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The effect of scurvy on glycosaminoglycans of granulation tissue and costal cartilage.

1. The effect of ascorbic acid deficiency on glycosaminoglycans of granulation tissue and cartilage of guinea pigs was investigated by determination of the changes in the glucosamine and galactosamine contents 12 days after tendonectomy. 2. In normal granulation tissue, the glucosamine and galactosamine contents rose to a peak at 5 and 10 days respectively, whereas the hydroxyproline and proline contents continued to rise throughout the 20 days after tendonectomy. 3. The galactosamine in scorbutic granulation tissue, but not in that of pair-fed controls, decreased significantly in absolute amount and relatively to glucosamine, which remained practically unchanged; the cartilage galactosamine did not decrease during the 22 days of deficiency owing to the presence of excess of preformed galactosaminoglycans, which masked the small amount of newly formed glycosaminoglycans. 4. The chemical results were confirmed by radioactivity studies in vivo of incorporation of [U-(14)C]glucose into galactosamine and glucosamine of scorbutic granulation tissue and cartilage. The incorporation of (14)C into galactosamine decreased significantly in scurvy in both tissues. 5. The results indicated in both tissues a decreased formation of galactosamine during scurvy, although an increased degradation of polymerized glycosaminoglycans could not be entirely ruled out. It is concluded that, if lack of ascorbic acid causes an impaired galactosamine formation, the most likely position for the block may be in the UDP-N-acetylglucosamine 4-epimerase reaction.

Ascorbic Acid Deficiency↗

Effects of soluble fractions from untreated and SiO2-treated subcellular particles of macrophages on nucleic acid metabolism in isolated nuclei of experimental granulation tissue.

Nuclei isolated from proliferating granulation tissue were incubated with 20 000 g supernatants from untreated and SiO2-treated subcellular particles of rat peritoneal macrophages in the presence of radioactive nucleic acid precursors. The supernatant from SiO2-treated subcellular particles increased the incorporation of [3H]CTP into nuclear RNA maximally by 26% at 5 min, and that of [methyl-3H]dTTP into DNA by 16% at 20 min. The release of radioactivity from labeled DNA was suppressed simultaneously. An RNase preparation from rat peritoneal macrophages enhanced the release of radioactivity from labeled DNA similarly as the soluble fraction from untreated subcellular particles of macrophages. The results suggest that the effects of the soluble fractions upon DNA metabolism of granuloma cells are at least partly independent of the effects on RNA metabolism and that the soluble fraction from SiO2-treated subcellular particles of macrophages stabilizes DNA through inhibition of nuclease activity.

Animals↗

The effect of local methylprednisolone on granulation tissue formation. II. Mechanisms of action.

The mechanism of action of a locally applied depot preparation of methylprednisolone on the granulation tissue was studied. Various connective tissue components were analysed in the developing granulation tissue induced by subcutaneously implanted viscose cellulose sponges in rats. Methylprednisolone treatment at the time of sponge implantation reduced the content of both hydroxyproline and hemoglobin and caused a clearly reduced RNA/DNA ratio in the new connective tissue. The total number of cells infiltrating the sponge was not affected by the treatment, but there were significantly fewer lymphocytes, monocytes and macrophages 4 and 7 days post-implantation. In the non-treated sponges there were significantly more fibroblastoid cells, which sticky cells usually died during the releasing procedure. When the corticosteroid was given after the third postoperative day it did not affect the developing granulation tissue. These results indicate that methylprednisolone mainly affects the initial inflammatory phase of granulation tissue formation and wound healing.

Animals↗

Two- and three-dimensional ultrastructural observation of two cell angiogenesis in human granulation tissue.

