Proteoglycan modifications by granulation tissue in culture

To study the process of tissue remodeling that occurs during wound healing, radioactive proteoglycan ([35S]-PGS) was used to assay for enzymatic activities present in the extracellular fluid of healing tissue. Mice, wounded by removal of a 2 x 1.5 cm patch of skin from the dorsal surface, were sacrificed after 3 days of healing. Granulation tissue (1 cm2) was removed, spread onto a sterile wire mesh support and placed in the center well of an organ culture dish. To each well was added 1 ml MCDB medium supplemented with 10% fetal calf serum and antibiotics and 5-20 microliters of [35S]-PGS (100,000 cpm/10 microliters). Medium, removed from the well by aspiration after 24 and 48 h of culture, was boiled 5 min at 100 degrees C and stored frozen at -20 degrees C. Alterations of the PGS were assayed with a Sepharose 4B column (1 x 50 cm) which had an excluded and included volume of 17 and 46 ml, respectively. PGS, incubated without cells or with tissues from unwounded animals, eluted at 26 ml. PGS, incubated with granulation tissue and cultured for either 24 or 48 h, eluted from the Sepharose 4B at 29 ml, a 10% increase in elution volume, suggesting that the size or shape of the PGS has been altered by enzymes secreted by the cells of the granulation tissue. In contrast, PGS incubated with tissues from unwounded animals or without granulation tissue showed no changes. These data suggest that enzymatic activities secreted by cells of granulation tissue may be involved in remodeling during healing.     

Tumor necrosis factor stimulates gelatinase and collagenase production by granulation tissue in culture

Tumor necrosis factor (TNF, 2.6 X 10(-11)-10(-9) M) caused an increase in the production of active gelatinase and latent collagenase by granulation tissue in culture. As determined by SDS-substrate polyacrylamide gel electrophoresis, granulation tissue produced mainly two species of gelatinase with molecular weights of 64 kDa and 57 kDa, and an additional 80-kDa gelatinase was produced by TNF treatment. The results suggest that TNF may play a role in a rapid collagen turnover in the carrageenin-induced granulation tissue in rats.     

Vascular changes in maturing granulation tissue

Granulation tissue was examined in rats on the 20th and 40th day after wounding of the back. It was shown that by both the 20th and the 40th day the vessels with impaired typical structure could be revealed together with the normal ones in the granulation tissue. Specific rearrangement was observed in the structure of these vessels, consisting in the impairment of their wall integrity, separation of the constituent cells and their free position among the other cells and fibrous structures. This process is especially marked by the 40th day. 3H-thymidine was actively incorporated by some fibroblasts and rather often by the cells of both normal and degenerating vessels. The authors suggest the existence of an earlier unknown phenomenon of transformation of capillary vessels, common for both normal dermal and reparative processes after injury. The essence of the phenomenon consists in the fact that small dermal vessels get permanently disintegrated and are included into the composition of cellular elements of the interstitial tissue, and form again, providing for the physiological regeneration of dermal cells and fibrous structures.     

Stimulation of fibrillogenesis in granulation tissue]

RNA synthesis by fibroblasts in healing wounds of mice and passage of the newly-formed RNA from the nucleus into the cytoplasm were studied in control mice and those treated by potassium orotate. Potassium orotate accelerated the passage of the RNA from the nucleus into the cytoplasm of the fibroblasts. Formation and maturation of the collagen fibrils in the wounds were studied by scanning electron microscope.     

Collagen polymorphism in experimental granulation tissue.

Dermal granulation tissues produced either in response to an acute inflammation by turpentine injection, or to a chronic inflammation by sponge implantation, have been shown to contain a high proportion of Type III collagen in marked contrast to the small amount normally present in mature skin. Although the acute granuloma is rapidly resorbed the synthesis of Type III is maintained in long-term sponge implants. The results demonstrate a reversion to embryonic collagen in proliferating granulation tissue, a fact of considerable importance in understanding wound healing.     

