Lysyl oxidase-mediated crosslinking in granulation tissue collagen in two models of hyperglycemia

Enzymatically mediated crosslinks and nonenzymatic glycation were quantified in granulation tissue collagen in two models of hyperglycemia, diabetes and galactosemia, that have opposite effects on collagen solubility. The effects of castration, which alters collagen solubility, was also investigated. Collagen from both diabetic and galactosenic rats had significantly increased levels of dihydroxylysinonorleucine (DHLNL), a difunctional reducible crosslink. Galactosemic rats had significantly decreased levels of hydroxypyridinium, a trifunctional product of DHLNL and hydroxylyse, relative to control values, while diabetic rats had normal levels. Values for all other detectable crosslinks in collagen from hyperglycemic rats were indistinguishable from control values. Nonezymatic glycation was increased in both groups of hyperglycemic rats. In diabetic rats, but not in galactosemic rats, nonenzymatic glycation was strongly correlated DHLNL content. Castration had no effect on crosslink content of collagen from diabetic or galactosemic rats. This study demonstrates that (1) collagen crosslinking is abnormal in granulation tissue collagen in both experimental diabetes and galactosemia, (2) these changes are similar to those observed in skin collagen from insulin-dependent diabetic subjects and (3) the crosslinking abnormalities are not correlated with alterations in collagen solubility. We conclude that hyperglycemia-associated increases in immature crosslinks cannot acount for altered collagen solubility, although impaired maturation of such crosslinks may be partially responsible for the lathyrogenic effect of galactosemia.     

The presence of partially degraded collagen fragments in the resorbing granulation tissue in rats.

Insoluble collagen of granulation tissue produced by carrageenin injection was solubilized by pepsin treatment and purified. The pepsin-solubilized insoluble collagen contained partially degraded collagen fragments and the amounts of these small fragments of collagen were much greater in the resorbing granulation tissues than in the growing tissues, suggesting that these small fragment were formed in the course of resorption of granulation tissue, including collagen breakdown.     

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.     

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.     

Purification of an active gelatinase from collagen fiber fraction of granulation tissue in rats

Gelatinase was extracted at 60 degrees C from the collagen fiber-rich fraction of granulation tissue induced by carrageenin in rats. A large part of the extracted gelatinase was unbound to Zn-chelating Sepharose. The unbound gelatinase gave a single band corresponding to a molecular mass of 57 kDa on SDS-substrate PAGE, but showed a much higher molecular mass (greater than 200 kDa) on Sephadex G-150 gel filtration. In addition, that unbound fraction contained gelatin fragments was revealed by SDS-PAGE. When the unbound fraction of Zn-chelating Sepharose was incubated at 37 degrees C, the gelatin fragments disappeared and the apparent molecular mass of gelatinase in gel filtration decreased. This gelatin degradation of the unbound fraction was enhanced by treatment with a 4-aminophenylmercuric acetate (APMA). The results suggest that the gelatinase is bound to gelatin fragments in the unbound fraction. After the treatment with APMA, the gelatinase was purified to to homogeneity; the purified gelatinase gave a single band corresponding to a molecular mass of 57 or 67 kDa on SDS-PAGE under nonreducing or reducing conditions, respectively. The purified gelatinase is a metalloproteinase, and extensively degraded gelatin, but showed no proteolytic activity toward alpha-casein or types I and IV collagens. The results suggest that the 67-kDa active gelatinase is bound to collagen fibers and plays an important role in a rapid degradation of collagen fibers in granulation tissue.     

Exogenous non-crosslinked collagen enhances granulation tissue formation in dermal excision wounds in guinea pigs

Based on previous observations indicating a role for collagen peptides in eliciting a positive feedback for collagen biosynthesis, this study was initiated to elucidate the effect of non-crosslinked collagen on granulation tissue formation in dermal excision wounds. The wounds were treated with either non-crosslinked or crosslinked native collagen, or left untreated as controls. Granulation tissue was analyzed for collagen type I mRNA, for levels of interstitial collagen and for the number of blood vessels. The results indicated significant increases in procollagen type I mRNA, in interstitial collagen, in the number of blood vessels and in epithelial advance in the non-crosslinked collagen-treated wounds relative to the untreated controls. It is assumed that the presence of non-crosslinked collagen in a healing wound enhances both procollagen type I biosynthesis and the repair process of dermal wounds, due to the more readily released collagen peptides derived from this exogenous collagen dressing.     

