Mechanical behavior of fibroblasts included in collagen lattices

Striae distensae are characterized by linear, smooth bands of atrophic-appearing skin. Excessive steroid activity, genetic and mechanical factors and inherited defects of connective tissues are the most frequent causes of this disease. Fibroblasts derived from women presenting striae distensae lesions were included into collagen gels to study their mechanical behavior: capacity to contract free-floating lattices and to produce isometric force in tense lattices. To measure the retracted lattice diameter, the culture dishes were placed on a transparent metric scale. An isometric force system was used to study quantitatively the forces developed during lattice contraction. alpha 2 beta 1 integrins expression (transmembrane receptors) was evaluated by flux cytometry. Striae distensae fibroblasts contract collagen gels slower than normal human fibroblasts but the final contraction is similar. They produce a greater isometric force which is associated with enhanced alpha 2 beta 1 integrins expression. By their mechanical properties, striae distensae fibroblasts appear as a different population from normal fibroblasts.     

Effects of varying density patterns and passes on depth of penetration in facial skin utilizing the carbon dioxide laser with automated scanner

Histologic data describing the depth of penetration that occurs with the CO2 laser using variations in delivered energy, number of passes, and density settings have been limited. Most of the initial studies used a 3-mm collimated handpiece; few studies used the computerized pattern generator, which is now standard in treatment. This study examined histologic depth of thermal damage with varying density settings and number of passes using the computerized pattern generator. The Coherent UltraPulse 5000 CO2 laser with a computerized pattern generator was used. Preauricular (sun-exposed) and postauricular (sun-protected) facial skin samples were used from seven rhytidectomy patients aged 37 to 68 years. Preoperative skin treatment regimen included Retin-A and hydroquinone application for 3 weeks before the procedure. Three adjacent sites from the preauricular and postauricular regions were chosen from each ear (12 sites per patient). A density setting of 5 (30 percent overlap) was chosen for the right ear, and a density setting of 9 (60 percent overlap) was used for the left ear. Each region was subjected to one, two, and three passes. The laser delivers approximately 7.5 J/cm2 of fluence at a spot size of 2.25 mm and 300 mJ. Excisional biopsies were performed at the time of cervicofacial flap redraping. All specimens were evaluated for depth of thermal damage by a dermatopathologist who was blinded to the treatment parameters for each test site. Histologic examination of the treated test sites consistently demonstrated that one pass at these settings obliterated most or all of the epidermis, with minimal invasion into the papillary dermis. Test sites treated with two or three passes resulted in increased cumulative depth of penetration and thermal injury into the papillary dermis. Only one sample site showed any thermal injury extending into the reticular dermis. Depth of penetration was greater at the postauricular sites. Additionally, depth of penetration was greater with a density of 9 (60 percent overlap) than one of 5 (30 percent overlap) in both the preauricular and postauricular sites. Our study supported previous observations that the cumulative depth of penetration is greater with increasing levels of energy and additional passes. Additionally, we saw a greater average depth of penetration as density overlap increased, as one would predict. However, at these settings, fewer passes at a higher density setting did not achieve the same depth of penetration as more passes at a lower density setting. Furthermore, we found that the margin of safety using these settings is high: only 1 of 84 sites extended into the reticular dermis.     

The interaction of epidermal keratinocytes and fibroblasts during the formation of a live skin equivalent]

The effect of human fetal fibroblasts and adult keratinocytes on collagen contraction was studied. Keratinocytes embedded in collagen lattices did not spread and produced only a slight contraction. When keratinocytes were seeded on the surface of tht gel, the contraction began within 24 h and correlated with the formation of epithelial colonies. Transplantation of multilayered epithelial sheets on the gel significantly accelerated the onset of contraction. Keratinocytes seeded on and fibroblasts grown in collagen lattices cooperatively contracted the gel, and keratinocytes were able to stimulate gel contraction even when they had no contact with the collagen roughly populated with fibroblasts. Swiss 3T3 cells remained spherical in collagen lattices and did not contract the gel but when cultivated with keratinocytes they stimulated gel contraction. In their turn, keratinocytes influenced the behaviour of Swiss 3T3 cells which elongated and produced processes. We suggest that both keratinocytes and mesenchymal cells can affect gel contraction 1) by a direct contact with collagen lattices, and 2) through potentiation of the ability of another cell type to contract the gel.     

