Enzyme activities in granulation tissue: Energy for collagen synthesis.

Sympathectomy was produced in 8 dogs by the intravenous administration of 6-hydroxydopamine (6-OHDA). The animals were studied at rest and during exercise, prior to and after administration of the drug. The hemodynamic patterns observed during this experiment are similar to the previously reported patterns obtained after extensive surgical denervation or cardiac transplantation.     

Topical vanadate improves tensile strength and alters collagen organisation of excisional wounds in a mouse model

Wound dehiscence, oftentimes a result of the poor tensile strength of early healing wounds, is a significant threat to the post-operative patient, potentially causing life-threatening complications. Vanadate, a protein tyrosine phosphatase inhibitor, has been shown to alter the organisation of deposited collagen in healing wounds and significantly improve the tensile strength of incisional wounds in rats. In this study, we sought to explore the effects of locally administered vanadate on tensile strength and collagen organisation in both the early and remodelling phases of excisional wound healing in a murine model. Wild-type mice underwent stented excisional wounding on their dorsal skin and were divided equally into three treatment conditions: vanadate injection, saline injection control and an untreated control. Tensile strength testing, in vivo suction Cutometer analysis, gross wound measurements and histologic analysis were performed during healing, immediately upon wound closure, and after 4 weeks of remodelling. We found that vanadate treatment significantly increased the tensile strength of wounds and their stiffness relative to control wounds, both immediately upon healing and into the remodelling phase. Histologic analysis revealed that these biomechanical changes were likely the result of increased collagen deposition and an altered collagen organisation composed of thicker and distinctly organised collagen bundles. Given the risk that dehiscence poses to all operative patients, vanadate presents an interesting therapeutic avenue to improve the strength of post-operative wounds and unstable chronic wounds to reduce the risk of dehiscence.     

Wound matrix attachment regulates actin content and organization in cells of the granulation tissue

Actin cytoskeletal polymerization is associated with a pro-proliferative, pro-survival state. We hypothesized that the actin polymerization of wound cells is increased in the presence of wound matrix attachment and is decreased after disruption of this attachment. Musculocutaneous flap and wound splinting models were used to investigate the effect of wound matrix attachment on the actin cytoskeleton. Disruption of wound matrix attachment was accomplished by incision of the wound matrix/dermis interface (wound matrix release) and/or desplinting. Polymerized actin was assayed with phalloidin labeling of wound specimens 24 hours after disruption of attachment and a method to quantify the content and organization of polymerized actin in granulation tissue was used. Disruption of wound matrix attachment decreased the content of polymerized actin, the actin staining intensity, and the actin fiber organization in the granulation tissue of both the flap and splint models. Disruption of wound matrix attachment decreased actin polymerization and fiber organization in the granulation tissue. Our data support the concept that the state of wound matrix attachment regulates the actin cytoskeleton of wound cells.     

Chronic administration of growth hormone-releasing factor increases wound strength and collagen maturation in granulation tissue

The effects of chronic administration of growth hormone-releasing factor (GRF) on wound healing were studied in rats. Cutaneous wound strength was measured by tensometry at 5, 10, and 14 days postwounding in rats implanted with a slow-release pellet which contained a compressed mixture of a fatty acid and [desamino Tyr1, D-Ala2, Ala15]hGRF(1-29)NH2 or the fatty acid alone. There was a significant increase in wound tensile strength in GRF-treated rats compared to controls at each measurement: Day 5, 130 +/- 12 vs 97 +/- 14 g; Day 10, 402 +/- 18 vs 280 +/- 11 g; Day 14, 830 +/- 17 vs 614 +/- 14 g (P less than 0.01 for each value). Granulation tissue obtained from subcutaneously implanted polyvinyl alcohol sponges encased in silicone tubing was also studied. The amount of collagen deposited in the granulation tissue was estimated by measuring the hydroxyproline (Hyp) content of sponges retrieved 5, 10, and 14 days postinsertion from GRF-treated and control rats. Hyp content (nmole/mg sponge) was similar in both treated and control animals at each measurement: Day 5, 1.7 +/- 0.2 vs 2.2 +/- 0.2; Day 10, 31.9 +/- 4.1 vs 26.7 + 0.4; and Day 14, 41.6 +/- 7.3 vs 38.5 +/- 4.4. Hyp/proline, Hyp/glycine, and glycine/total amino acid ratios, evaluated after 10 days, were also similar in both groups. Collagen from the granulation tissue of sponges retrieved after 14 days from treated and control rats was studied by electron microscopy (magnifications, 7,100 and 22,720).(ABSTRACT TRUNCATED AT 250 WORDS)     

Elastin and granulation tissue.

