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Biomedical subjects

R F Diegelmann

Publications and source records attributed to R F Diegelmann.

At least 37 records · Page 2Linked to original sources
PubMed1995

Corticosteroids increase procollagen gene expression, synthesis, and secretion by human intestinal smooth muscle cells.

BACKGROUND & AIMS: Collagen synthesis by smooth muscle cells plays an important role in intestinal fibrosis. Corticosteroids inhibit collagen synthesis in fibroblasts. The aim of this study was to examine the effect of corticosteroids on the expression of collagen by human intestinal smooth muscle (HISM) cells in vitro. METHODS: Collagen synthesis was determined by the sensitivity of radiolabeled protein to collagenase. Secretion was determined by polyacrylamide gel electrophoresis of radiolabeled procollagen in the medium. Procollagen messenger RNA was determined by Northern blot analysis. RESULTS: Collagen synthesis by confluent HISM cells was not affected by corticosteroids at 10(-10) to 10(-5) mol/L but, in subconfluent cultures, was nonspecifically increased 50% at 10(-5) mol/L. Procollagen secretion was nonspecifically increased 60% at 10(-6) mol/L dexamethasone without any effect on the type III/I ratio. Procollagen I and III messenger RNA levels responded in a biphasic manner: a 45%-65% increase at 10(-10) mol/L and a 15% and 30% decrease at 10(-8) and 10(-6) mol/L. In fibroblasts, collagen synthesis was inhibited 85% by dexamethasone, procollagen secretion was decreased 70%, the type III/I ratio decreased from 70:1 to 18:1, and procollagen messenger RNA was inhibited 25% and 60% (types I and III). CONCLUSIONS: Collagen expression by HISM cells is refractory to corticosteroids and, at certain concentrations, is augmented.

Blotting, Northern↗
PubMed1995

Hyaluronic acid metabolism in keloid fibroblasts.

Hyaluronic acid (HA), an important component of the tissue extracellular matrix, is a ubiquitous glycosaminoglycan (GAG) that forms a pericellular coat on the surface of cells. It has been speculated that this pericellular HA boundary may localize cytokines, such as transforming growth factor-beta 1, which is known to stimulate collagen production. The purpose of this study was to examine the role of HA and its cell surface receptor (CD44), an active participant in HA degradation, as they relate to keloid formation. Dermal excisions from both normal patients (n = 13) and keloid patients (n = 13) were analyzed for HA content using an alcian blue staining technique. Fibroblast cell cultures were used to quantitate HA synthesis and CD44 receptor density. Histological analyses showed a greater HA content in keloid tissue compared with normal dermal tissue. In agreement with this observation, keloid fibroblasts were found to synthesize significantly more HA than normal dermal fibroblasts (2469 +/- 483 cpm versus 1122 +/- 256 cpm, P = .02). Treatment of keloid fibroblasts with triamcinolone acetonide reduced the level of HA synthesis to that of normal fibroblasts (1560 +/- 477 cpm versus 1293 +/- 264 cpm, P = .6). However, there was no significant difference in HA receptor density on keloid cells compared with normal skin fibroblasts. Therefore, the increased HA deposits found in keloids are attributable to increased synthesis rather than to decreased degradation mediated by the CD44 receptor.

Adolescent↗
PubMed1995

Aggregatory characteristics and expression of the collagen adhesion receptor in fetal porcine platelets.

