Title: Proliferation and Mitogenic Response to PDGF-BB of Fibroblasts Isolated from Chronic Venous Leg Ulcers is Ulcer-Age Dependent11Presented in part at the Gordon Research Conference on Wound Repair July 2–7 1995 and at an IBC meeting on Fibrosis in Washington, DC, April 18–19 1996.
Abstract: Several pathophysiologic mechanisms have been proposed to explain slow-healing leg ulcers, but little is known about the growth behavior of cells in these wounds. Platelet-derived growth factor-BB applied topically to chronic wounds has shown beneficial effects, although the effects have been less pronounced than would have been expected based on studies on acute wounds. The objective of this study was to compare fibroblasts in culture obtained from chronic wounds (non-healing chronic venous leg ulcers), acute wounds and normal dermis regarding growth, mitogenic response to platelet-derived growth factor-BB and levels of platelet-derived growth factor α-receptor and β-receptor. Fibroblasts were obtained by an explant technique and expanded in vitro using fibroblast growth medium supplemented with 10% fetal bovine serum and used for the assays at their third passage. Growth of chronic wound fibroblasts (n = 8) was significantly (p < 0.05) decreased compared with those from acute wounds (n = 10) and normal dermis (n = 5). Fibroblasts from ulcers older than 3 y grew significantly (p < 0.01) slower than those from ulcers that had been present for less than 3 y. Morphology and size of fibroblasts from the oldest chronic wounds deviated substantially from those of acute wounds and normal dermis, and resembled in vitro aged or senescent fibroblasts. Mitogenic response of chronic wound fibroblasts to human recombinant platelet-derived growth factor-BB was also reduced with ulcer age. No significant differences were found in the amount of either platelet-derived growth factor α-receptor or β-receptor among the three groups. The features decreased growth related to ulcer age, altered morphology, and reduced response to platelet-derived growth factor, indicating that fibroblasts in some chronic wounds have approached or even reached the end of their lifespan (phase III). This might provide one explanation for the non-healing state and therapy resistance to topical platelet-derived growth factor-BB of some venous leg ulcers. Several pathophysiologic mechanisms have been proposed to explain slow-healing leg ulcers, but little is known about the growth behavior of cells in these wounds. Platelet-derived growth factor-BB applied topically to chronic wounds has shown beneficial effects, although the effects have been less pronounced than would have been expected based on studies on acute wounds. The objective of this study was to compare fibroblasts in culture obtained from chronic wounds (non-healing chronic venous leg ulcers), acute wounds and normal dermis regarding growth, mitogenic response to platelet-derived growth factor-BB and levels of platelet-derived growth factor α-receptor and β-receptor. Fibroblasts were obtained by an explant technique and expanded in vitro using fibroblast growth medium supplemented with 10% fetal bovine serum and used for the assays at their third passage. Growth of chronic wound fibroblasts (n = 8) was significantly (p < 0.05) decreased compared with those from acute wounds (n = 10) and normal dermis (n = 5). Fibroblasts from ulcers older than 3 y grew significantly (p < 0.01) slower than those from ulcers that had been present for less than 3 y. Morphology and size of fibroblasts from the oldest chronic wounds deviated substantially from those of acute wounds and normal dermis, and resembled in vitro aged or senescent fibroblasts. Mitogenic response of chronic wound fibroblasts to human recombinant platelet-derived growth factor-BB was also reduced with ulcer age. No significant differences were found in the amount of either platelet-derived growth factor α-receptor or β-receptor among the three groups. The features decreased growth related to ulcer age, altered morphology, and reduced response to platelet-derived growth factor, indicating that fibroblasts in some chronic wounds have approached or even reached the end of their lifespan (phase III). This might provide one explanation for the non-healing state and therapy resistance to topical platelet-derived growth factor-BB of some venous leg ulcers. fibroblast basal medium fibroblast growth medium Several pathophysiologic mechanisms have been proposed to explain slow/non-healing leg ulcers. Overproduction of reactive oxygen species, hypoxia, imbalances in levels of cytokines and proteolytic enzymes, excessive fibrin deposition, and failure of keratinocyte migration on the chronic wound bed have been presented as possible explanations for the defective healing of leg ulcers (Coleridge Smith, 1994Coleridge Smith P.D. Venous ulcer.Br J Surg. 1994; 81: 1404-1405Crossref PubMed Scopus (15) Google Scholar;Falanga et al., 1994Falanga V. Grinell F. Gilchrest B. Maddox Y.T. Moshell A. Workshop on the pathogenesis of chronic wounds.J Invest Dermatol. 