The contraction of granulation tissue is an important factor in wound healing. The tract of an abdominal drainage tube closes at the rate of 1 mm/h once the tube is removed. Specialized myofibroblasts are generally believed to be responsible for this process and the presence of elastin in granulation tissue has been debated for many years. A 3-mm polyethylene tube was inserted through the left lobe of the liver in 41 rats. A tube of granulation tissue formed around the plastic. This granulation tissue was isolated, after periods ranging from 8 to 20 days, by removing the left lobe of the liver and scraping away the surrounding liver tissue. Biochemical assay for elastin revealed that normal hepatic tissue does not contain any detectable amount of elastin. All 41 samples of granulation tissue contained elastin with a mean value of 8.5 X 10(-6) g of elastin per milligram of tissue. Normal rat aorta contains 31.4 X 10(-6) g of elastin per milligram of tissue. These tubes of granulation tissue were histologically normal and the presence of elastin was recognized using elastin stains. The exact role of elastin in granulation tissue is uncertain but this experiment clearly demonstrates its generation and presence there.     

Wound splinting regulates granulation tissue survival

PURPOSE: Fibroblast survival within an in vitro collagen matrix is dependent on matrix anchorage to a rigid substratum. The purpose of this study was to determine whether granulation tissue survival in vivo also is dependent on matrix anchorage. We hypothesized that splinting an excisional wound (i.e., anchoring the wound edges) would promote granulation tissue survival and that desplinting a splinted wound would produce granulation tissue apoptosis. METHODS: Eighteen Wistar rats (3 months, 350 g) underwent excisional wounding (2 x 2 cm, dorsal skin) with immediate wound splinting (a metal template affixed with sutures) on day 0. On day 6, rats (n = 6 per group) underwent splint removal (desplinted), splint removal with circumferential incision of the wound edge (desplint/release), or no intervention (splinted); sacrifice of all animals was on day 7. Frozen sections of granulation tissue were stained with TUNEL or H and E; data were analyzed with ANOVA and the unpaired t test. RESULTS: The cross-sectional and surface area of the desplinted and desplint/release granulation tissue both decreased compared to the splinted granulation tissue (*P < 0.05). The nuclear density of the desplint/release granulation tissue was 25% less compared to the splinted granulation tissue (*P < 0.05). The desplinted and desplint/release apoptotic rates were twice and >10x greater than the splinted apoptotic rate, respectively (*P < 0.05). CONCLUSIONS: The rate of cell death in a splinted wound (an in vivo equivalent of an anchored FPCM) is minimal to nil, which is consistent with our hypothesis. Desplinting and releasing the wound edge of a previously splinted wound (the in vivo equivalent of a detached FPCM) results in granulation tissue regression and a large increase in apoptosis. Desplinting a wound alone results in changes somewhat intermediate to the splinted and desplint/release conditions. Loss of wound anchorage acutely promotes granulation tissue apoptosis.     

Differential regulation of collagen gene expression in granulation tissue and nonrepair connective tissues in vitamin C-deficient guinea pigs

Poor wound healing during vitamin C deficiency is thought to be due to decreased hydroxylation of proline residues in collagen. In non-repair connective tissues of guinea pigs, however, procollagen gene expression is not decreased until weight loss occurs during the third and fourth weeks of scurvy (phase II) with only a moderate decrease in proline hydroxylation. Decreased procollagen gene expression is related to the induction of insulin-like growth factor binding proteins 1 and 2 that inhibit insulin-like growth factor-I action. We examined wound healing and granulation tissue formation during phase I of vitamin C deficiency. Synthetic sponges were implanted on day 7 of vitamin C deficiency and analyzed at 6 and 10 days after surgery, when there was no weight loss or induction of insulin-like growth factor binding proteins. Healing of incisions was almost complete at 10 days after surgery in normal controls but not in scorbutic animals. The area around the incision and implant exhibited excessive angiogenesis and hemorrhaging of vessels in the scorbutic animals at 6 and 10 days after surgery. At 10 days after surgery, collagen synthesis in the implants of scorbutic guinea pigs was 36% lower than control values, with a normal extent of proline hydroxylation. Concentrations of messenger RNAs for types I and III procollagens were slightly increased by scurvy at 6 days after surgery but were decreased by 26% and 40%, respectively, at 10 days. Fibronectin mRNA levels were unaffected by scurvy at both time points. Our results suggest that poor wound healing in phase I of scurvy may be related to defective interstitial procollagen gene expression and defective blood vessel formation, but it does not involve inhibition of proline hydroxylation or induction of insulin-like growth factor binding proteins. mRNA for insulin-like growth factor-II, transforming growth factor-beta(1), and transforming growth factor-beta(2) were significantly expressed in implants, but their patterns of expression did not correlate with changes in procollagen gene expression.     