Fetal wound healing differs significantly from that of the adult by its rapidity, the paucity of an inflammatory response, and the lack of scarring. In the adult, activation and aggregation of platelets at the site of injury result in the release of cytokines and inflammatory mediators that stimulate wound healing by initiating an acute inflammatory response. The aim of this study was to characterize the activity of midtrimester (day 60) and third-trimester (day 95) fetal porcine platelets (full term, 114 days) compared with that of adults in an attempt to understand the lack of inflammation in fetal wounds. The aggregatory capabilities of adult and fetal platelets were analyzed after exposure to adenosine diphosphate (ADP) concentrations of 10 mumol/L and 40 mumol/L concentrations, collagen of 0.19 mg/mL, and arachidonic acid of 0.5 mg/mL. Expression of the alpha 2 subunit of the collagen receptor (alpha 2 beta 1) was evaluated by Western blot analysis. The aggregation of day-60 fetal platelets when exposed to ADP (10 mumol/L and 40mumol/L) and collagen was significantly lower than that of the adult. The aggregation of third-trimester platelets to 10 mumol/L of ADP was similar to that of the adult and significantly greater than that of midtrimester fetuses at higher concentrations (40 mumol/L). Both fetal groups responded suboptimally to collagen, and the response was significantly less than that of adults. In contrast, arachidonic acid caused rapid and complete aggregation of both fetal platelet groups, suggesting that both mid- and late-trimester fetal platelets possessed the ability to fully aggregate with the appropriate stimulus. The different aggregatory responses to collagen could not be explained by differences in collagen receptor expression, because these were found to be similar in adults and midtrimester fetuses. It is concluded that although fetal platelets have the potential to aggregate effectively, they aggregate poorly to collagen and exhibit improved aggregation to ADP with increasing maturity. There is a transition to "adultlike" platelet aggregatory activity in the third trimester, which correlates with the period of transition to adultlike wound healing in utero. Similar expression of the alpha 2 beta 1 collagen receptor in the fetus and adult cannot explain the differences observed in their responses to collagen.

Animals↗
PubMed1995

Topical application of epidermal growth factor onto partial-thickness wounds in human volunteers does not enhance reepithelialization.

Recent reports have suggested that human epidermal growth factor will accelerate the healing of corneal wounds and donor sites of burned patients undergoing split-thickness skin grafts. In one report using human epidermal growth factor in a silver sulfadiazine cream, these data were gathered from patients of various ages who had various depths of donor sites and degrees of burn. Therefore, despite the fact that these data were prospective, randomized, and double-blinded, we questioned the validity of the study. To test the hypothesis that topical application of human epidermal growth factor will enhance the healing of partial-thickness wounds, we utilized healthy volunteers (n = 17) and created bilateral split-thickness skin wounds (4 cm2) of the same depth (0.014 in) on each flank and then studied time until total epithelialization occurred. One side was treated with silver sulfadiazine as a control and the opposite side with silver sulfadiazine and human epidermal growth factor. There was no significant difference in the healing times between the human epidermal growth factor-treated sites and the silver sulfadiazine cream controls.

Administration, Topical↗
PubMed1994

Platelet-derived growth factor induces fetal wound fibrosis.

The fetal response to cutaneous injury differs markedly from that of the adult, proceeding with only minimal inflammation, minimal fibroblast proliferation, and only essential collagen deposition. Although the sequence of events in adult wound healing is well defined and thought to be controlled in part by potent polypeptide cytokines, relatively sparse information exists regarding growth factor involvement in fetal wound repair. Thus, the authors sought to examine the effect of platelet-derived growth factor (PDGF), a putative adult wound healing regulator, on the cellular and extracellular matrix events at a fetal wound site. SILASTIC wound implants containing 0, 1.0, 5.0, or 10.0 ng of human PDGF were placed subcutaneously on the backs of 24-day-gestation fetal rabbits (full term, 31 days) and then harvested after either 1, 3, or 5 days in utero. The specimens underwent standard histological processing and were evaluated in a blinded fashion. Compared with controls, PDGF-treated implants had a marked increase in acute inflammation, fibroblast recruitment, and collagen and hyaluronic acid deposition; these differences appeared to be largely time- and PDGF dose-dependent. Thus, the fetal system is responsive to an adult wound healing mediator, and these data suggest that fetal repair proceeds in the absence of PDGF.

Animals↗
PubMed1994

Biology of fetal wound healing: hyaluronate receptor expression in fetal fibroblasts.

