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Biomedical subjects

R F Diegelmann

Publications and source records attributed to R F Diegelmann.

At least 73 records · Page 4Linked to original sources
PubMed1986

A subcutaneous implant for wound healing studies in humans.

A method for studying wound healing in humans is described. The technique is based on the production of a standard subcutaneous injury during implantation of a retrievable sponge. The injury is produced by introducing a small device (PVA implant) which consists of a 5.7-cm piece of perforated silicone tubing containing two pieces of polyvinyl alcohol sponge. The sponge provides a site for attracting inflammatory cells with subsequent fibroblast infiltration. The sterile PVA implant is inserted subcutaneously in the upper arm by means of a 12-gauge needle and remains there for 14 days. Upon removal, one sponge has hydroxyproline deposition quantitated using a high-performance liquid chromatography technique. The other sponge may be processed for light and electron microscopy or specialty staining. Other collagen determinations such as percentage neutral salt-soluble collagen are also possible. Using the PVA implant has made it possible to follow the kinetics of collagen deposition in the rat. There was a marked increase in collagen accumulation from Day 2 (0.89 nmole/mg sponge) to Day 14 (18 nmole/mg sponge) in the rat. Collagen deposition was also measured in human control subjects (5.07 nmole/mg sponge, n = 12) and compared to trauma patients (2.04 nmole/mg sponge, n = 5). Histologic staining showing fibroblast infiltration and collagen deposition correlated well with the biochemical findings. This implant, coupled with recent HPLC technology, provides a safe, acceptable technique to study human wound healing parameters and overcomes many of the limitations of previous methods.

Adult↗
PubMed1986

Identification of a low molecular weight, macrophage-derived chemotactic factor for fibroblasts.

Fibroblasts migrate in vitro in response to a variety of chemoattractants including cellular, humoral, and connective tissue components. This report describes a low molecular weight, macrophage-derived chemotactic substance for fibroblasts (MDCF-F). Thioglycolate-elicited peritoneal exudate cells were collected from mice 4 days postinjection. Platelets were removed and glass-adherent macrophages were selected in the absence of fetal calf serum. The macrophage culture medium was then tested for chemotactic activity for 3T3 mouse fibroblasts using a modified Boyden chamber. Chemotactic activity was observed in the macrophage medium after a 48-hr culture period and increased during the next 24 hr. The activity was sensitive to heat treatment at 56 degrees C for 30 min, could be effectively absorbed out of the culture medium by preincubation with 3T3 fibroblasts, and was not recognized by human fibroblasts. Molecular-sieve chromatography indicated a molecular weight below 10,000. This low molecular weight, macrophage-derived chemoattractant for fibroblasts may represent a rapidly diffusable substance involved in the recruitment of fibroblasts to sites of inflammation.

Animals↗
PubMed1985

Cellular sources of fibrotic collagen.

Every somatic cell within vertebrate species contains genetic information and therefore the potential to synthesize collagen. Normally, when there is a requirement for new connective tissue following injury, cells of mesodermal origin are activated to meet that demand and supply sufficient amounts of collagen to return structure and function to the damaged tissue or organ. Under these conditions, nonconnective tissue cells either do not synthesize collagen or may produce only minute quantities which are barely detectable by our most sensitive techniques. However, when these nonmesodermal cells are exposed to an abnormal or toxic environment, collagen expression may become greatly enhanced. For example, when hepatocytes are removed from their normal in vivo environment and placed in cell culture, they demonstrate a dramatic increase in their potential to produce collagen. Hepatocytes may also express this ability to produce significant quantities of collagen when they are exposed to toxins or injury in vivo. The list of cells shown to produce collagen or contain enzymes related to collagen biosynthesis has expanded in recent years and now includes such diverse and specialized cells as glomerular mesangial, adipocytes, aortic endothelial, smooth muscle, Schwann cells, and lymphocytes. Therefore, the loss of structure and function in a tissue or organ due to fibrosis may result from excessive deposition of collagen from nonmesodermal sources as well as from connective tissue cells.

Animals↗
PubMed1984

Quantitation of hydroxyproline isomers in acid hydrolysates by high-performance liquid chromatography.

A method has been developed to rapidly separate and quantitate levels of hydroxy-L-proline isomers in tissue hydrolysates. The procedure incorporates derivatization of the imino acids with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole chloride followed by separation by high-performance liquid chromatography employing two C18 reverse-phase columns connected in series. Conditions for the derivatization procedure have been optimized for the selective reactivity of imino acids. The derivatized imino acid fractions are then quantitated spectrophotometrically at 495 nm. Using this technique, quantities above 40 pmol are readily detected for trans-4-hydroxyl-L-proline, trans-3-hydroxyl-L-proline, proline, and other imino acid analogs. The method is applicable to a wide range of clinical and experimental tissues.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Intrinsic tendon fibroplasia: documentation by in vitro studies.