HETEROGENEITY IN RADIOSENSITIVITY OF HUMAN DIPLOID FIBROBLASTS FROM KELOIDS AND NORMAL SKINS

Colony-forming ability was employed in evaluating the susceptibility toin vitrogamma ionizing radiation in human diploid skin fibroblasts (HDF). Twelve pairs of HDF, each composed of fibroblasts from excised keloid lesion and local normal skin tissue as its control, were studied in patients with clinically persistent keloids. Parameters of radiosensitivity, both D₀and D₁₀, and growth kinetics were examined. The radiosensitivity in three of the 12 keloids (25%) were demonstrated significantly increased than their counterpart controls, even though no difference in growth kinetics in between. Moreover, a broad range in the radiosensitivity of fibroblast cells was demonstrated and it is suggested that there is a great heterogeneity of cellular response to radiation in HDF.     

Temperature dependence of ultraweak photon emission in fibroblastic differentiation after irradiation with artificial sunlight

Yield of ultraweak photon emission in a cell culture model for biophotonic measurements using fibroblastic differentiation depended on the temperature of photonic measurement. The ultraweak photon emission of medium was significantly higher at 37 degrees C than at 25 degrees C and after UVB-irradiation this difference was even more pronounced. While with cells in the medium no temperature dependence could be determined in unirradiated samples, after UVB-irradiation of cells an increase of biophotonic emission was observed in postmitotic fibroblasts. While after several UVB exposures normal cells begin to absorb the ultraviolet light, cells from patients with the disease Xeroderma Pigmentosum loose this capacity. In view that fibroblasts play an essential role in skin aging, skin carcinogenesis and wound healing, the biophotonic model using the fibroblastic differentiation system provides to be a new and powerful non-invasive tool for the development of skin science.     

Effect of low dose rate irradiation on the division potential of cells in vitro. IV. Embryonic and adult human lung fibroblast-like cells

Early and late passage human embryonic lung fibroblasts were compared with early passage adult lung fibroblasts with regards to their survival (number of population doublings), after low dose rate ionizing radiation. It was found that early passage embryonic cells are quite resistant to this type of radiation. Late passage embryonic and early passage adult fibroblasts are more sensitive to ionizing radiations. The results suggest that cell aging is accompanied by an increased sensitivity to low dose rate ionizing radiation and favor the idea that aging in vitro, expressed as a function of the fibroblast division potential, is correlated with aging in vivo.     

Tissue origin and extracellular matrix control neutral proteinase activity in human fibroblast three-dimensional cultures

Remodeling of the extracellular matrix by fibroblasts is an important step in the process of wound healing and tissue repair. We compared the behavior of fibroblasts from two different tissues, dermis and gingiva, in three-dimensional lattices made of two different extracellular matrix macromolecules, collagen and fibrin. Cells were grown in monolayer cultures from normal skin or gingiva and seeded in three-dimensional lattices made of either collagen or fibrin. Photonic and scanning electron microscopy did not reveal any morphological differences between the two types of fibroblasts in both sets of lattices. Both types of fibroblasts retracted collagen lattices similarly and caused only a slight degradation of the collagen substratum. By contrast, when seeded in fibrin lattices, gingival fibroblasts completely digested their substratum in less than 8 days, whereas only a slight fibrin degradation was observed with dermal fibroblasts. The ability of gingival but not dermal fibroblasts to express high levels of tissue plasminogen activators (tPA) when cultured in fibrin lattices was assessed on an immunological basis. Also, deprivation of plasminogen-contaminating fibrinogen preparations or use of tPA inhibitors markedly inhibited both fibrinolysis and retraction rates of fibrin lattices by gingival fibroblasts. Casein-zymography confirmed the intense proteolytic activity induced by fibrin in gingival fibroblasts. It was inhibited by aprotinin and phenyl methylsulfonyl fluoride (PMSF), two non-specific inhibitors of serine proteinases, and by η-amino-caproic acid (ηACA), an inhibitor of plasminogen activators. Monolayer cultures exhibited only trace amounts of caseinolytic activity. Our results demonstrate that the expression of proteinases by fibroblasts is dependent not only on their tissue origin but also on the surrounding extracellular matrix. The intense fibrinolytic activity of gingival fibroblasts in fibrin lattices may explain partially the high rate of healing clinically observed in gingiva. © 1996 Wiley-Liss, Inc.     

