Sustained microgravity reduces intrinsic wound healing and growth factor responses in the rat.

Spaceflight is known to diminish bone mass and reduce immune function, suggesting that repair of connective tissue might be impaired in a microgravity environment. Fisher 344 rats were used to test wound healing responses in the orbiting Space Shuttle Endeavour by preflight implantation of polyvinyl acetal sponge disks in which pellets were placed to release either platelet-derived growth factor (PDGF-BB), basic fibroblast growth factor (bFGF), or placebo. Control groups on the ground included a matched environment group in similar housing modules and temperature control groups in cages at 22 degreesC and 28 degreesC. After 12 days of implantation and 10 days in orbit, the removed sponges were analyzed for histological and biochemical responses. Growth factor responses were histologically evident after release of PDGF-BB and bFGF in ground controls, whereas only immediate-release bFGF and delayed-release PDGF-BB showed significant responses in microgravity. Biochemical data confirmed that cellularity was increased by both factors in ground sponges; however, this response was significantly blunted in flight sponges (P<0.005, ANOVA), irrespective timing of factor release. Collagen content was 62% lower in sponges from animals with 10 days of microgravity exposure (P<0.01, ANOVA) and further reduced by bFGF. These data suggest that orbital exposure retards the capacity of wounds to heal and respond to exogenous stimuli.

Pancreatic one-carbon metabolism in early folate deficiency in rats.

An amino acid-defined, folate-deficient diet was used to investigate the regulation of pancreatic glycine N-methyltransferase in vivo. This enzyme modulates the ratio of S-adenosylmethionine to S-adenosylhomocysteine and is inhibited by bound folate in vitro. Rats were fed either a folate-deficient diet, a folate-supplemented diet (pair-fed to the deficient group), or a folate supplemented diet ad libitum and measurements were made after 2, 3, and 4 wk. Folate concentrations were greatly reduced in the folate-deficient pancreas after only 2 wk and pancreatic glycine N-methyltransferase activity was elevated but the amount of immunologically measured enzyme protein was the same. The ratio of S-adenosylmethionine to S-adenosylhomocysteine was rapidly reduced in the deficient pancreas. This ratio was also reduced with age in the ad libitum control rats. The pancreas of deficient rats had more immature secretory granules and the ducts were devoid of secreted material.

The influence of harvesting technique on endothelial preservation in saphenous veins.

To study optimal conditions of preparation of saphenous veins as coronary artery bypass grafts, segments of saphenous veins were obtained from 29 consecutive patients undergoing coronary artery bypass grafting. The saphenous vein segments were divided into three groups. In Group I, 10 saphenous vein segments were harvested using a "no-touch" technique without any other preparation aids. In Group II, 10 saphenous vein segments were removed while distended at 70-120 mm Hg with a balanced pH electrolyte solution at 37 degrees C. In Group III, consisting of 10 saphenous vein segments, nitroglycerin (1 microgram/ml) was added to the distending solution used in Group II. Samples of saphenous vein were assessed in a blind study using light and scanning electron microscopy to estimate endothelial cell preservation by the three harvest techniques. Saphenous veins receiving only a no-touch dissection technique without distention solution (Group I) revealed significantly better endothelial preservation (P less than 0.005). The administration of distention solution alone, or with nitroglycerin, to saphenous veins in situ using our operative technique during harvest does not appear to protect endothelial-integrity and may be harmful.

Monospecific antibodies implicate basic fibroblast growth factor in normal wound repair.

Exogenous polypeptide growth factors influence the rate of wound healing and other biological processes, but there is no direct evidence that these peptides have an intrinsic role. To test whether basic fibroblast growth factor is involved in wound repair, rats were implanted with subcutaneous polyvinyl alcohol sponges containing slow-release pellets releasing either a polyclonal neutralizing antiserum directed against basic fibroblast growth factor, preimmune IgG, or nothing. Histological and biochemical evaluation of the granulation tissue that infiltrated the sponges showed anti-basic fibroblast growth factor to cause significant reductions in DNA, protein, and collagen content when compared with either preimmune IgG or placebo at the early stages of wound repair.