Fetal tissue repair is rapid, relatively scarless, and proceeds in an environment rich in hyaluronic acid. Understanding the interaction of hyaluronic acid (HA) with the reparative cells may provide important insight into the remarkable process of fetal wound healing. Therefore, the purpose of this study was to characterize the HA receptor (a member of the CD44 family of cell surface glycoproteins) and its density on the cell surface of fetal fibroblasts. HA receptor expression on both adult and fetal fibroblasts was first studied by Western blot analysis. Autoradiographs of the blots were assessed by densitometry to quantitate the relative amounts of the HA receptor. It appears that the HA receptor expressed on both adult and fetal rabbit dermal fibroblasts is a 56-kd protein. After normalizing for total protein concentration using the Bradford protein assay, the fetal HA receptor density was found to be approximately four-fold greater than that of the adult. The same adult and fetal fibroblasts were then studied by flow cytometry, using a fluorescence-activated cell sorter (FACS). Relative fluorescence was representative of HA receptor density. Corroborating the authors' earlier result with the Western blot analysis, the FACS analysis showed that the fetal fibroblasts had 2.5 times the fluorescence of the adult cells. It is concluded that, in comparison to adult fibroblasts, fetal fibroblasts have an increased density of cell-surface HA receptor.

Animals↗
PubMed1994

The role of inflammatory cytokines in wound healing: accelerated healing in endotoxin-resistant mice.

UNLABELLED: The role of cytokines in normal wound healing remains poorly defined. In vitro, tumor necrosis factor alpha (TNF-alpha) decreases collagen accumulation, perhaps by increasing collagenase activity. The current study was undertaken to test the hypothesis that cytokines impair wound healing and collagen production. Wounds in C3H/HeJ (J) mice, which are characterized by a genetic defect in macrophage production of TNF and other cytokines in response to endotoxin, were compared with wounds in normal endotoxin-sensitive C3H/HeN (N) mice. METHODS: TNF (by murine ELISA) and collagenolytic activity (CA by in vivo labelled collagen fibril degradation assay) were measured in wound fluid from silicone reservoirs on post-wounding days 1, 3, and 5 (n = 5 per group). Hydroxyproline (HOP, nmol/mg sponge) incorporation (by HPLC) in polyvinylacohol sponges and breaking strength (BS, as determined by tensiometry) in linear wounds were assessed on days 5, 7, 10, and 14 (n = 5 per group). The data demonstrate significantly increased BS at 5 and 7 days in endotoxin-resistant J mice compared with that in endotoxin-sensitive N mice. An early, significant reduction in TNF production in J mice corresponded with a significant increase in CA on day 1, increased collagen production at day 7, and increased procollagen gene transcription at early time points. Conclusion. The reduced production of inflammatory cytokines including TNF in the wound fluid of J mice corresponded with an early improvement in wound tensile strength. An accelerated accumulation of collagen in the wounds of J mice, perhaps resulting from a significant decrease in collagenolytic activity or increased collagen production, are potential mechanisms. The data suggest that cytokines produced in normal healing of clean wounds may contribute to a delay in increased tensile strength.

Animals↗
PubMed1994

Morphological and immunochemical differences between keloid and hypertrophic scar.

There are two types of excessive scarring, keloid and hypertrophic scar. Contrary to hypertrophic scars, keloids do not regress with time, are difficult to revise surgically, and do not provoke scar contractures. These two lesions require different therapeutic approaches but are often confused because of an apparent lack of morphological differences. We have investigated the collagen organization and the possible presence of alpha-smooth muscle (SM) actin-expressing myofibroblasts in these conditions. Keloids contain large, thick collagen fibers composed of numerous fibrils closely packed together. In contrast hypertrophic scars exhibit modular structures in which fibroblastic cells, small vessels, and fine, randomly organized collagen fibers are present. We confirm that such nodular structures are always present in hypertrophic scar and rarely in keloid. Furthermore, only nodules of hypertrophic scars contain alpha-SM actin-expressing myofibroblasts. Electron microscopic examination supports the above-mentioned differences in collagen organization and in fibroblastic features and shows the presence of an amorphous extracellular material surrounding fibroblastic cells in keloid. The presence in hypertrophic scar myofibroblasts of alpha-SM actin, the actin isoform typical of vascular SM cells, may represent an important element in the pathogenesis of contraction. Interestingly, when placed in culture fibroblasts from hypertrophic scars and keloid express similar amounts of alpha-SM actin, suggesting that local microenvironmental factors influence in vivo the expression of this protein. Thus several morphological and immunohistochemical differences exist between hypertrophic scar and keloid that are useful for the biological and pathological characterization of the two lesions.