Combined rhytidectomy and full-face laser resurfacing

A series of patients undergoing a combined face lifting procedure with simultaneous laser resurfacing is described. Although resurfacing is accepted as safe for deep-plane face lifts and forehead lifts, there are reports of preauricular skin loss with a standard face lift. In this series, 26 consecutive cases are described. A superficial musculoaponeurotic system (SMAS) face lift technique was used. A skin flap was developed no more than 3.0 cm from the preauricular area, and most of that was excised. Full-face laser resurfacing was done with the SilkTouch laser. An 8-mm square pattern was used at 16 to 18 W. Three to four passes were done. Care was taken, however, to do very superficial lasering of the periphery, especially over the small amount of undermined skin that remained. There was no preauricular skin loss. This series demonstrates the safety of combining laser resurfacing with the SMAS technique face lift in regard to preauricular skin loss.     

Slowed growth of cultured fibroblasts from human radiation wounds

To study radiation effect separate from the microcirculation, fibroblasts were cultured from four patients with radiation wounds. Cells could be grown from irradiated tissue near the ulcer and from control normal tissue, but no cells could be cultured from the ulcers. The ability of radiation-treated fibroblasts to attach to the substrate and form colonies was less than that of unirradiated cells. Irradiated skin fibroblasts from the four patients had significantly longer mean generation times than did control cells. During log-phase growth (1 to 9 days), the population doublings of damaged cells were significantly reduced compared to colonies from normal cells. These data suggest a permanent intrinsic radiation effect on fibroblasts or a selective ablation of faster-growing fibroblast subpopulations that is not dependent on decreased blood supply.     

Protection from solar simulated radiation-induced DNA damage in cultured human fibroblasts by three commercially available sunscreens

Exposure to solar radiation can produce both acute and chronic changes in the skin, including sunburn, edema, immunosuppression, premature skin aging, and skin cancer. At the cellular level, solar radiation can produce adverse structural and functional changes in membrane proteins and lipids and in chromosomal and mitochondrial DNA. The increasing awareness of these adverse effects has led the public to demand better photoprotection. In this study, the alkaline comet assay was used to evaluate the photoprotective effects of three commercially available sunscreens at sun protection factors (SPF) 15 and 30. Human fibroblasts were used as target cells to conveniently study the effects of solar simulated radiation on DNA damage in the presence and absence of sunscreens. When human fibroblasts were exposed to various doses of solar simulated radiation, DNA damage, as measured in sunscreen-protected cells by the comet assay, was not significantly different from that detected in unexposed cells. At 1.0 and 1.5 minimal erythemal doses (MED), all sunscreens, at both SPF 15 and 30, provided nearly 100% photoprotection to the fibroblasts. Further studies are required to elucidate the role of UVA in the production and repair of DNA damage in cells exposed to sunlight.     

Gamma-interferon inhibits extracellular matrix synthesis and remodeling in collagen lattice cultures of normal and scleroderma skin fibroblasts.