The diabetic rat as an impaired wound healing model: stimulatory effects of transforming growth factor-beta and basic fibroblast growth factor.

Two models of wound repair compared the effect of defined, recombinant growth factors on the rate of wound repair in both normal and streptozotocin-induced diabetic rats: subcutaneous implantation of polyvinyl alcohol sponges and incisional wounding. Transverse incisional wounds were made on the dorsal surface of rats and closed with steel sutures. Three days postwounding the rats received a single injection of either transforming growth factor-beta or vehicle alone directly into the wound site. Animals were sacrificed 7, 14, and 21 days postwounding, and fresh and formalin-fixed wound tensile strength were measured. Diabetic rats had expected defects in wound repair, including decreased granulation tissue and reduced amounts of collagen, protein, and DNA. Fresh tensile strength of the diabetic incisions was 53% of normal on Day 7 (p < or = .01) and 29% of normal on Day 21. Fixed tensile strength was 41% of normal on Day 7 (p < or = .01) and fell to 78% of normal by Day 21 (p < or = .01), suggesting that collagen concentrations of diabetic wounds increased towards normal but did not undergo maturation. TGF beta produced a moderate increase in tensile strength of fresh and fixed wounds of diabetic rats, but not to the levels of wounds in untreated normal rats. Sponges fill with granulation tissue, their reproducible rate of organization being measured by histological and biochemical methods. A single injection into sponges 3 days postimplantation of basic fibroblast growth factor, transforming growth factor-beta, or vehicle only, was evaluated at 7 and 9 days postimplantation. In the sponge model, bFGF and TGF beta were each able to induce significant increases in the accumulation of granulation tissue in both diabetic and normal rats. TGF beta increased the collagen content of sponges by 136% in sponges from diabetic animals (p < or = .001), thereby raising the collagen content to that of normal control wounds, while stimulating a 49% (p < or = .02) increase in sponges from normal animals on Day 9. By contrast, the response to bFGF was predominantly an increase in the protein and DNA content of the sponges. These results emphasize the differential effects of the two cytokines in accelerating healing under conditions of defective wound repair.

Differential stimulation of collagenase and chemotactic activity in fibroblasts derived from rat wound repair tissue and human skin by growth factors.

Epidermal growth factor and cartilage-derived basic fibroblast growth factor (EGF and CD-bFGF) are mitogens shown to increase the rate of wound repair in animal models. In addition to being a mitogen for granulation tissue, CD-bFGF stimulates the recruitment of cells to the wound site. CD-bFGF and a closely-related chondrosarcoma-derived fibroblast growth factor stimulated chemotaxis of granulation tissue cells in vitro, each factor having a maximum activity at a concentration of 55 pM. Epidermal growth factor was also a potent chemoattractant for rat granulation tissue fibroblasts; however, maximum activity was obtained at 1.7 nM. Cells from all stages of wound repair were chemotactically responsive to these factors, but there was some attenuation of the response to bFGF in cells derived from fully-organized day 28 granulation tissue. Collagenase-catalyzed restructuring of collagen, an additional significant feature of wound repair, is probably critical to cell movement in an extracellular matrix. Cells derived from organizing (6-day old) sponge granulation tissue secreted latent collagenase constitutively in vitro. In the presence of serum, the production of collagenase was stimulated three-four fold by 1.8 nM bFGF derived either from cartilage or chondrosarcoma. When serum was present, as at a wound site, collagenase production was not enhanced by the addition of EGF. Cells from fully organized, day 21 sponge granulation tissue did not secrete latent collagenase constitutively and could not be stimulated to do so by the addition of EGF, bFGF, or phorbol ester. Human skin fibroblast collagenase production was also stimulated by bFGF and was refractory to EGF. While both classes of growth factor have the ability to promote wound healing, the varying responses they elicit in cell populations from the wound site emphasize the different pathways of cellular activation.

Recombinant basic fibroblast growth factor accelerates wound healing.