Type I collagen messenger RNA levels in experimental granulation tissue and silicosis in rats

A complementary DNA (cDNA) clone was constructed for chick pro alpha 2(I) collagen mRNA. This and previously constructed cDNA clones for chick and human pro alpha 1(I) collagen mRNAs were used to measure levels of type I procollagen messenger RNAs in two experimental models: viscose cellulose sponge-induced experimental granulation tissue and silica-induced experimental lung fibrosis in rats. Both Northern RNA blot and RNA dot hybridizations were used to quantitate procollagen mRNAs during formation of granulation tissue. The period of rapid collagen synthesis was characterized by high levels of procollagen mRNAs, which were reduced when collagen production returned to a low basal level. The rate of collagen synthesis and the levels of procollagen mRNAs during the period of rapid reduction in collagen production did not, however, parallel with each other. This suggests that translational control mechanisms are important during this time in preventing overproduction of collagen. In silicotic lungs, the early stages of fibroblast activation follow a similar path but appear faster. At a later stage, however, the RNA levels increase again and permit collagen synthesis to continue at a high rate, resulting in massive collagen accumulation.     

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.     

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.     

The influence of biosynthetic human growth hormone on biomechanical properties and collagen formation in granulation tissue

Biomechanical properties and collagen formation in the granulation tissue of cellulose sponges, implanted subcutaneously in male rats for 7, 10 and 16 days, were tested after treatment with biosynthetic human growth hormone given subcutaneously in a dose of 0.5 mg/kg body weight/day. At each implantation period, one group started hormone treatment at the day of implantation and another group started hormone treatment 7 days prior to implantation. After 7 days of implantation, increases in maximum stress (36 per cent), relative failure energy (48 per cent) and strain at maximum stress (25 per cent) were found when treatment was started 7 days prior to implantation. After 10 days of implantation an increase in relative failure energy (60 per cent) was found when treatment was started 7 days prior to implantation. No differences were found after 7 and 10 days of implantation when treatment was started at the day of implantation. After 16 days of implantation, no influence on mechanical strength was found in any of the hormone treated groups. The collagen deposition after 7, 10 and 16 days did not differ in any of the hormone treated groups compared to controls.     

Glycoproteins from experimental granulation tissue and their effects on collagen synthesis in embryonic chick tendon cells

Buffer-soluble and pronase-liberated glycoproteins from experimental granulation tissue were fractionated by gel filtration and DEAE-cellulose chromatography. The age of the granuloma was reflected in the gel filtration pattern. Two glycoproteins were isolated, purified to homogeneity and analyzed for their carbohydrate and amino acid compositions.The collagen synthesis in embryonic chick tendon cells was measured in the presence of these fractions, which were found to be inhibiting even at 10^?6 M. These glycoproteins may be significant in the feedback regulation of the development of granulation tissue and fibrosis.     

Lysyl oxidase dependent synthesis of a collagen cross-link containing histidine

To date, collagen appears unique among proteins in that it contains histidine in certain of its cross-links. Synthesis of histidine containing collagen cross-links was studied in vitro with lathyritic L-¹⁴C histidine or L-¹⁴C lysine labelled bone collagen fibrils and purified lysyl oxidase. Synthesis of the tetrafunctional cross-link, dehydrohistidinohydroxymerodesmosine occurred with lysyl oxidase and was inhibited by β-aminopropionitrile. Synthesis began after a lag period of sixteen hours and then proceeded linearly for four days. These data indicate that enzyme dependent synthesis of dehydrohistidinohydroxymerodesmosine occurs in vitro in a biochemically defined system. Biosynthesis in vivo might occur under similar conditions.     

交联对胶原降解速率的影响

目的：研究交联反应对胶原降解速率的影响。方法：以热交联(DHT)、1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)化学交联以及EDC／DHT交联三种方法对胶原海绵材料进行处理,并测定材料在处理前后的降解速率。结果：各种交联反应均不同程度地提高了胶原的生物稳定性．降低了胶原的降解速率。     

Mechanochemical melting of collagen fibers. 3. Cooperativity induced by crosslinking

Mechanical inciting experiments performed on collagen fibers were used to study the effect of crosslinking on contraction kinetics. With the aid of a two-state model it is shown that the degree of cooperativity of this dematuration process is increasing with the degree of crosslinking.     

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.