Three-dimensional collagen lattice cultures of fibroblasts mimic the in vivo situation better than monolayer cultures. Here, skin fibroblasts from scleroderma patients and healthy controls were cultivated in collagen lattices, and the effects of recombinant human gamma-interferon (IFN-gamma) on these cultures investigated. IFN-gamma inhibited collagen lattice retraction in a dose-dependent way at concentrations ranging from 10 to 10,000 U/ml. This effect was independent of any alteration to the cell proliferation within the lattices. The inhibition was of the same order of magnitude in normal and pathological fibroblasts. The synthesis of collagen and non-collagen proteins, particularly fibronectin, was increased in scleroderma cultures. It was inhibited in both normal and scleroderma fibroblasts by IFN-gamma, with a maximal effect at the concentration 1000 U/ml, but the inhibition of protein synthesis was far more intense in scleroderma than in normal cells. In situ hybridization, Northern blot and dot blot analyses showed that mRNA coding for pro alpha 1(I) collagen was decreased in IFN-gamma-treated cells, indicating an effect at the pretranslational level. IFN-gamma also inhibited glycosaminoglycan synthesis, but in scleroderma cells only. This study shows that IFN-gamma regulates cell behavior in three-dimensional collagen matrices: (i) it decreases protein and specifically glycosaminoglycan synthesis in scleroderma fibroblasts, (ii) it modulates the interactions between cells and matrix that lead to the retraction of the lattice. Whereas collagen synthesis is largely decreased in lattice cultures like in vivo, it remains increased in the case of scleroderma compared to normal fibroblasts and may be down-regulated by IFN-gamma. Similar conclusions may be drawn for fibronectin and glycosaminoglycans. The inhibitory effect of IFN-gamma on the retraction capacity of fibroblasts and on their ability to synthesize increased amounts of extracellular matrix macromolecules may be of potential interest for therapeutic use of IFN-gamma in scleroderma patients.     

Increased retraction of collagen lattices by fibroblasts from patients with scleroderma

Skin fibroblasts from eight scleroderma patients were seeded in collagen lattices, and their capacity of retraction was compared to that of fibroblasts from normal volunteers. In all cases, pathological fibroblasts retracted collagen lattices earlier and more intensively than controls. This in vitro feature may be related to the cutaneous retraction which characterizes scleroderma lesions in vivo.     

Different regulation of collagen I gene transcription in three-dimensional lattice cultures.

Human skin fibroblasts were cultivated in confluent monolayers, retracting collagen lattices, retracting fibrin lattices and non-retracting fibrin lattices and the expression of messenger RNA specific for the alpha 1 chain of type I procollagen comparatively studied by Northern blot and dot blot hybridization. Two factors contribute to the lower level of procollagen messenger RNA in collagen lattices: the retraction and the nature of the fibrillar protein that constitutes the lattices. Fibrin lattices, when they do not retract, make as much collagen and procollagen mRNA as monolayer confluent cells.     

The modulation of contraction of fibroblast populated collagen lattices by types I, II, and III collagen

Human dermal fibroblasts incorporated in a polymerized collagen lattice reduce the size of that matrix. When cell number, collagen concentration, and medium are identical, lattices made with type III collagen contract faster and to a greater degree than those made with type I collagen. The latter contract faster and to a greater degree than those made with type II collagen.     

The in vitro photosensitivity of systemic lupus erythematosus skin fibroblasts

To investigate the role of DNA damage in the pathogenesis of systemic lupus erythematosus (SLE), we studied the ability of skin fibroblasts derived from SLE patients to recover from ultraviolet (UV) light radiation of varying wavelengths. Four of five SLE cell strains were more sensitive to UV-C (254 nm), sun lamp, and UV-A (320 to 400 nm) light than were normal cells. SLE cellular recovery was most sensitive to broad spectrum, long wavelength light. This hypersensitivity did not appear to result from the UV light activation of a clastogenic factor. Experiments which examined the DNA repair capacity of irradiated cells indicated that SLE fibroblasts may be able to excise certain DNA lesions as well as normal cells. The mechanisms responsible for the hypersensitivity of SLE cells remain under investigation.     

The postauricular fascial flap as an adjunct to Mustardé and Furnas type otoplasty.