Actins↗
PubMed1993

Biology of fetal repair: the presence of bacteria in fetal wounds induces an adult-like healing response.

The minimal acute inflammatory response to tissue injury is one of the most dramatic differences between fetal and adult wound healing. Considering the prominent role of inflammation in adult tissue repair, this study tested the hypothesis that the minimal fetal inflammatory response to tissue injury plays a central role in the "scarless" fetal repair process. Sponge implants were treated with lethally irradiated or live bacteria and placed subcutaneously in fetal rabbits to test the ability of the fetus to mount an acute inflammatory response to bacterial antigens present at the wound site and to analyze the effects of this inflammatory response on fetal fibroplasia and neovascularization. After harvest, these implants were examined histologically for inflammation, fibroblast infiltration, collagen deposition, and neovascularization, and collagen deposition was measured using hydroxyproline quantitation by high-performance liquid chromatography. Bacteria-treated implants showed dose-dependent acute inflammatory responses and significant increases in collagen deposition compared with control sponges. Implants containing live bacteria demonstrated maximal fibroplasia and neovascularization. These findings suggest that, despite neutropenia and immaturity of the fetal immune system, the fetus is capable of mounting an acute inflammatory response to avirulent bacteria present at the wound site. Fetal inflammatory cells which respond to this bacterial stimulus appear capable of initiating an adult-like healing response. Thus, by failing to provide a bacterial stimulus for leukocyte recruitment at the site of tissue injury, the sterile fetal environment appears to play a role in effecting "scarless" fetal wound healing.

Acute-Phase Reaction↗
PubMed1993

Hyaluronic acid modulates proliferation, collagen and protein synthesis of cultured fetal fibroblasts.

Fetal wound healing is characterized by minimal inflammation, mild fibroplasia and rapid, but organized collagen deposition such that scarring is not apparent. The matrices of fetal wounds differ greatly from adult wounds in that fetal wounds are persistently enriched with hyaluronic acid (HA). It has been shown that a reduction in fetal rabbit wound HA results in an adult-like healing response with increased fibroplasia and neovascularization. These observations suggest that HA can modulate cellular activity in fetal repair. Therefore, this study was designed to define the effect of HA on fetal fibroblast function. Fibroblasts from the skin of fetal rabbits were isolated and maintained in culture medium containing either no HA (controls), 1 microgram/ml, 10 micrograms/ml or 100 micrograms/ml of HA (n = 6 for each group). Fibroblast proliferation was quantitated by DNA content in each culture, and collagen and noncollagen protein synthesis were analyzed by incorporation of [3H] proline into collagenase-digestible and collagenase-nondigestible protein, respectively. At all concentrations tested, HA significantly inhibited fetal fibroblast proliferation (p < 0.02), but stimulated collagen (p < 0.002) and noncollagen protein (p < 0.005) synthesis. These findings provide further evidence that HA affects the function of fetal fibroblasts. Moreover, this study in conjunction with previous in utero findings suggests that HA may have a regulatory influence in scarless fetal healing by affecting cellular function during the repair process.

Animals↗
PubMed1993

In vitro and in vivo analysis of the inability of fetal rabbit wounds to contract.

Fetal rabbit wounds that are sutured show excellent repair without obvious scarring. In contrast, an unsutured wound in a rabbit fetus does not close, and it appears that the process of wound contraction does not occur. Experiments were carried out to illustrate the mechanisms responsible for the noncontraction of open fetal rabbit wounds. Results showed that the lack of wound contraction was not an artifact caused by rapid fetal growth. With regard to the ability of cultured fetal fibroblasts to show cytoplasmic muscle-induced cell contraction, we found that, in cultured fetal fibroblasts, cell contraction was induced by adenosine triphosphate. Contractile abilities of fetal-derived fibroblasts were equivalent to those of adult-derived fibroblasts. The fetal fibroblasts also demonstrated the generation of superior contractile activity when examined in a fibroblast-populated collagen lattice model. Finally, the ability of amniotic fluid to alter wound contraction was addressed by means of the fibroblast-populated collagen lattice in vitro model. Increasing concentrations of amniotic fluid inhibited fetal fibroblast lattice contraction. Therefore, rabbit amniotic fluid contains an inhibitor that may be partially responsible for the noncontraction of fetal rabbit wounds in utero.