There has been considerable controversy concerning the capacity of tendons to heal intrinsically without adhesion formation. To help clarify this issue, a tissue culture model was developed to study the potential of tendon fibroblasts to proliferate and migrate. Avian flexor tendons, isolated free of sheath and all synovial components, were maintained in sterile culture plates with Dulbecco's Modified Eagle Medium. After 5 days in culture, the 2 mm tendon biopsies were analyzed for DNA synthesis [( 125I])iododeoxyuridine incorporation) and migration of fibroblasts out of the explant (planimeter analysis). DNA synthesis was maximal on day 5 in culture, and tendon fibroblasts were observed migrating from the biopsy within 48 h. Addition of cells from plasma resulted in a significant increase in fibroblast migration, and the presence of fibrin in the plasma clot was an absolute requirement for the migratory process. These findings further support the hypothesis that injured tendons can heal by an intrinsic healing process in addition to healing by adhesion formation.

Animals↗
PubMed1984

Isolation and culture of human intestinal smooth muscle cells.

Intestinal smooth muscle cells were isolated from human bowel and maintained in culture through several passages. These cells were obtained by enzyme digestion of slices taken from the circular layer of the muscularis propria of human jejunum. When subcultured, they initially flattened out and then began proliferating after 3 days. After 3 weeks in culture, they began aggregating into ridges. Fluorohistochemical staining revealed numerous prominent actin stress fibers. When these cells were exposed to the C-terminal octapeptide of cholecystokinin they contracted in a dose-dependent fashion. The availability of human intestinal smooth muscle cells in culture will considerably enhance our ability to study the contractile, proliferative and connective tissue responses of the smooth muscle of the human gastrointestinal tract.

Cell Division↗
PubMed1984

An in vitro model of fibroplasia: simultaneous quantification of fibroblast proliferation, migration, and collagen synthesis.

Previous studies of fibroblast proliferation, migration, and collagen synthesis have been limited in their ability to define the interrelationship among these events in response to various inflammatory mediators. We have now defined an in vitro tissue culture model for the synchronous quantification of these parameters of fibroplasia. Biopsies (2 mm) of chicken flexor tendons are embedded in a fibrin matrix and exposed to various factors for 5 days in tissue culture. The availability of the fibrin matrix surrounding the tendon biopsy satisfies the need for a solid support medium for fibroblast migration. Multiple measurements of tendon fibroblast proliferation, migration into the fibrin matrix, and relative collagen synthesis are then made on these preparations. Fetal calf serum stimulated tendon fibroblast proliferation and migration in a dose responsive fashion, whereas the selective expression of collagen synthesis was decreased. Platelet lysate stimulated fibroblast proliferation at low concentration, but migration only at high concentration and was without effect on relative collagen synthesis. This model now provides a means of more clearly defining the influence of various inflammatory factors on the events of fibroplasia.

Animals↗
PubMed1984

Glucocorticoids suppress formation of collagen by the hepatocyte. Studies in primary monolayer cultures of parenchymal cells prepared from adult rat liver.

We have established previously that the adult rat hepatocyte produces collagen under conditions of primary monolayer culture or under pathologic conditions in vivo. During the initial 3 days of incubation of hepatocytes in serum-free culture medium, collagen synthesis represented only 0.01%-0.05% of the synthesis of noncollagen proteins. Thereafter, rates of collagen synthesis rose dramatically, and after 9 days of incubation were increased an average tenfold in cultures derived from normal hepatocytes and 30-fold in cultures derived from regenerated liver. The spontaneous increase in the rates of collagen synthesis was almost completely blocked (94%) when cultures were incubated for 8 days in medium containing the synthetic glucocorticoid dexamethasone (10(-7) M). Dexamethasone was without effect on the synthesis of noncollagen proteins. Inhibition of collagen synthesis by dexamethasone exhibited a sigmoidal dose-response curve with 50% inhibition observed at a concentration of 10(-9) M. Collagen synthesis was diminished similarly by other glucocorticoids (hydrocortisone, methylprednisolone, triamcinolone, prednisone, all 10(-6) M) but not by other hormonal or nonhormonal steroids (progesterone, estradiol, testosterone, all 10(-5) M). We excluded increased collagen degradation as an explanation for these results by demonstrating that dexamethasone failed to increase the incorporation of radioactive proline into trichloroacetic acid-soluble radioactive hydroxyproline. Exposure of hepatocytes to dexamethasone during the first 48 h of incubation produced inhibition of collagen synthesis in 9-day-old cultures, even though washout studies with [3H]dexamethasone revealed negligible retention of the steroid by the cells. This observation suggests that among the initial, culture-adaptive responses of the hepatocyte are glucocorticoid-sensitive events that culminate in increased rates of collagen synthesis days later. We conclude that glucocorticoids may serve as natural suppressors of the potential of the hepatocyte to produce collagen.

