The effect of mechanical force on mRNA levels of collagenase, collagen type I, and tissue inhibitors of metalloproteinases in gingivae of dogs.

Orthodontic force causes an injury to and subsequent degradation of the attachment apparatus, thus leading to the transposition of the tooth. The gingiva, however, is compressed and sometimes becomes hypertrophic with tooth movement and often shrinks after treatment. To study the effect of force on the gingiva, we applied orthodontic force in dogs and analyzed gingival tissues 1, 2, 3, 7, 14, and 28 days later as well as after removing the force. The effect of force on mRNA levels of collagen type I (col-I), matrix metalloproteinase-1 (MMP- 1), and tissue inhibitors 1 and 2 (TIMPs) genes was analyzed by RT-PCR, and MMP-1 activity was determined by zymography. The results showed that force significantly increased both the mRNA levels of MMP-1 and its interstitial activity. After the removal of force, MMP-1 gene expression was significantly decreased. The results could partly explain the clinically observed shrinkage and adaptation of the gingiva during tooth movement.

Retinoic acid enhances the effect of collagen on bone union, following induced non-union defect in guinea pig ulna.

OBJECTIVE AND DESIGN: The aim of the present study is to evaluate the involvement of retinoic acid and collagen in wound healing, by combining them in a therapeutic modality for treating a non-union bone defect in a guinea-pig ulnar-bone model. METHODS: a 4-mm disc was excised from the guinea-pig's ulnar-bone, and the space formed between the two ulnar fragments was filled with either collagen solution, retinoic acid solution or a combination of both. The guinea-pigs were sacrificed 2 or 6 weeks later, and the defected ulnar bones were studied by X-ray, by histology and by computerized histomorphometry. RESULTS: After 6 weeks, the long bone area fraction within the histological sections of the bone, was increased after treatment with this mixture by 180%, as compared to the untreated controls. The cartilage area in those sections was decreased by 44% after the combined treatment, as compared to increases of 133% and 182% following treatments with collagen alone. CONCLUSION: These findings demonstrate that addition of 500 IU of retinoic acid to collagen at a site of a bone defect, is superior to either agent in enhancing regeneration of new bone, achieving union across the defect and leading to its complete repair.

Gingival response to orthodontic force.

Orthodontic tooth movement is brought about by prolonged application of force on the attachment apparatus. This results in cellular and extracellular changes within the periodontium. As shown in numerous studies, tooth movement is achieved after the remodeling of alveolar bone and the response of the periodontal ligament to the mechanical force. Although gingival changes have also been found to be an important factor in the overall response, the effect of orthodontic tooth movement on the gingiva has been investigated to a lesser extent. Unlike bone and periodontal ligament, which regain their original structure after removal of force, the gingival tissue does not regain its pretreatment structure, a fact on which a hypothesis has been made that tooth relapse after removal of retention may be associated with changes in the gingiva. The present review summarizes available data on the effect of orthodontic force on collagen, elastin, and collagenase in the gingiva and its relevance to understanding the mechanism of tooth relapse.

The effect of centrifugal force on the transcription levels of collagen type I and collagenase in cultured canine gingival fibroblasts.

An orthodontically treated tooth is often destabilized in its newly corrected location and relapses towards its original position. Hitherto, the explanation for this phenomenon was that orthodontic force brings about "stretching" of gingival collagen fiber, which "pull back" the tooth towards its pretreatment position. A previous ultrastructural study showed that after force application the gingival collagen fibres were torn, laterally spaced and of increased diameter. Therefore, they could not "pull back" the tooth and be the cause of the relapse. In the present study, in order to find a more plausible explanation at the molecular level, the effect of pressure on the gene transcription of collagen type I and tissue collagenase was examined by semiquantitative, reverse transcriptase-polymerase chain reaction assay. Attached buccal gingiva was excised from anaesthetized dogs and gingival fibroblasts were grown in culture. Following application of pressure (0.167 kg/l g cell mass), the transcription of collagen type I was increased while that of tissue collagenase was decreased. These results corroborate the ultrastructural in vivo findings that orthodontic force is associated with larger amounts of collagen type I in the gingiva.

Reduction in basic fibroblast growth factor mediated angiogenesis in vivo by linomide.

Linomide (N-phenylmethyl-1,2-dihydro-4-hydroxyl-1-methyl-2-oxoquinoline-3-carboxa mide) is a novel immunomodulator with a potent anti-tumoral activity. This study was undertaken to test the effect of Linomide on basic fibroblast growth factor (bFGF) induced angiogenesis in vivo, which manifests itself in an increased number of blood vessels per unit of cell infiltrated area. Subcutaneously implanted polyvinyl alcohol sponges (PVS) in guinea pigs were used as a model system to quantitate angiogenesis in vivo. Oral treatment with Linomide was able to reduce significantly the bFGF induced blood vessel growth and proliferation within the implanted PVS, relative to untreated controls. In addition, Linomide significantly reduced the bFGF mediated augmentation of protein and collagen content in the implanted PVS, indicating an inhibition in the deposition of extracellular matrix (ECM). We conclude that the potent inhibition of bFGF induced angiogenesis by Linomide in vivo in addition to immunomodulatory effects may have potentially important clinical applications.