Basic fibroblast growth factor (bFGF) stimulates extracellular matrix metabolism, growth, and movement of mesodermally derived cells. We have previously shown that collagen content in polyvinyl alcohol sponges increased after bFGF treatment. We hypothesized that bFGF-treated incisional wounds would heal more rapidly. After intraperitoneal pentobarbital anesthesia, male, 200- to 250-g, Sprague-Dawley rats (n = 27) each underwent two sets of paired, transverse, dorsal incisions closed with steel sutures. On Day 3 postwounding, 0.4 ml of bFGF (recombinant, 400 ng. Synergen) or normal saline was injected into one of each paired incisions. Animals were killed with ether on postwounding Days 5, 6, and 7 and their dorsal pelts were excised. Fresh or formalin-fixed wound strips were subjected to tensile strength measurements using a tensiometer. Breaking energy was calculated. Wound collagen content (hydroxyproline) was measured in wound-edge samples following hydrolysis using high-performance liquid chromatography. There was an overall significant increase in fresh wound tensile strength (13.7 +/- 1.06 vs 19.1 +/- 1.99 g/mm, P less than 0.01) and wound breaking energy (476 +/- 47 vs 747 +/- 76 mm2, P less than 0.001) in bFGF-treated incisions. There was an increase in wound collagen content which was not statistically significant and there was no difference in fixed incisional tensile strength. Histologic examination showed better organization and maturation in bFGF wounds. Recombinant bFGF accelerates normal rat wound healing. This may be due to earlier accumulation of collagen and fibroblasts and/or to greater collagen crosslinking in bFGF-treated wounds.

Growth factors bFGF and TGB beta accelerate the rate of wound repair in normal and in diabetic rats.

The effects of basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF beta) on the rate of wound repair in both normal and streptozotocin-induced diabetic rats were investigated using two model systems of wound repair, namely incisional wounding and subcutaneous implantation of polyvinyl alcohol (PVA) sponges. Both models showed the expected wound-healing defects of the diabetic rats. Granulation tissue collected from the implanted PVA sponges showed that the diabetic rats had reduced amounts of collagen, DNA and protein present at the wound site at two time points tested (7 and 9 days post-implantation). Fresh tensile strength of the incisional wounds, a measure of the collagen organization in a wound, was reduced to 53% of normal in diabetic rats on day 7 post-wounding, and was only 29% of normal by day 21. Formalin-fixed tensile strength, a measure of collagen content of the wound, was 41% of normal on day 7, and 78% of normal by day 21, giving evidence that while the collagen concentration of the diabetic wounds approached that of normal wounds, it did not undergo the normal maturation process. A single injection of 2 micrograms of TGF beta directly into the incision three days after wounding resulted in little difference in the fresh and fixed tensile strength of diabetic wounds when tested at 7, 14 and 21 days post-wounding. Normal rats, however, responded well, resulting in a greater than 30% increase in both fresh and fixed tensile strength.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Characterization of a potent inhibitor of platelet aggregation and release reaction isolated from allium sativum (garlic).

When added to platelet-rich plasma, aqueous extracts of garlic inhibited platelet aggregation and the release reaction. Subsequent experiments designed to characterize the inhibitory component revealed that the inhibitory activity was i) associated with small molecular-weight components, ii) the inhibitory component possessed the typical garlic odor and contained an abundance of sulfur, iii) the inhibitory activity could be extracted with organic solvents, and iv) temperatures above 56 degrees C and alkaline pH above 8.5 quickly destroyed the inhibitory activity. The Rf value of the major inhibitory component after thin-layer chromatographic separation was similar to that of allicin, an unique thiosulfinate in garlic previously shown to possess strong antibiotic and antifungal properties. Allicin was synthesized. On thin-layer chromatographic plates, allicin co-migrated with the inhibitory component in garlic. At 10 microM concentration, allicin inhibited completely platelet aggregation and the release reaction. Comparative studies suggest that the major platelet aggregation and release inhibitor in garlic may be allicin.