Journal Article↗
PubMed1992

Collagen production by human smooth muscle cells isolated during intestinal organogenesis.

The extracellular matrix influences organogenesis by modulating cell behavior. In humans, collagen is the major matrix constituent of the adult intestinal wall and is synthesized by smooth muscle cells. The objective of the current study was to examine collagen production by fetal human intestinal smooth muscle cells isolated during intestinal morphogenesis. Techniques were developed for the isolation and culture of human fetal intestinal smooth muscle cells. The cultured cells were confirmed as muscle by immunohistochemical stains for cytoskeletal filaments and documentation of contractile behavior. In culture, these cells stained for mesenchymal and muscle cytoskeletal proteins: vimentin, actin, and desmin, and did not stain for neural or epithelial markers. The muscle cells contracted in response to acetylcholine, in contrast to human fetal dermal fibroblasts which did not contract appreciably. Collagen production was assayed by the uptake of [3H]-proline into collagenase-digestible protein. Collagen production was greatest at 11 weeks gestation, the youngest age studied. By 20 weeks gestation, collagen production had decreased to adult levels. However, when compared to another matrix-producing fetal mesenchymal cell, the dermal fibroblast, intestinal smooth muscle cells produced twice as much collagen. Collagen types were determined by polyacrylamide slab gel electrophoresis. Smooth muscle cells predominantly produced types I and III collagen alpha chains. Therefore, collagen production is a significant function of human fetal intestinal smooth muscle cells, and probably plays a major role in the development of intestinal structure. The in vitro model presented here provides a means of studying the regulation of this collagen production throughout intestinal organogenesis.

Collagen↗
PubMed1992

Biology of fetal wound healing: collagen biosynthesis during dermal repair.

The rapid restoration of tissue integrity and breaking strength in healing fetal wounds is mainly a function of fetal wound collagen. In this study, the fetal and adult tissue responses to injury were characterized in terms of changes in collagen biosynthesis. Linear wounds and unwounded skin were incubated with radioactive proline, and collagen synthesis was measured as isotope incorporation into collagenase-sensitive protein. Likewise, noncollagen protein synthesis was represented by isotope incorporation into collagenase-resistant protein. Adult wounds demonstrated a preferential stimulation of collagen as compared with noncollagen protein synthesis after wounding. In contrast, both collagen and noncollagen protein synthesis were significantly elevated in the fetus during the first 5 days postwounding. Despite marked increases in fetal wound collagen synthesis above both unwounded fetal skin and adult wound levels, fetal wounds exhibited no evidence of excessive collagen deposition or scar formation after wounding. These findings suggest that the fetal response to tissue injury is a function of the distinctive qualities of fetal fibroblasts associated with the extracellular wound matrix and may involve rapid collagen turnover and degradation.

Animals↗
PubMed1992

In vivo degradation of fetal wound hyaluronic acid results in increased fibroplasia, collagen deposition, and neovascularization.

Fetal tissue repair occurs without acute inflammation, prominent fibroplasia, or marked neovascularization. The fetal wound extracellular matrix is rich in hyaluronic acid (HA), while collagen is deposited in an organized normal dermal pattern. In various biologic systems, including regeneration and development, the controlled accumulation and subsequent degradation of hyaluronic acid is associated with distinct cellular and matrix events. Therefore, it is hypothesized that the abundance of hyaluronic acid in fetal wounds may influence cellular and/or matrix events such that tissue repair is highly organized and adult-like scarring does not occur. To test this hypothesis, the hyaluronic acid content of fetal rabbit wounds was reduced by specific degradation with Streptomyces hyaluronidase. Control wounds were treated with either enzyme buffer (n = 12) or denatured enzyme solution (n = 8) and exhibited a normal fetal healing response with scattered peripheral fibroblasts, a matrix of hyaluronic acid, and no infiltrating collagen. In marked contrast, the hyaluronidase-treated wounds (n = 14) demonstrated increased fibroblast infiltration, collagen deposition, and capillary formation. A significant reduction in the hyaluronic acid content of the hyaluronidase-treated wounds was confirmed biochemically. Since the degradation of hyaluronic acid resulted in an altered healing response, this study demonstrates that hyaluronic acid affects the cellular and matrix events in fetal healing and may be partially responsible for the unique qualities of this regenerative repair process.