Thalidomide reduces vascular density in granulation tissue of subcutaneously implanted polyvinyl alcohol sponges in guinea pigs.

The efficacy of thalidomide in the treatment of erythema nodosum leprosum is a well established fact; there is also accumulating evidence of its therapeutic value in a number of other inflammatory and immune-mediated conditions. In addition, thalidomide has been shown to be an inhibitor of angiogenesis induced by basic fibroblast growth factor (bFGF). Nevertheless, its mechanism of action remains speculative. Using guinea pigs, orally administered thalidomide significantly enhanced the response of multinucleated foreign body giant cells (p<0.05) in subcutaneously implanted polyvinyl alcohol sponges. Furthermore, the drug exerted a dual effect in that it reduced vascular density (p<0.05), which was not abolished by recombinant human bFGF, and at the same time amplified the granulomatous response with and without bFGF (p<0.05 and p<0.01, respectively). The results of our experiments represent a further step toward understanding the mechanism of action of thalidomide, with implications for its potential use in wound healing and scar formation as well as in the control of tumorigenesis.

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.

Lack of influence of cyclosporin A on levels of gingival procollagen types I and III mRNAs in rats of different ages.

Previous studies showed that gingival overgrowth following cyclosporin A (CsA) administration is not associated with an increase in interstitial collagen. It also was shown that CsA causes a significant decrease in collagen content within the gingival stroma. In order to determine whether this decrease is caused by down-regulation of collagen mRNA, the procollagen mRNA level in gingiva of young and old rats was measured correlated with the ratio of interstitial collagen to DNA in these regions. Hybridization of 32P-labelled cDNA probes for procollagen types I and III with total RNA extracted from the molar gingiva showed that administration of Csa did not change the steady-state levels of mRNAs for both procollagens in the gingiva of either young or old rats. The ratio of gingival interstitial collagen to DNA was significantly reduced in the CsA-treated animals (4.2 +/- 0.85) relative to the controls (7.8 +/- 1.6). It is concluded that the reduction in interstitial collagen following CsA treatment is not age-related, and is most probably caused by increased degradation rather by decreased biosynthesis.

Gingival response to a new multipurpose dental adhesive: a histologic study in dogs.

The biocompatibility of cementing materials is a prerequisite for any dental procedure. In this study, the tolerance of gingival tissue to an advanced fourth-generation dental adhesive (High-Q-Bond) was tested in dogs. The results from High-Q-Bond adhesive were compared with those obtained from Superbond C&B adhesive. Buccal class V subgingival cavities were restored with either High-Q-Bond or Superbond C&B adhesive Untreated teeth served as normal intact controls. The teeth with the attached buccal gingivae were extracted and processed for histologic examination. The histologic observations showed an inflammatory response in the gingiva of the Superbond C&B adhesive-treated teeth, whereas the High-Q-Bond fillings exhibited no noticeable adverse effect on the gingival tissue.

Reduction in pulmonary fibrosis in vivo by halofuginone.

Pulmonary fibrosis is a disorder causing a high mortality rate for which therapeutic options are limited. Therefore, the effect of halofuginone, a novel inhibitor of collagen type I synthesis, on bleomycin-induced pulmonary fibrosis was studied in rats. Pulmonary fibrosis was induced by intraperitoneal injections of bleomycin for seven consecutive days, and halofuginone was administered intraperitoneally every second day during the entire experimental period of 42 d. Collagen determination in the lungs and the examination of histologic sections showed that halofuginone significantly reduced fibrosis relative to the untreated control rats. We conclude that halofuginone is a potent in vivo inhibitor of bleomycin-induced pulmonary fibrosis, and that it may potentially be used as a novel therapeutic agent for the treatment of this dysfunction.

The response of supraalveolar gingival collagen to orthodontic rotation movement in dogs.