Sustained release of epidermal growth factor accelerates wound repair.

Epidermal growth factor (EGF) is a potent mitogen in vitro, but its biological role is less clear. The vulnerary effects of EGF were evaluated in a model of wound repair, the polyvinyl alcohol sponge implanted subcutaneously in rats. EGF was purified to homogeneity by reverse-phase HPLC and quantified by receptor binding assay and amino acid analysis. Preliminary data showed moderate promotion of granulation tissue formation by daily injections of 10 micrograms of EGF. To test the hypothesis that long-term exposure to EGF is required for complete cellular response, the factor was incorporated into pellets releasing 10 or 20 micrograms of biologically active EGF per day, and the pellets were embedded within the sponges. Slow release of EGF caused a dramatic increase in the extent and organization of the granulation tissue at day 7, a doubling in the DNA content, and 33% increases in protein content and wet weight, as compared with placebo controls. Although collagen content was also increased by almost 50%, the relative rate of collagen synthesis remained the same, suggesting that the morphological and biochemical increase in collagen resulted from increased numbers of fibroblasts rather than a specific stimulation of collagen synthesis. These results indicate that the local sustained presence of EGF accelerates the process of wound repair, specifically neovascularization, organization by fibroblasts, and accumulation of collagen.

Accelerated wound repair, cell proliferation, and collagen accumulation are produced by a cartilage-derived growth factor.

Cartilage-derived growth factor (CDGF), a cationic polypeptide of approximately 18,000 mol wt, was prepared from bovine articular cartilage; other sources were bovine and human scapular and costal cartilage. Previous studies have shown that CDGF stimulates the proliferation of cultured mouse fibroblasts as well as chondrocytes and endothelial cells from various sources. In this study, CDGF was shown to stimulate dose-dependently the accumulation of DNA and collagen by rat embryo fibroblasts and a population of fibroblasts derived from granulation tissue. CDGF also stimulated the proliferation of cultured bovine capillary endothelial cells dose-dependently. To evaluate the effects of CDGF in vivo, we implanted polyvinyl alcohol sponges subcutaneously in rats. 6 d postimplantation, sponges were injected with 300 micrograms of partially purified CDGF, a dose which takes into account the cell numbers in the sponges as compared with cell cultures. CDGF rapidly disappeared from the sponges and only approximately 10% of the initial dose was present at 4 h. Despite its transient presence, CDGF caused a relative increase in sponge DNA content of 2.6-fold at 48 h and 2.4-fold at 72 h. We repeated the sponge experiment by using 500-ng injections of CDGF purified to near homogeneity by heparin-Sepharose chromatography. Purified CDGF caused significant increases in sponge collagen, protein, and DNA content at 48 and 72 h after a single injection. The effects of CDGF were abolished by heat and unaffected by reduction of disulfide linkages. Morphologically, CDGF did not evoke an inflammatory response, and its effect on proliferating endothelial cells and fibroblasts was, therefore, probably direct. However, increases in DNA content of sponges could not be fully accounted for by increased DNA synthesis, which suggests that recruitment may be an important component of the in vivo response. Taken together, the effects of CDGF on cultured cells and granulation tissue suggest that the sustained presence of CDGF in vivo may greatly enhance its effects upon wound repair.

The intracellular degradation of poly(epsilon-caprolactone).

Poly(epsilon-caprolactone) [PEC], a biodegradable aliphatic polyester, undergoes a two-stage degradation process: The first lengthy phase involves nonenzymatic hydrolytic cleavage of ester groups, the second phase beginning when the polymer is more highly crystalline, and of low molecular weight. The cellular events of the second phase were examined by implanting gelatin capsules containing 25 mg of low molecular weight (Mn 3000) PEC powders, 106 to 500 micron, in rats. PEC fragments ultimately were degraded in phagosomes of macrophages and giant cells, the process requiring less than 13 days for completion at some sites. PEC was also identified within fibroblasts. These studies support the intracellular degradation of PEC as the principal pathway of degradation once the molecular weight of the aged polymer is reduced to 3000 or less.