Glycosaminoglycans in granulation tissue and hypertrophic scars

The glycosaminoglycans of granulation tissue from regenerating tissue of severely burned patients was investigated and compared with those of (1) hypertrophic scars and keloids, (2) mature scars and (3) normal skin. Results clearly show that granulation tissues have high levels of chondroitin-4-sulphate relative to the other glycosaminoglycans as compared to low levels in mature scar and normal skin. The chondroitin-4-sulphate levels of children's granulation tissue was significantly higher than the levels from hypertrophic scars of children, however the chondroitin-4-sulphate levels of adults' granulation tissue was not significantly different from those of hypertrophic scars. The dermatan sulphate levels of granulation tissue are significantly lower than the dermatan sulphate levels of hypertrophic scars, mature scars or normal skin. This suggests that chondroitin-4-sulphate is related to proliferation of collagen in granulation tissue, and that as the collagen matures and the normal scar forms, chondroitin-4-sulphate levels decrease and dermatan sulphate increases. When the hypertrophic scar develops, the high chondroitin-4-sulphate levels remain high, dermatan sulphate increases, but does not reach the levels of those in the mature scar. Neither hypertrophic scar nor mature scar have levels of hyaluronic acid as high as normal skin. However, our data suggest that adult granulation tissue may have relatively high hyaluronic acid levels. It is suggested that the different distribution of glycosaminoglycans in granulation tissue influences the size and shape of collagen filaments and may also influence the rate of synthesis of collagen.     

创面肉芽组织细胞的原代培养

目的　探索开放创面肉芽组织细胞培养的可能性 ,比较早、晚期肉芽组织中培养细胞成分的差别。方法　用酶消化法对早 ( 7天 )、晚 ( 17天 )期开放创面的肉芽组织进行原代培养 ,并进行形态学及免疫组化检查。结果　开放创面早、晚期肉芽组织中的培养细胞成分有差异。结论　开放创面的肉芽组织可以进行细胞培养 ,早期以成纤维细胞为主 ,晚期肌成纤维细胞较多。     

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.     

Obstructive suprastomal granulation tissue following percutaneous tracheostomy

Percutaneous dilatational tracheostomy is frequently performed as an alternative to traditional surgical open tracheostomy with many reported benefits. Despite its relative safety and widespread acceptance, complications can be associated with the procedure itself or long-term. We present four cases where there was difficulty with decannulation because of exuberant obstructive granulation tissue. In each case, the percutaneous tracheostomy involved the cricoid cartilage.     

创面肉芽组织细胞的原代培养

目的探索开放创面肉芽组织细胞培养的可能性,比较早、晚期肉芽组织中培养细胞成分的差别.方法用酶消化法对早(7天)、晚(17天)期开放创面的肉芽组织进行原代培养,并进行形态学及免疫组化检查.结果开放创面早、晚期肉芽组织中的培养细胞成分有差异.[HT5”H〗结论开放创面的肉芽组织可以进行细胞培养,早期以成纤维细胞为主,晚期肌成纤维细胞较多.     

The contractile properties of wound granulation tissue

A systematic examination of the contractile properties of wound granulation tissue is presented. Shortening of and tension generated by granulation tissue in the presence of 30 mM diphenhydramine HCl have been measured. Analysis of the stress (load per unit area)/strain (extent of shortening) results from isotonic shortening studies showed that over the range of 0 to 2.5% shortening there was an approximately linear relationship between stress and strain with a high modulus of elasticity. At lower stresses, wide variations in the amount of shortening occurred with little change in stress. Our interpretation of these findings is that diphenhydramine HCl caused an active shortening of the granulation tissue by 2.5% of its length and contractions greater than this were the result of secondary effects such as coiling and bending of the strips. It is shown that the granulation tissue would have to shorten by 2% once every 3 days to account for observed in vivo rates of contraction for large human wounds and once every 13 hr for rapidly contracting experimental rabbit wounds. The time course of the development of isometric tension by the granulation tissue is shown to be consistent with the proposal that each contractile cell contributes equally to the overall tension developed and that each cell is individually activated by a critical concentration of diphenhydramine HCl which is transported through the tissue by diffusion.     