An orthodontically rotated tooth relapses toward its pretreatment position. Explanations for this phenomenon have been given after light microscopic studies, according to which it had been assumed that stretched supraalveolar gingival fibers pulled back the tooth and brought about relaxation of the stretched fibers. The rotational relapse, however, can be prevented by supraalveolar fiberotomy of the gingiva around the tooth. This investigation was initiated to reevaluate the validity of the hitherto assumed causes for the relapse, by obtaining ultrastructural data on the response of collagen fibers after orthodontic intervention. Lateral maxillary incisors in the dog were rotated with bonded fixed appliances. The teeth were divided into groups according to different orthodontic procedures. Scanning and transmission electron microscopic analyses were performed on gingival samples after proper processing. Analyses of the untreated control samples showed well-organized, parallel, and densely packed thick bundles of collagen fibers, interconnected with thin fibers. After rotation-followed-by-retention, the gingival fibers were torn, ripped, disorganized, and laterally spaced and of increased diameter. Thus it was concluded that all these patterns are incompatible with stretching. Also, an increased number of elastic fibers were seen in proximity to the torn collagen fibers. After gingival fiberotomy, most fibers resumed the appearance of the organized pattern of large fiber bundles similar to those seen in the controls.

Levels of cytokines and collagen type I and type III as a function of age in human gingivitis.

The objective of this study was to examine the age-dependent relationships between levels of inflammatory cytokines and collagen in human gingival inflammation. The gingival biopsies were obtained from 142 patients, divided into the following age groups: 6 to 14 years (prepubertal children); 18 to 35 years (young adults); 36 to 54 years (mature adults); and 55 years or above. The patients were also divided according to the severity of gingivitis. The tissues were analyzed for the contents of the inflammatory cytokines interleukin (IL)-1 beta, IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) using specific ELISA kits, and interstitial collagen type I and type III using the ELISA method and specific antibodies. We found that in young adults, levels of IL-1 beta and IL-6 were significantly higher in inflamed than in non-inflamed gingiva. Total collagen in the young adults, however, was lower in inflamed than in non-inflamed gingiva. There was no significant difference in the levels of either IL-8 or TNF-alpha between inflamed and non-inflamed gingiva independent of age. No difference in the level of collagen type I between the inflamed and non-inflamed gingiva was found in any age groups. The level of collagen type III was lower in inflamed than in non-inflamed gingiva in both children and > or = 55 year group. The results indicate a disparity in the effect of age on the levels of cytokines and of collagen type I and type III in both clinically normal and inflamed gingiva.

Topically applied halofuginone, an inhibitor of collagen type I transcription, reduces peritendinous fibrous adhesions following surgery.

To test in vivo the effect of previously observed inhibition of collagen type I transcription by the plant alkaloid Halofuginone, deep flexor tendons of 12 chickens were severed and sutured, and Halofuginone was applied topically at the site of surgery. Intact tendons, and severed but untreated tendons served as controls. The effect of the treatment was assessed by histological, biochemical, and biomechanical examinations of the operated and intact tendons three weeks after surgery. The results indicated an almost complete absence of fibrous peritendinous adhesions in the histologic sections of the Halofuginone treated tendons. There was a statistically significant decrease in collagen contents of and in both force and energy required to pull out the Halofuginone treated tendons from their sheath, relative to the untreated controls. Halofuginone had no effect on the cellularity of the healing tissue. We conclude that Halofuginone is a potent inhibitor of fibrous peritendinous adhesions with potentially important clinical applications.

Hereditary leukoencephalopathy and palmoplantar keratoderma: a new disorder with increased skin collagen content.

We report a new neurocutaneous syndrome of apparent autosomal recessive inheritance consisting of early-childhood-onset palmoplantar keratoderma followed in adulthood by progressive tetrapyramidal syndrome and cognitive impairment. Of the four affected siblings, two were available for evaluation. Investigation disclosed cerebral white-matter involvement on MRI and arylsulfatase A pseudodeficiency carrier state, which was also identified in clinically unaffected family members. Since skin biopsies showed dermal connective tissue abnormalities, we studied collagens I, III, and VI biosynthesis. Northern blotting of RNA extracted from cultured skin fibroblasts revealed an increased steady-state messenger RNA (mRNA) level of alpha 1(VI) collagen, whereas no differences were detected for pro alpha 1(I), pro alpha 1(III), and tropoelastin mRNAs. The skin content of collagen and total protein was higher in the patients than in controls. We suggest that an extracellular matrix abnormality may be involved in the pathogenesis of this disorder.

Of/Lb collagen promotes chemoinvasion of breast-cancer cells and directs epithelial-cell migration into granulation-tissue of experimental dermal wounds.

OF/LB collagen is a recently described once-fetal form of collagen, with laminin-binding properties, composed of three alpha(1)(I)-sized chains, one of which displaying an unusually acidic pI. This collagen appears able to direct the migration of breast cancer cells through Matrigel, and of injury-activated epithelial cells into the underlying granulation stromal tissue. The effect exerted by OF/LB collagen in vitro appears preferentially linked to its acidic chain. The data reported strongly support the hypothesis that the presence and accumulation of OF/LB collagen in cancer may play a fundamental role in the invasive growth.