Amino acid metabolism of experimental granulation tissue in vitro

1. The intracellular volume in granulation tissue was about 15% of the total urea space. 2. The experimental granuloma has a greater ability to retain amino acids during the proliferation phase than later during the synthesis of collagen. 3. The synthesis of collagen and other proteins by granulation tissue is related to the concentrations of proline and glutamic acid in the medium. 4. The rate of synthesis of proline from glutamic acid in granulation-tissue slices is greatest during collagen synthesis. It is enhanced by lactate. 5. Extracellular cations influence the synthesis of collagen and ouabain is inhibitory. Synthesis of other proteins is less sensitive in this respect. 6. It is suggested that the synthesis of collagen is related to the supply of certain amino acids, especially proline, and hence to the redox balance, and also to the function of the cell wall.     

Wound splinting modulates granulation tissue proliferation.

Attachment of the extracellular matrix to a substratum is important for fibroblast survival and proliferation in three-dimensional in vitro culture systems. We hypothesized that wound matrix attachment in a wound splinting model would modulate wound cell proliferation in vivo. Male rats were excisionally wounded on the dorsum, and a splint was sutured to the wound edge. In one experiment (N = 12), 6 rats were desplinted on day 5, and then all were sacrificed 24 h later, 6 h after 5-bromo-2'-deoxyuridine (BrdU) injection. In the second experiment (N = 18), 6 rats each were desplinted, desplinted with wound edge release, or not disturbed, followed by BrdU injection and sacrifice 24 h later. BrdU-labeled nuclei were quantified on frozen sections of granulation tissue, cut at three different levels. In the first experiment, the percentage of BrdU-positive nuclei per high power field (hpf) in the splinted vs. desplinted animals was 6.15 +/- 2.45 (S.D.) vs. 3.03 +/- 1.58%* p<0.001, ANOVA. In the second experiment, the number of BrdU-positive per hpf was 33.1 +/- 17.4 vs. 14.5 +/- 17.1 vs. 10.2 +/- 9.1* (splinted vs. desplinted vs. desplinted/released); *p<0.001 [analysis of variance (ANOVA)]. Removal of the wound splint decreased the rate of BrdU-labeled cells in the granulation tissue by approximately 50%; complete disruption of wound matrix attachment may have decreased this rate even further. Wound cell proliferation is modulated by lateral attachment of the wound matrix.     

Metabolic phases during the development of granulation tissue

1. The metabolism of incubated slices of sponge-induced granulation tissue, harvested 4-90 days after the implantation, was studied with special reference to the capacity of collagen synthesis and to the energy metabolism. Data are also given on the nucleic acid contents during the observation period. Three metabolic phases were evident. 2. The viability of the slices for the synthesis of collagen was studied in various conditions. Freezing and homogenization destroyed the capacity of the tissue to incorporate proline into collagen. 3. Consumption of oxygen reached the maximum at 30-40 days. There was evidence that the pentose phosphate cycle was important, especially during the phases of the proliferation and the involution. The formation of lactic acid was maximal at about 20 days. 4. The capacity to incorporate proline into collagen hydroxyproline in vitro was limited to a relatively short period at 10-30 days. 5. The synthesis of collagen was dependent on the supply of oxygen and glucose, which latter could be replaced in the incubation medium by other monosaccharides but not by the metabolites of glucose or tricarboxylic acid-cycle intermediates.     

Effects of interleukin-8 on granulation tissue maturation

The inflammatory alpha-chemokine, interleukin-8 (IL-8), affects the function and recruitment of various inflammatory cells, fibroblasts, and keratinocytes. Gap junctions are anatomical channels that facilitate the direct passage of small molecules between cells. The hypothesis is that IL-8 enhances gap junctional intercellular communication (GJIC) between fibroblasts in granulation tissue, which increases the rate of granulation tissue maturation. In vitro, human dermal fibroblasts were incubated with IL-8 prior to scrape loading, a technique that quantifies GJIC. Polyvinyl alcohol (PVA) sponges were implanted within subcutaneous pockets in rats and received local injections of either IL-8 or saline and were harvested on day 11. In vitro, IL-8 treated fibroblasts demonstrated an increase in GJIC by scrape loading compared to saline treated controls. In vivo, IL-8 treated PVA sponges demonstrated a decrease in cell density and an increase in vascularization compared to saline controls by H&E staining. Polarized light viewed Sirius red-stained specimens demonstrated greater collagen birefringence intensity, indicating thicker, more-mature collagen fibers. IL-8 increases GJIC in cultured fibroblasts and induces a more rapid maturation of granulation tissue.