Anterior riberation methods of otoplasty have been criticized because of the risk of anterior hematoma that can cause anterior skin necrosis, scarring, and even cartilage destruction caused by infection. As a result, cartilage-sparing otoplasty such as the Mustardé and Furnas types has been increasingly popular. However, postauricular suture extrusion may result, and recurrence rates of up to 25 percent have been recorded. In this study, cartilage-sparing otoplasty is refined by the addition of a postauricular fascial flap to reduce suture extrusion and recurrence rates. Fifty-one patients underwent otoplasty (45 bilateral, six unilateral). This technique involves the elevation of a fascial flap from the postauricular region. A new antihelical fold is then created by Mustardé sutures, and the conchal bowl is rotated by Furnas-type concha-mastoid sutures. The fascial flap is then advanced to cover the sutures with a supplementary vascularized layer to prevent suture extrusion. In addition, the advancement of the flap acts as a postauricular support to prevent recurrence. A natural-looking antihelical fold and helical rim is created by this technique. There were no hematomas. There was recurrence in eight ears (8 percent) in six patients. Two patients requested further surgery. No patients developed suture extrusion or granuloma. This is a simple and intrinsically safe procedure and does not cause irreparable complications such as anterior scarring or skin necrosis. The postauricular fascial flap seems to prevent suture extrusion. It may also help to reduce recurrence rates to acceptable levels.     

Accumulation of DNA damage and cell death after fractionated irradiation

The purpose of this work was to determine how fractionated radiation used in the treatment of tumors affects the ability of cancer as well as normal cells to repair induced DNA double-strand breaks (DSBs) and how cells that have lost this ability die. Lymphocytic leukemia cells (MOLT4) were used as an experimental model, and the results were compared to those for normal cell types. The results show that cancer and normal cells were mostly unable to repair all DSBs before the next radiation dose induced new DNA damage. Accumulation of DSBs was observed in normal human fibroblasts and healthy lymphocytes irradiated in vitro after the second radiation dose. The lymphocytic leukemia cells irradiated with 4 × 1 Gy and a single dose of 4 Gy had very similar survival; however, there was a big difference between human fibroblasts irradiated with 4 × 1.5 Gy and a single dose of 6 Gy. These results suggest that exponentially growing lymphocytic leukemia cells, similar to rapidly proliferating tumors, are not very sensitive to fraction size, in contrast to the more slowly growing fibroblasts and most late-responding (radiation therapy dose-limiting) normal tissues, which have a low proliferation index.     

Patients with myotonic dystrophy,a possible segmental progeroid syndrome,and duchenne muscular dystrophy have fibroblasts with normal limits for in vitro lifespan and growth characteristics

Myotonic dystrophy (MyD) has been suggested to be a segmental progeroid syndrome in man, as this syndrome has some clinical manifestations of premature aging. Fibroblasts from patients with other progeroid syndromes have been shown to have diminished in vitro lifespans or growth characteristics; therefore, it was of interest to study cellular senescence in fibroblasts from patients with MyD. Fibroblast cultures from patients with Duchenne muscular dystrophy (DMD) were used as additional controls, as premature aging is not associated with this genetic disorder. Primary skin fibroblast cultures obtained from patients with MyD or DMD and from age-sex matched controls were grown in DMEM plus 10% FBS. The in vitro lifespan was determined by either a 1:4 split ratio or with a constant initial inoculum of 1 × 10⁴ cells/cm², followed by determination of the final density at weekly intervals. Our results demonstrate that there is no difference in the limits of the in vitro lifespan for either the MyD or DMD fibroblast strains compared to the controls. Likewise, no difference could be detected in the growth characteristics of these cells. The only observable difference was that the pooled age-matched controls and MyD cultures had a shorter in vitro lifespan than the DMD group and their pooled controls, a finding expected because of the age of the patients in each group. Unlike the other progeroid syndromes, MyD fibroblasts have normal limits for in vitro lifespan. MyD is probably not closely related to the other premature aging syndromes, although there is an increasing phenotypic expression as a function of age.