Topological differences in the expression of collagen type I and collagen type III mRNAs in the rat gingiva.

Collagen mRNA levels in the gingival cells of molars and incisors in rats were measured and correlated with the ratio of interstitial collagen to DNA in these regions. Hybridization of 32P-labeled specific cDNA probes for collagen types I and III with total RNA isolated from gingival tissue of rat molars and incisors showed that the steady-state levels of mRNAs of type I was significantly higher in the molars than in the incisors (molars/incisors = 2.12 +/- 0.12, P < 0.004). However, the ratio of interstitial collagen to DNA in the gingiva of the molars was significantly lower than that found in the incisors (collagen/DNA = 4.13 +/- 0.90 and 12.89 +/- 1.24 respectively, P < 0.001). It is suggested that the difference between the mRNA levels and those of interstitial collagen may reflect an intrinsic characteristic presumably associated with the different modes of mastication between molars and incisors of the rat.

Healing process of laser-welded intestinal anastomosis.

Intestinal welding by means of low-power laser has been reported as an efficient method for intestinal anastomosis. We designed an experimental model in rats to investigate collagen and DNA concentrations in CO2 laser-welded anastomoses as compared with those in sutured anastomoses on the 4th, 7th, and 10th postoperative days. The results revealed that DNA, total collagen, and insoluble collagen concentrations were significantly lower in the lased anastomoses than in the sutured anastomoses on the 4th postanastomotic day. On the 7th and 10th postanastomotic days, collagen concentrations increased in the laser-treated group attaining significantly higher levels than in the sutured group at that time. These findings are compatible with other studies demonstrating that laser-welded intestinal anastomoses are more prone to dehiscence during the first 4 postanastomotic days, but become at least as effective as the sutured ones with passage of time.

Type I and type III collagen mRNA levels in kidney regions of old and young rats.

Interstitial fibrosis is a common feature of renal aging. The steady-state levels of type I and type III collagen mRNAs as well as DNA, protein and collagen deposition were investigated in the cortex, inner and outer medulla of aged (22 months old) rats in comparison to young (5 months old) controls. Our data show that the cortex and outer medulla of old rats expressed significantly higher percentage of type I collagen mRNA compared to the respective regions in the young rat kidneys. Moreover, within the group of the old rats, the cortex expressed significantly higher percentage of type I collagen mRNA compared to the inner medulla whereas in the group of the young rats the expression was similar in all kidney regions. The ratio of extracellular collagen to DNA was significantly higher in the cortex, inner and outer medulla of old compared to young rats. The ratio of collagen to total protein, although showing a similar age-related difference, attained statistical significance in the cortex only. Thus, the present study indicates a close relationship between the expression of the mRNA for type I collagen, the major structural constituent of fibrotic tissues, and the deposition of collagen in both the cortex and outer medulla of the kidney. Moreover, the clear differences found between old and young rat kidneys can serve as markers for renal aging and might explain at least some of the kidney impairments caused by fibrosis during senescence.

Bone marrow stromal elements in murine leukemia: decreased CSF-producing fibroblasts and normal IL-1 expression by macrophages.

A study of bone marrow stromal elements in murine acute myeloid leukemia (AML) was carried out. Our previous studies had indicated marrow stromal deficiency in murine AML. In the current investigation, separate stromal cells were cultured and the results obtained have shown that, while marrow stromal macrophages are normal in leukemia and express adequate amounts of IL-1, the fibroblasts are markedly reduced. However, if sufficient fibroblasts are pooled in vitro, they produce adequate amounts of CSF. Test of TNF alpha in leukemic cells CM, as possible cause of marrow stromal inhibition in leukemia, had not disclosed this cytokine. Further, it was observed that total body lethal irradiation of leukemic mice aggravates the stromal deficiency, confirming results of our previous investigations. It is concluded that bone marrow stromal deficiency in murine AML is due to decreased fibroblasts and, implicitly, reduced CSF production.

Prolonged systemic administration of cyclosporin A affects gingival epithelium.

Gingival biopsies were obtained from 12 patients suffering from Behcet's disease who were treated with Cyclosporin A (CsA) for up to 20 months. Preparations were made for examination with both light and scanning electron microscopy (SEM). Along with known changes in the gingival epithelial structure observed following CsA treatment, we also found unusual clusters of needle-like crystallites embedded in the epithelium, mostly at the base of the acanthotic projections. Toluidin blue staining revealed increased numbers of both intact and degranulated mast cells in the attached epithelium. It is concluded that CsA affects the gingival epithelium and that the clinically observed enlargement of gingival tissue following prolonged treatment with CsA is due primarily to CsA-epithelial interaction.