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      Cardiac tissue engineering: Cell seeding, cultivation parameters, and tissue construct characterization

      Biotechnology and Bioengineering

      Wiley

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          Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development.

          The switch from myocyte hyperplasia to hypertrophy occurs during the early postnatal period. The exact temporal sequence when cardiac myocytes cease dividing and become terminally differentiated is not certain, although it is currently believed that the transition takes place gradually over a 1-2-week period. The present investigation has characterized the growth pattern of cardiac myocytes during the early postnatal period. Cardiac myocytes were enzymatically isolated from the hearts of 1, 2, 3, 4, 6, 8, 10, and 12-day-old rats for the measurements of binucleation, cell volume and myocyte number. Almost all myocytes were mononucleated and cell volume remained relatively constant during the first 3 days of age. Increases in cell volume and binucleation of myocytes were first detected at day 4. Myocyte volume increased 2.5-fold from day 3 to day 12 (1416 +/- 320 compared to 3533 +/- 339 microns 3). The percentage of binucleated myocytes began to increase at day 4 and proceeded at a high rate, reaching the adult level of approximately 90% at day 12. Myocyte number increased 68% during the first 3 days (from 13.6 +/- 3.5 x 10(6) at day 1 to 22.9 +/- 5.6 x 10(10) at day 3) and remained constant thereafter. To confirm that no further myocyte division exists after 4 days, bromodeoxyuridine (Brdu) was administered to 4-day-old rats and the fate of DNA-synthesizing myocytes was examined 2 h and 2, 4, 6 and 8 days after Brdu injection. About 12% of myocytes were labeled with Brdu at 2 h and all were mononucleated at that time. Gradually, these Brdu-labeled myocytes became binucleated. However, the percentage of labeled myocytes in all groups was identical, indicating that DNA-synthesizing myocytes were becoming binucleated without further cell division after 4 days of age. Within 8 days after injection, approximately 82% of total labeled myocytes were binucleated, while the others remained mononucleated. Sarcomeric alpha-actinin was fully disassembled in dividing myocytes of 2-day-old rats, while typical alpha-actinin striations were present in dividing myocytes of 4-day-old rats. The results from this study suggest that a rapid switch from myocyte hyperplasia to hypertrophy occurs between postnatal day 3 and 4 in rat hearts.
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            Fluorometric assay of DNA in cartilage explants using Hoechst 33258.

            A simple two-step fluorometric assay of DNA in cartilage explants, utilizing the bisbenzimidazole dye Hoechst 33258, is described. Cartilage explants were prepared for assay by digestion with papain. Aliquots of the digest were mixed with dye solution, and the fluorescence emission measured. The enhancement in fluorescence of dye was specific for DNA, as demonstrated by 97% sensitivity to DNase and resistance to RNase. In addition, little or no interference was caused by non-DNA tissue components, since DNA caused an equal enhancement in fluorescence independent of the presence of papain-digested cartilage. By performing the assay on isolated chondrocytes, the cellular content of DNA was computed to be 7.7 pg per chondrocyte. The assay was stable for at least 2 h and sensitive to as little as 6 ng of DNA or equivalently less than 1000 cells. This procedure offers advantages over other established DNA assays of cartilage and may be especially useful in metabolic studies of cartilage explants.
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              A simple rapid and sensitive DNA assay procedure

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                Author and article information

                Journal
                10.1002/(SICI)1097-0290(19990905)64:5<580::AID-BIT8>3.0.CO;2-X

                http://doi.wiley.com/10.1002/tdm_license_1.1

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