The growth of endothelial sprouts from capillary walls in human granulation tissue has been examined by two- and three-dimensional electron microscopy using a serial sectioning method. Within the parent capillary wall endothelial sprouts composed of two layers of relatively immature endothelium was demonstrated. Three-dimensional reconstruction revealed that the two layered endothelial projections extended and/or migrated outward in a bicellular configuration, the slit-like lumen of the endothelial sprout connecting with the parent capillary lumen. These ultrastructural appearances have not been reported previously with sequential composition to the morphological progression of the sprout. In the cytoplasm of the endothelial sprout, abundant intermediate filaments were assumed to play a mechanical role, tension resistance, in the development of the endothelial sprout. The active endothelial sprout in granulation tissue was considered to be at least partially responsible for the growth of the capillary network and subsequent development of granulation tissue.

Endothelium, Vascular↗

Physicochemical properties of extracellular matrix proteins in post-burn human granulation tissue.

Wound healing is a finely controlled biological process involving a series of complex cellular interactions. Following inflammation, the wound bed matrix is gradually replaced by granulation tissue followed by the long slow process where collagen accumulates and restores tensile strength. The studies revealed that human granulation tissue varied in many aspects in comparison with normal skin. In granulation tissue the molecular organization of collagen showed an increased amount of type III collagen resembling embryonic tissue. The presence of type V collagen with three distinct chains was the characteristic feature of granulation tissue. The physicochemical properties of collagen extracted from granulation tissue showed the influence of proteoglycans during collagen aggregation and these proteoglycans from the major non-collagenous proteins during the proliferative phase of healing.

Burns↗

Wound contraction. Correlations between the tension generated by granulation tissue, cellular content and rate of contraction.

The tension generated in vitro by samples of granulation tissue taken from wounds at various times after wounding has been measured. Throughout the period when in vivo contraction was occurring with a constant linear rate of movement of the wound margins the in vitro tension produced by samples of the granulation tissue remained constant at approximately 80 mg/mm2. With cessation of contraction in vivo, the tension generated by the granulation tissue fell to 40 mg/mm2. Morphometric studies demonstrated that total cellularity paralleled the tension developed. However, the decrease in cellularity preceded by approximately 4 d the decrease in the tension developed. Strong correlation between the rate of contraction and the maximum thickness of the granulation tissue was demonstrated.

Animals↗

Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts.

Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilament bundles and the expression of alpha-SM actin, the actin isoform typical of vascular SM cells. Myofibroblasts have been proposed to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. We show here that the subcutaneous administration of transforming growth factor-beta 1 (TGF beta 1) to rats results in the formation of a granulation tissue in which alpha-SM actin expressing myofibroblasts are particularly abundant. Other cytokines and growth factors, such as platelet-derived growth factor and tumor necrosis factor-alpha, despite their profibrotic activity, do not induce alpha-SM actin in myofibroblasts. In situ hybridization with an alpha-SM actin probe shows a high level of alpha-SM actin mRNA expression in myofibroblasts of TGF beta 1-induced granulation tissue. Moreover, TGF beta 1 induces alpha-SM actin protein and mRNA expression in growing and quiescent cultured fibroblasts and preincubation of culture medium containing whole blood serum with neutralizing antibodies to TGF beta 1 results in a decrease of alpha-SM actin expression by fibroblasts in replicative and non-replicative conditions. These results suggest that TGF beta 1 plays an important role in myofibroblast differentiation during wound healing and fibrocontractive diseases by regulating the expression of alpha-SM actin in these cells.

Actins↗

Amelioration of dural granulation tissue growth for primate neurophysiology.

Four methods were tried in order to reduce the growth of granulation tissue on the dura. The best results were obtained using white petrolatum jelly, which almost completely suppressed the growth of granulation tissue when the recording chamber was filled with petrolatum. Collagen and acrylic seals were very effective in one monkey. Panalog ointment slowed the growth of granulation tissue; preformed silicon sheets had no apparent effect. We conclude that long-term application of petrolatum jelly has no adverse effects and achieves striking suppression of the growth of granulation tissue.

Acrylic Resins↗

Successful resection of obstructing airway granulation tissue following lung transplantation using endobronchial laser (Nd:YAG) therapy.

