslinked collagen sponges. Crosslinking did not appear to influence this cell-free contraction of collagen sponges. Treatment of sponges with EDAC markedly increased the resistance of sponges to collagenase digestion. The penetration of both keratinocytes and fibroblasts was retarded by crosslinking the sponges. Fibroblasts penetrated through the sponges to a greater extent than keratinocytes, and their proliferation rate was faster. The total number of cells populating the crosslinked sponges after 10 days culture was approximately 50% of that on untreated collagen sponges. The mechanism responsible for this effect was different with the two crosslinkers used. Diaminohexane appeared to inhibit cell growth, whereas EDAC may have caused a decrease in cell adhesion to the sponges, without an apparent inhibition of growth rate. In terms of morphology, fibroblasts were elongated to a greater extent on crosslinked sponges, and alligned themselves along the collagen fibers. Keratinocytes grew in colonies on untreated sponges, but on crosslinked sponges they grew in isolation, with minimal cell-cell interactions. It may be necessary to reach a compromise to obtain the best combination of properties for using collagen sponges as substrata for artificial skin substitutes.

The effect of certain variables on the tumor and tissue distribution of tracers. Part 1; Carrier.

Buffalo rats bearing thigh-implanted strain-7777 Morris hepatomas were used as a model for studying the effect of carrier material on the body distribution, tumor uptake, excretion, and tumor-to-background ratios of 67Ga and 54Mn. An effort was also made to observe the changes in 67Ga and 54Mn concentrations induced by carrier in viable tumor and skeletal muscle, relative to their interstitial fluid space. This value is referred to as the Tissue Distribution Index. Carrier manipulation resulted in striking changes in the distribution of the two ions from the carrier-free state. The data also indicated a difference in the pharmacodynamics of 67Ga and 54Mn in malignant and healthy tissues which could be of importance to nuclear medicine and oncology.

Distributions of 137Cs, 201Tl, 203Hg, 203Pb and 57Co in a rat hepatoma model. Comparison with 67Ga.

The distribution of carrier-free 203Pb-acetate, 203HgCl2, 57 CoCl2, 137CsCl and 201TlCl was investigated in rats bearing thigh-implanted Morris 7777 hepatomas. Viable and nonviable tumor tissue was collected in order to determine the relative affinities of the radiopharmaceuticals for these tissues. The animals were sacrificed at 4, 24, 48, 72 and 96 hrs following intravenous injection. Washout of the radioisotope from the viable tumor tissue was rapid, the maximum concentration being reached on or before 4 hrs following injection. In contrast, residual activity within the nonviable tumor tissue decreased much more slowly and in some cases even increased with time. Viable tumor-to-muscle and nonviable tumor-to-muscle ratios for 203Pb, 203Hg and 57Co were comparable to the analogous ratios reported for 67Ga. However, none of these isotopes approached 67Ga as a potential tumor imaging agent because the large ratios were the result of low muscle uptake rather than high tumor uptake. Blood clearance of 67Ga was faster than any of the five cations, while viable and nonviable tumor affinity for 67Ga was greater than for any of the radiopharmaceuticals studied.

Comparison of viable and nonviable tumor uptake of Sc-46, Mn-54, Zn-65, In-111 and Au-195 with Ga-67 citrate in a hepatoma model.

The tissue distribution of Sc-46, Mn-54, Zn-65, In-111 and Au-195 were studied in a rat hepatoma model at various time intervals over a 96 h period. The tumor localizing properties of these isotopes were evaluated by examining their incorporation and clearance from viable and nonviable tumor tissue and determining the critical tissue ratios formed with blood and muscle. In general, the results showed greater uptake in viable than nonviable tumor tissue at early time periods (4-24 h). By 96 h, however, the activity remaining in the nonviable tumor tissue exceeded the quantity in viable tumor tissue. This trend was previously noted for Ga-67. When compared with Ga-67, only Mn-54 among the isotopes studied showed remarkably higher viable tumor/blood ratios (4-24 h, 45:1-83:1 respectively). Manganese-54 also showed highly significant accumulation in cardiac muscle with a heart/blood ratio at 4 h superior to comparable values previously reported for Cs-137 and Tl-201. It is suggested that tumor and heart imaging may be feasible utilizing radioactive manganese (Mn-51 or Mn-52) with the new positron imaging systems.

Tissue distribution studies with radioactive manganese: a potential agent for myocardial imaging.

Manganese, a trace metal, is known to localize in mitochondria. Because mitochondria are abundant in heart muscle, the possible utility of radioactive manganese as a myocardial imaging agent was examined in 25 rats and six dogs. Myocardial uptake of Mn-54 in rats was found to exceed that of thallium-201; myocardium-to-blood ratios averaged 306:1 versus 48:1 for Tl-201. In the dog, uptake of Mn-54 by ischemic myocardium was reduced by 17-75% compared with normal myocardium. Thus, radioactive manganese appears promising as an intravenous myocardial imaging agent, and might be useful in studying the function of myocardial mitochondria by external imaging.