Transfer RNA changes in rat granulation tissue possibly related to collagen synthesis

Transfer RNAs for glycine, proline, lysine, serine and leucine were compared in developing rat granulation tissue 6 and 15 days after sterile subcutaneous implantation of pieces of cellulose sponge. The acceptance of glycine, proline and lysine by unfractionated tRNAs were ca. 30 per cent greater in tRNA derived from 15-day granulation tissue, whereas those of serine and leucine were unaltered. Cochromatography on benzoylated DEAE-cellulose of the ³H- and ¹⁴C-labeled aminoacyl-tRNAs from the two sources revealed a significant increase in the relative amount of one of the three glycyl-tRNA fractions in the 15-day granulation tissue, whereas the elution profiles for prolyl-, lysyl-, seryl-, and leucyl-tRNAs were unaltered. The changes observed suggest a causal relation to the enhanced synthesis of collagen in the late-stage granulation tissue.     

Measurement of changes in amino acids related to total collagen in fibrotic human liver

In the liver, total collagen accumulation during the fibrotic or cirrhotic process was measured using a methodology based on the determination of collagen amino acids in liver biopsies from adults with alcoholic liver diseases or children with biliary atresia. The results obtained with this methodology were compared to histopathological findings. Thus, it was shown that generally the severity of hepatic injury was dependent on collagen accumulation. In biliary atresia, collagen accumulation increased with the children's age despite reconstructive surgery and restoration of biliary flow.     

Quantitative histology of changes with age in rat bone cortex

Changes with age in bone cortex of the rat were investigated by establishing histological parameters which could be quantitated to estimate age at death. Decalcified cross sections of mandible, femur and tibia were prepared from rats two to 120 days old, and measurements were made of: (1) number of osteons, (2) average number of lamellae per osteon, (3) average Haversian canal diameter, and (4) number of non-Haversian (primary) canals. Multiple regression techniques were used to estimate age at death from several combinations of these variables. With age, the number of osteons per unit area of bone and the number of lamellae per osteon increased, but Haversian canal diameter and the number of primary canals decreased. Multiple regression analyses indicated that age at death could be estimated to ± 3 days of the true value in 95% of the cases. Nomographs based on histological measurements of each bone were prepared which can provide accurate estimates of age between two and 120 days in the Sprague-Dawley female rat. It was concluded that microstructure of bone cortex can not only be quantitated to provide accurate estimates of age but it may also constitute a sensitive measure of the metabolic state of the organism. The techniques utilized should prove useful in anthropology as well in studies of bone aging.     

Quantitative changes of the capillary bed in aging human skin

The present study focuses on the quantitative changes of the capillary bed in aging human skin. Forty-five skin samples were excised from the anterior thoracic region of cadavers of caucasian origin in the age range 33-82 years. The immunohistochemical method with anti-human CD34 was used for the detection of the capillary endothelium. Morphometric analysis was done by Vision Assistant software. The capillary bed was quantified by two parameters: capillary area (CA) and intercapillary distance (ID) in 6 age groups. Results revealed no quantitative changes of the capillary bed up to the age of 60 years. In the papillary dermis a significant reduction of the capillary area was seen in the 7th, 8th and 9th decennium. A considerable decrease, by 33%, was determined in the 7th decennium. During the 8th and 9th decennium the capillary area was reduced by a further 19% and 13%. In total from the 4th till the 9th decennium, the capillary bed in the papillary dermis was diminished by 65%. The intercapillary distance in the papillary dermis singnificantly increased during the 8th decennium. On the basis of the mutual evaluation of both the observed parameters, CA and ICD, the authors supposed that the reduction of the capillary bed in the papillary dermis during the 7th decennium was probably caused only by the shortening of the capillary loops, which copied flattened dermal papillae, and during the 8th decennium also by the decreased number of the capillary loops. In the reticular dermis the capillary bed remained unchanged.     

Developmental changes in glycosaminoglycans,collagen and collagenase activity in embryonic chick skin

In order to study remodelling of connective tissue during development, changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin at various stages have been studied.Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20).At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis.Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Developmental changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin.

In order to study remodeling of connective tissue during development, changes in glycosaminoglycan, collagen and collagenase activity in embryonic chick skin at various stages have been studied. Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20). At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum, indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis. Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Analysis of age-associated changes in collagen crosslinking in the skin and lung in monkeys and rats

The present study was designed to address a specific question: can we define collagen aging in vivo in terms of alterations in collagen crosslinking? In order to assess the complete spectrum of change throughout life, tissues from rats, monkeys and (where available) humans were examined at ages ranging from fetal to old. Skin and lung were selected in order to include all of the crosslinks derived from lysyl oxidase-generated aldehydes that have been identified thus far, both reducible and nonreducible. Crosslinks analyzed included hydroxylysinonorleucine, dihydroxylysinorleucine, histidinohydroxymerodesmosine, hydroxypyridinium, lysyl pyridinium, and a deoxy analogue of hydroxypyridinium found in skin that differs structurally from lysyl pyridinium. Tissues from both a short-lived species (rats) and a long-lived species (monkeys) were analyzed to test further the hypothesis that changes in crosslinking are linked predominantly to biological age of the animal, rather than temporal aging. We found that biological aging seems to regulate certain predictable changes during the first part of the lifespan: the disappearance postnatally of dihydroxylysinonorleucine in skin, the rapid decrease in difunctional crosslink content in lung and skin during early growth and development, and the gradual rise in hydroxypyridinium and lysyl pyridinium in lung tissue. Changes in crosslinking were far less predictable during the second half of the lifespan. Although hydroxypridinium content continued to rise or reached a plateau in rat and monkey lungs, respectively, it showed a decrease in human lungs. The analogous trifunctional crosslink in skin, the so-called 'pyridinoline analogue', decreased dramatically in both rats and monkeys in later life. Our data suggest that caution must be taken in drawing inferences about human connective tissue aging from experiments performed in short-lived species such as rodents. Furthermore, the finding that there may be fewer total lysyl oxidase-derived crosslinks per collagen molecule in very old animals as compared with young animals suggests that we may need to expand our concepts of collagen crosslinking.     

Minoxidil-induced changes in the contraction of collagen lattices by human skin fibroblasts.

This investigation has studied the effect of minoxidil on the contraction of hydrated collagen lattices by human dermal fibroblasts. Type I collagen was mixed with a fibroblast suspension and polymerized, and minoxidil 10 to 800 micrograms/ml (0.05 to 4 mM) was added at the time the lattices were released. Minoxidil at concentrations from 100 to 600 micrograms/ml inhibited contraction in a dose-dependent manner, whereas 800 micrograms/ml prevented contraction completely, most cells remaining rounded. Considerable inhibition was already evident within 24 hours. Visualization of living cells with MTT and cell counts showed that inhibition in the first 48 hours was not due to fibroblast death. Exchange of minoxidil to normal medium led to a resumption of contraction and a return to an elongate morphology. Minoxidil at 10 micrograms/ml had no significant effect on lattice contraction, whereas at 100 micrograms/ml it slowed contraction without affecting proliferation or morphology, as observed under the light microscope. The inhibitory effect of minoxidil should be investigated further in relation to the control of contraction of wounds in vivo.