OBJECTIVE: Airway obstruction due to an excessive growth of granulation tissue at the level of the anastomosis is an important complication following lung transplantation which requires early diagnosis and treatment. We report encouraging experience in the management of this condition using endobronchial Nd:YAG laser therapy. METHODS: Four adult lung transplant recipients developed airway anastomotic obstruction secondary to granulation tissue formation at 9, 10, 32 and 32 days following bilateral sequential lung transplantation (2 patients), en bloc double lung transplantation (1 patient) and single lung transplantation (1 patient). The diameter of the airways at the level of the anastomoses was reduced by 75, 30, 60, 60, 50 and 90%, respectively. Endobronchial Nd:YAG laser was applied via a fiberoptic bronchoscope introduced through a rigid bronchoscope. The granulation tissue was visualised and resected with photocoagulation with the laser using between 1000-2000 J depending on the amount of tissue present. Necrotic tissue was removed with large forceps. If the obstruction extended to the orifice of a lobar bronchus resection was undertaken in a staged fashion. RESULTS: Airway patency was fully restored at two anastomotic sites, and restored to 90% patency at two and 80 and 75% at one each, respectively. This was associated with a significant improvement in pulmonary function in 3 patients. One patient had a subsequent bougie dilatation of a stenotic area and 2 patients received an endobronchial stent for tracheo or broncho-malacia. One patient died from a gastrointestinal haemorrhage. Three patients are well at 10, 17 and 18 months following transplantation and have no further granulation tissue recurrence. There were no complications directly attributable to laser therapy. CONCLUSION: Our encouraging early experience leads us to suggest that Endobronchial Nd-YAG laser therapy should be considered in the management of airway anastomotic obstruction due to excessive granulation tissue formation after lung transplantation.

Adult↗

Human granulation-tissue fibroblasts show enhanced proteoglycan gene expression and altered response to TGF-beta 1.

Granulation-tissue fibroblasts are phenotypically unique cells that play an important role in wound repair and the development of chronic inflammatory lesions in connective tissue. In the present study, we compared proteoglycan, type I, and type III procollagen gene expression by granulation-tissue fibroblasts from wound and chronically inflamed tissues with normal gingival fibroblasts. We also analyzed the effect of TGF-beta 1 on proteoglycan mRNA levels and macromolecule production by these cells. One granulation-tissue fibroblast strain that was composed exclusively of alpha-smooth-muscle actin-positive cells (myofibroblasts) expressed strongly elevated basal levels of biglycan, fibromodulin, and versican (the large chondroitin sulphate proteoglycan), as well as type I and III procollagen mRNA. TGF-beta 1 enhanced more potently the expression of types I and III procollagen, biglycan, and versican mRNA by these cells as compared with normal fibroblasts. Other granulation-tissue fibroblast strains, in which about half of the cells expressed alpha-smooth-muscle actin, also showed enhanced proteoglycan and types I and III procollagen expression as compared with normal fibroblasts. These results suggest that alterations in matrix composition during inflammation and wound healing are regulated partly by altered phenotypes of the cells that produce the matrix, and partly by altered responses of these cells to TGF-beta 1.

Biglycan↗

Fibroblasts from the inner granulation tissue of the pseudocapsule in hips at revision arthroplasty induce osteoclast differentiation, as do stromal cells.