Thiamine influence on collagen during the granulation of skin wounds

To determine the effect of thiamine (vitamin B₁) on collagen production during wound healing, hydroxyproline content (HP), lysyl oxidase activity (LO), and pepsin-solubilized collagen components were examined in dermis, wound fluid, and artificially induced granulation tissue from surgically wounded rats. Rats were divided into three groups: thiamine-deficient diet (?B₁); thiamine-deficient diet supplemented with 3 mg thiamine-HCl (+3B₁); and thiamine-deficient diet supplemented with 1 mg thiamine-HCl (+B₁) (pair fed). When rats from the ?B₁ group were demonstrated to be deficient in urinary thiamine, all of the experimental animals had one polyvinyl alcohol wound chamber implanted subcutaneously. Differences were observed in: HP concentration between ?B₁ and both +3B₁ and +B₁ in granulation tissue and wound fluid, and LO between ?B₁ and +B₁ in skin and granulation tissue. Both high- and low-molecular-weight collagenous components extracted from granulation tissue were greater for +3B₁ and +B₁, than for ?B₁. The most dramatic findings were in the higher molecular weight component characterized as Type III collagen. The alterations observed in the collagen contents and maturation of granulation tissue demonstrate an involvement of thiamine in wound repair and scar development.     

Influence of human growth hormone on granulation tissue formation, collagen deposition, and the aminoterminal propeptide of collagen type III in wound chambers in rats

The influence of growth hormone on granulation tissue formation was investigated in wire mesh cylinders implanted subcutaneously in rats. Two groups of 10 rats (study 1) and 1 group of 12 rats (study 2) were used for the investigation. Growth hormone, 0.02 and 0.2 IU (study 1), 0.05 and 0.2 IU (study 2), or vehicle only, was injected into the cylinders every third day for 16 days. In study 2, wound fluid was aspirated before injection of growth hormone and saved for later analysis of the aminoterminal propeptide of collagen type III. In both studies, growth hormone significantly increased the formation of granulation tissue and of total collagen content dose-dependently, whereas the relative amount of collagen was unaffected by growth hormone treatment. Wound fluid aminopropeptide increased significantly after implantation of the cylinders until day 7, before declining slightly, with no difference between the groups. We conclude that growth hormone stimulated granulation tissue formation and collagen deposition dose-dependently in the wound cylinders when injected every third day. The results suggest that growth hormone treatment does not cause excessive collagen deposition in newly formed granulation tissue.     

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.     

Transgenic mice overexpressing CD109 in the epidermis display decreased inflammation and granulation tissue and improved collagen architecture during wound healing

Transforming growth factor‐β (TGF‐β) is a multifunctional growth factor involved in all aspects of wound healing. TGF‐β accelerates wound healing, but an excess of its presence at the wound site has been implicated in pathological scar formation. Our group has recently identified CD109, a glycophosphatidylinositol‐anchored protein, as a novel TGF‐β coreceptor and inhibitor of TGF‐β signaling in vitro. To determine the effects of CD109 in vivo on wound healing, we generated transgenic mice overexpressing CD109 in the epidermis. In excisional wounds, we show that CD109 transgenic mice display markedly reduced macrophage and neutrophil recruitment, granulation tissue area, and decreased Smad2 and Smad3 phosphorylation, whereas wound closure remains unaffected as compared with wild‐type littermates. Futhermore, we demonstrate that the expression of the proinflammatory cytokines interleukin‐1α and monocyte chemoattractant protein‐1, and extracellular matrix components is markedly decreased during wound healing in CD109 transgenic mice. In incisional wounds, CD109 transgenic mice show improved dermal architecture, whereas the tensile strength of the wound remains unchanged. Taken together, our findings demonstrate that CD109 overexpression in the epidermis reduces inflammation and granulation tissue area and improves collagen organization in vivo.     

Epidermal growth factor increases granulation tissue formation dose dependently

Recent studies have shown that epidermal growth factor (EGF) stimulated the rate of formation of granulation tissue in a model of wound repair (A. Buckley, et al., Proc. Nat. Acad. Sci. USA 82: 7340, 1985). Because pharmacologic doses of EGF were used previously, the relationship of EGF concentration to physiologic effects was determined in this study. Rats were implanted with subcutaneous polyvinyl alcohol sponges containing slow-release pellets formulated to release 0, 0.1, 1.0, or 10 micrograms of EGF/day. Tissue response was judged by the degree of histologic organization and vascularity, as well as several quantitative parameters: wet weight, hydroxyproline content, protein content, and DNA concentration. Each of these parameters showed consistent increases by Day 5 after implantation, when inflammation and edema had subsided. Compared with placebo controls, hydroxyproline (collagen) content was significantly increased by as little as 1 microgram/day of EGF, and DNA content was significantly increased by all dose levels of EGF. Endogenous EGF concentration in experimental granulation tissue was found to be fairly constant (30-40 ng/g wet wt); however, the increasing cellularity of the sponges may have reduced the local concentration of free EGF to low levels. Pellets releasing as little as 4 ng/hr of EGF into the surrounding tissue were able to accelerate wound healing, suggesting that the availability of this growth factor may be a rate-limiting step in wound repair.