Animals↗
PubMed1983

Collagen formation by the hepatocyte in primary monolayer culture and in vivo.

Immunohistochemical techniques were used to confirm biochemical evidence that parenchymal cells isolated from adult rat liver and maintained in nonreplicating monolayer culture for 2 days synthesized type IV basement membrane collagen. On continued incubation in serum-free medium, the hepatocytes also synthesized the interstitial collagens, types I and III. Consistent with these results in culture, type IV collagen was localized to the hepatocytes in slices of pathologic rat liver. Hence collagen formation is a previously unrecognized function of the hepatocyte that may be important in the pathogenesis of liver fibrosis or cirrhosis.

Animals↗
PubMed1981

Collagen synthesis by the hepatocyte: studies in primary cultures of parenchymal cells from adult rat liver.

Based on the finding that prolyl hydroxylase, a key enzyme in collagen biosynthesis, is a constituent of the hepatic parenchymal cell, we have suggested that the hepatocyte may synthesize collagen (Exp. Cell Res. 123: 269-279, 1979). We now report that, consistent with this idea, collagen formation has been detected in primary nonproliferating cultures of isolated rat hepatocytes prepared from either normal liver or regenerated liver four days after partial hepatectomy. The characteristics of the radiolabeled collagen formed in two-day old cultures incubated for 24 hours in the presence of either [3H]-proline or [35S]-cystine were its resistance to pepsin and its susceptibility to degradation by highly purified, protease-free bacterial collagenase. The presence of fibroblasts in the hepatocyte cultures was excluded as an explanation for these results because we detected no type I collagen, a universal product of the cultured fibroblast. The initial low rates of synthesis of collagen relative to total cellular protein (0.1-0.4 percent) increased dramatically upon continued incubation of the cells reaching 0.31 and 0.81 percent in nine-day old cultures of normal or regenerated hepatocytes, respectively. This change was accompanied by the synthesis of an additional 100,000 molecular weight from of collagen, possibly type I or A, B. Morphologically, the hepatocytes progressively flattened and overlapped adjacent cells with time in culture. However, their identify as hepatocytes was confirmed by the fact that synthesis of fibrinogen, a liver-specific function, was maintained above initial levels throughout the experiment. We conclude that synthesis of collagen is a constitutive function of the hepatocyte. This function is linked to hepatocyte replication, is subject to phenotypic change in culture, and may be important in the pathogenesis of hepatic fibrosis or cirrhosis.

Age Factors↗
PubMed1981

Intrinsic tendon cell proliferation in tissue culture.

This study examines the potential of tendon cells to participate in tendon repair. Explants were prepared from chicken sublimis tendons and studied in tissue culture. Under defined culture conditions, in the absence of tendon sheath, tendon cells were observed to migrate in a limited concentric fashion around the margin of a 2 mm window in the explant. When a plasma clot was present in the window, tendon cell migration and proliferation were accelerated and continued until the window was confluent with cells. Electron micrographs demonstrated that these tendon cells were fibroblasts associated with extracellular collagen fibrils. Specific biochemical analyses confirmed that these tendon fibroblasts were synthesizing collagen. This in vitro study demonstrates that tendons contain active fibroblasts capable of proliferation, migration, and collagen synthesis. These findings support the hypothesis that tendons can heal by intrinsic processes.

Animals↗
PubMed1979

Quantitative assay of types I and III collagen synthesized by keloid biopsyes and fibroblasts.

Molecular sieve column chromatography was used to determine the amount of type I and III collagen synthesized by normal dermis and keloid biopsies and fibroblasts derived from these tissues. After incubation with radioactive proline, the collagen was extracted and separated into types I and III and then quantitated. There was no significant difference in the percent type III collagen synthesized by fresh keloid biopsies compared to normal dermis. Likewise, there was no significant difference in the percent type III collagen synthesized by keloid fibroblasts compared to normal dermal fibroblasts. However, fibroblasts from both keloid and normal dermis synthesized a lower percentage of type III collagen in cell culture compared to the original biopsies. These findings demonstrate that keloid collagen has the same type distribution as normal dermis and suggest that increased collagen synthesis in these lesions is not related to altered collagen types.

Adolescent↗