BACKGROUND: It has previously been shown that many osteoclast precursors are included in the granulation tissue within the pseudocapsule obtained at revision arthroplasty from hips with osteolysis. In vitro culture of only cells isolated from the granulation tissue has been previously shown to generate many mature osteoclasts. OBJECTIVE: To investigate the presence or otherwise of supporting cells, similar to stromal cells, which differentiate osteoclasts within the granulation tissue. METHODS: Cells isolated from the granulation tissue were cultured alone, and after four weeks fibroblast-like cells (granulation fibroblasts) remained. Rat non-adherent bone marrow cells (NA-BMCs) were co-cultured with the granulation fibroblasts with or without 1alpha,25(OH)2D3 (10(-8) M) or heat treated ROS 17/2.8 cell conditioned medium (ht ROSCM), or both. Multinucleated cells (MNCs), which formed, were assessed by biochemical and functional characterisation of osteoclasts. Receptor activator of NFkappaB ligand (RANKL) was investigated by immunohistochemistry. RESULTS: Co-culture of NA-BMCs and granulation fibroblasts caused the formation of tartrate resistant acid phosphatase (TRAP) positive MNCs, which had the calcitonin receptor (CTR), the Kat-1 antigen, which is specific to the surface of rat osteoclasts, and the ability to form pits in the presence of both 1alpha,25(OH)2D3 and ht ROSCM or in the presence of just ht ROSCM. RANKL was detected in fibroblast-like cells in the granulation tissue. CONCLUSION: These data suggest that granulation fibroblasts support osteoclast differentiation, as do osteoblasts/stromal cells, and may play a part in aseptic loosening.

Aged↗

Local hyperalimentation of experimental granulation tissue.

The effect of local hyperalimentation on developing granulation tissue was studied in rats. Cylindrical hollow viscose cellulose sponge implants were used subcutaneously as an inductive matrix fro the growth of granulation tissue. In the first, control group the implants were kept untouched while the second, "sham" group was treated daily by withdrawing 1 ml of wound fluid from the central dead space of the implant and then injecting the fluid back. In the third, hyperalimentation group the aspirated wound fluid was substituted with a corresponding volume of sterile, nonpyrogenic solution containing a mixture of amino acids (Le-7402 A) and glucose, electrolytes and vitamins (Le-7402 B). Within the first week of tissue growth daily application of these nutritional substances caused a changeover of local tissue from predominantly anaerobic towards more oxidative metabolism. Measurement of nucleic acid and hydroxyproline contents indicated enhanced accumulation of cells and collagen in tissues receiving local hyperalimentation. The results combined with earlier data from our laboratory strongly suggest that several types of wounds, especially those containing a marked dead space or large regenerative area, exist in chronic lack of oxygen and other nutrients. Therefore, the healing process in these wounds can be stimulated, to a certain extent, by exposure to increased oxygen tension and/or by local hyperalimentation.

Amino Acids↗

Enhancement of the granulation tissue formation in hairless mice by a potent vitamin D receptor agonist--KH 1060.

KH 1060 is a 20-epi analogue of 1,25-dihydroxyvitamin D3 and a potent agonist of the vitamin D receptor. Our recent finding that it stimulates glycosaminoglycan synthesis and transforming growth factor-beta 1 (TGF-beta 1) expression in normal skin provided a rationale for investigating its influence on the process of wound healing. Normal and betamethasone-impaired granulation tissue formation was studied in a polytetrafluoroethylene dead space model in hairless mice. The application of KH 1060 increased the indexes of fibroplasia and cellularity ([3H]thymidine incorporation and DNA concentration) of the betamethasone-impaired granulation tissue. Collagen production and deposition, measured as hydroxyproline synthesis and concentration in the granulation tissue, were also increased. The effect of KH 1060 on normal connective tissue repair was less pronounced; DNA and hydroxyproline concentrations in granulation tissue were unchanged. KH 1060 strongly stimulated the expression of TGF-beta 1 in betamethasone-impaired granulation tissue. Thus, it effectively reversed the deleterious effect of betamethasone on granulation tissue. The hyperproliferative response to this vitamin D analogue might be related to the direct stimulation of the vitamin D receptors in the granulation tissue, while the increased collagen synthesis and deposition was probably caused indirectly, via stimulation of TGF-beta 1.

Animals↗

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.

Animals↗

Collagen glycosylation in human granulation tissue and scar.

Some biochemical characteristics of collagen extracted from granulation tissue were studied and compared with those of normal skin and scar. By using electrophoretic techniques the type III collagen content was confirmed to be significantly greater in granulation tissue and lower in scar with respect to normal skin. The chromatographic determination of hydroxylysine (Hyl) glycosides in collagen extracted from granulation tissue showed a significant increase in both the degree of Hyl glycosylation and in the di-/monoglycoside ratio, while both parameters turned out to be lower in scar. These data suggest that the degree of Hyl glycosylation and the di-/monoglycoside ratio could represent an index of the degree of collagen fiber maturation.