Animals↗
PubMed1992

Scarless wound healing in the mammalian fetus.

Fetal wound healing is a remarkable process that is fundamentally different than postnatal healing. Healing of primarily closed, linear wounds occurs rapidly and without scarring. In late gestational age fetal sheep, a transition to adult-like healing occurs as evidenced by minimal scar formation. Acute inflammation is not involved, fibroblast recruitment and proliferation is minimal, the matrix of the wounds is enriched with HA and collagen deposition is highly organized so that scarring is minimal or nonexistent. Open wounds in several species do not contract, an observation that may be because of an inhibitor of contraction in amniotic fluid. The underlying mechanisms that regulate fetal wound repair are currently not well understood. An altered supply or activity of growth factors may be instrumental. Fibroblasts may preferentially produce HA secondary to a factor present within the fetal system, and HA may influence cellular and matrix events within the wound. As greater knowledge of the biologic factors of scarless healing in the fetus is gained, applications to abnormal adult healing may be developed. As the age of fetal surgery is approaching, it is essential to understand the injury response in these new patients.

Adult↗
PubMed1991

Hyaluronic acid is a major component of the matrix of fetal rabbit skin and wounds: implications for healing by regeneration.

The extracellular matrix (ECM) of fetal rabbit wounds contains an abundance of the glycosaminoglycan hyaluronic acid (HA) but is devoid of excessive collagen. Thus, fetal wounds heal without scarring, such that tissue repair grossly resembles regeneration. To obtain further insight into the process of fetal wound healing, the ECM of normal fetal rabbit skin was analyzed, thus providing a comparative endpoint for the ECM of healing fetal wounds. Similarities between the matrices would support the theory of healing by regeneration. The glycosaminoglycan (GAG) component of fetal rabbit skin from 24- and 29-day gestational age fetuses was extracted and then quantitated using an alcian blue binding assay. The extracted GAG was characterized by cellulose acetate electrophoresis and HA was identified by its selective digestion by Streptomyces hyaluronidase. The mean GAG content, measured as ng GAG per mg dry weight skin, was 260 +/- 200 for the 24-day group (n = 28) and 280 +/- 220 for the 29-day group (n = 26). The only GAG identified at both times of gestation was HA. This study has demonstrated that HA is the predominant GAG present in fetal rabbit skin and its quantity is stable during the period studied late in gestation. A major component of the ECM of both wounded and normal fetal skin is HA, indicating a close compositional similarity. These observations provide biochemical support for the hypothesis that the reparative process of injured tissue in the fetal rabbit proceeds in an attempt to reconstitute normality, i.e. regeneration.

Animals↗
PubMed1991

Analysis of skin grafting techniques in the fetal rabbit.

The experimental model reported here was developed initially to examine the possibility of in utero coverage of congenital soft tissue defects using several types of reconstructive techniques. To pursue this, full-thickness skin grafts, pedicle flaps, and skin "islands" were fashioned on the backs of fetal rabbits; equivalent adult control wounds were also created. While all pedicle flaps and skin islands remained viable, none of the full-thickness grafts survived in the fetus. All adult control flaps, skin islands, and skin grafts were viable. Angiogenesis is crucial to full-thickness skin graft survival. These observations suggest that the death of full-thickness fetal skin grafts may be related to a failure of neovascularization in the graft bed. Further analysis using this model may help elucidate the factors involved in fetal angiogenesis. Additionally, this model may permit testing of putative angiogenic factors applied under a full-thickness skin graft; graft survival offers an easy, objective, and quantifiable means of data analysis.

Animals↗