Cicatrix↗

Study of the accelerating effect of shikonin and alkannin on the proliferation of granulation tissue in rats.

The present study was carried out to compare the accelerating effect of shikonin and alkannin and to elucidate the expression of CD antigen and histological changes on the proliferation of granulation tissue in rats. Shikonin and alkannin produced a dose-dependent acceleration of the cotton pellet-induced granuloma formation and this accelerating potency of both compounds on the proliferation of granulation tissue was about the same 5 and 10 d after implantation of the cotton pellet. Also, both compounds increased the ratio of CD11b+ cells in the granulation tissue 5 and 10 d after implantation of the cotton pellet. Both compounds increased the expression of CD11b+ cells with granulocytes such as macrophages and histiocytes, and then accelerated the proliferation of fibroblasts and collagen fiber. On the other hand, neither compound increased the ratio of CD3+ cells in the granulation tissue after 5 and 10 d. These results suggest that shikonin and alkannin accelerate the proliferation of granulation tissue induced by the cotton pellet and this accelerating effect may be attributed to an increase in the expression of CD11b+ cells, and the acceleration of the proliferation of fibroblasts and collagen fiber in the granulation tissue.

Animals↗

Granulation tissue as a contractile organ. A study of structure and function.

CONTRACTING GRANULATION TISSUES CONTAIN FIBROBLASTS THAT DEVELOP CHARACTERISTICS TYPICAL OF SMOOTH MUSCLE: (a) They contain an extensive cytoplasmic fibrillar system. (b) They show immunofluorescent labeling of their cytoplasm with human anti-smooth muscle serum. (c) The nuclei show complicated folds and indentations, indicative of cellular contraction. (d) There are cell-to-cell and cell-to-stroma attachments. (e) It is possible to extract similar quantities of actomyosin (having the same adenosine triphosphatase activity) from granulation tissue and from pregnant rat uterus. (f) Strips of granulation tissue, when tested pharmacologically in vitro, behave similarly to smooth muscle. All these data support the view that, under certain conditions, fibroblasts can differentiate into a cell type structurally and functionally similar to smooth muscle and that this cell, the "myo-fibroblast," plays an important role in connective tissue contraction.

Actomyosin↗

Collagenase production at the border of granulation tissue in a healing wound: macrophage and mesenchymal collagenase production in vivo.

We demonstrated the cells producing collagenase and the time course of collagenase-production at early stages of wound healing, using histology and two immunohistochemical procedures on cross sections of rat skin harvested 0, 3, 5, 7 and 12 days after full-thickness incisions. A monospecific rabbit polyclonal antibody to neutral collagenase purified from rat myometrial cells was used to demonstrate collagenase production. Specificity of this reaction was confirmed by blocking the reaction with excess homogeneously purified antigen. Macrophages were simultaneously labelled using a mouse anti-rat monoclonal antibody recognizing exclusively mature macrophages. Intracellular collagenase was not reliably detectable at day 0, but was prominent at days 3 and 5 and thereafter declined. Double labeling technique showed occasional macrophages producing collagenase in the developing granulation tissue, but most cells labeled as macrophages were negative for collagenase. Most activity was found in fibroblasts adjacent to granulation tissue elements. Since the granulation tissue parallels revascularization in a dendritic pattern, a cross section at three days typically shows an annulus of collagenase-positive cells surrounding a branch of the active granulation tissue. At days 5, 7 and 12 after wounding the pattern of collagenase expression became indistinct as more tissue was involved in the granulation process. However, double-labelling for macrophages and collagenase showed the dichotomy between collagenase expression and presence of macrophages to persist. The finding that collagenase is produced in connective tissue adjacent to granulation tissue suggests an inductive process, possibly due to diffusion of cytokines produced by granulation tissue elements.

Animals↗