1994; 102: 125-127Abstract Full Text PDF PubMed Google Scholar). Little is known, however, about growth and other biologic activities of cells in these wounds. Fibroblasts are one of the key cells in wound repair. Apart from producing the major extracellular components of collagen, elastin, and proteoglycans, fibroblasts also make mitogens for keratinocytes, fibroblasts, and endothelial cells (Werner et al., 1994Werner S. Smola H. Liao X. Longaker M.T. Krieg T. Hofschneider P.H. Williams L.T. The function of KGF in morphogenesis of epithelium and reepithelialization of wounds.Science. 1994; 266: 819-822Crossref PubMed Scopus (490) Google Scholar). Fibroblasts probably migrate from neighboring tissues, although a bloodborne origin has also been proposed (Bouissou et al., 1988Bouissou H. Pieraggi M. Julian M. Uhart D. Kokolo J. Fibroblasts in dermal tissue repair. Electron microscopic and immunohistochemical study.Int J Dermatol. 1988; 27: 564-570Crossref PubMed Scopus (23) Google Scholar;Bucala et al., 1994Bucala R. Spiegel L.A. Chesney J. Hogan M. Cerami A. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.Mol Med. 1994; 1: 71-81Crossref PubMed Google Scholar). At the wound site, fibroblasts change into a less proliferative but a more contractile and collagen synthetic phenotype (Regan et al., 1991Regan M.C. Kirk S.J. Wasserkrug H.L. Barbul A. The wound environment as a regulator of fibroblast phenotype.J Surg Res. 1991; 50: 442-448Abstract Full Text PDF PubMed Scopus (52) Google Scholar;Germain et al., 1994Germain L. Jean A. Auger F.A. Garrel D.R. Human wound healing fibroblasts have greater contractile properties than dermal fibroblasts.J Surg Res. 1994; 57: 268-273Abstract Full Text PDF PubMed Scopus (95) Google Scholar;Ågren et al., 1996Ågren M.S. Haisa M. Grotendorst G.R. Differential expression of platelet-derived growth factor receptors in porcine fibroblasts cultured from skin and granulation tissue.Wound Repair Regeneration. 1996; 4: 288-296Crossref Scopus (2) Google Scholar). These functional alterations can be mimicked in vitro by adding wound fluid to cultured normal skin fibroblasts (Regan et al., 1991Regan M.C. Kirk S.J. Wasserkrug H.L. Barbul A. The wound environment as a regulator of fibroblast phenotype.J Surg Res. 1991; 50: 442-448Abstract Full Text PDF PubMed Scopus (52) Google Scholar). This finding indicates the control provided by factor(s) present in the wound environment. The main regulators of the cellular activities in wound repair are polypeptide growth factors (Steenfos, 1994Steenfos H.H. Growth factors and wound healing.Scand J Plast Reconstr Surg Hand Surg. 1994; 28: 95-105Crossref PubMed Scopus (159) Google Scholar). Platelet-derived growth factor (PDGF) is mitogenic and chemotactic for connective tissue cells such as fibroblasts, and appears to be an important autocrine and paracrine factor during wound repair (Grotendorst et al., 1985Grotendorst G.R. Martin G.R. Pencev D. Sodek J. Harvey A.K. Stimulation of granulation tissue formation by platelet-derived growth factor in normal and diabetic rats.J Clin Invest. 1985; 76: 2323-2329Crossref PubMed Scopus (217) Google Scholar;Ågren et al., 1996Ågren M.S. Haisa M. Grotendorst G.R. Differential expression of platelet-derived growth factor receptors in porcine fibroblasts cultured from skin and granulation tissue.Wound Repair Regeneration. 1996; 4: 288-296Crossref Scopus (2) Google Scholar). Controlled clinical trials with one of the three isoforms of PDGF, PDGF-BB homodimer have not produced the same beneficial effects, however, when applied topically to chronic wounds as when applied to acute, experimental wounds (Robson et al., 1992Robson M.C. Phillips L.G. Thomason A. Robson L.E. Pierce G.F. Platelet-derived growth factor BB for the treatment of chronic pressure ulcers.Lancet. 1992; 339: 23-25Abstract PubMed Scopus (298) Google Scholar;Pierce et al., 1994Pierce G.F. Tarpley J.E. Allman R.A. et al.Tissue repair processes in healing chronic pressure ulcers treated with recombinant platelet-derived growth factor BB.Am J Pathol. 1994; 145: 1399-1410PubMed Google Scholar;Robson, 1997Robson M.C. The role of growth factors in the healing of chronic wounds.Wound Repair Regeneration. 1997; 5: 12-17Crossref PubMed Scopus (115) Google Scholar). PDGF activates cells via specific cell surface receptors: α-receptor and β-receptor. The presence of PDGF receptors appears to be upregulated during human wound healing compared with normal skin, as demonstrated by in situ hybridization and immunohistochemistry (Reuterdahl et al., 1993Reuterdahl C. Sundberg C. Rubin K. Funa K. Gerdin B. Tissue localization of β receptors for platelet-derived growth factor and platelet-derived growth factor B chain during wound repair in humans.J Clin Invest. 1993; 91: 2065-2075Crossref PubMed Scopus (97) Google Scholar;Peus et al., 1995Peus D. Jungtäubl H. Knaub S. et al.Localization of platelet-derived growth factor receptor subunit expression in chronic venous leg ulcers.Wound Repair Regeneration. 1995; 3: 265-272Crossref PubMed Scopus (8) Google Scholar). Immunostaining for both types of receptors is predominantly localized to fibroblast-like cells in granulation tissue from both acute and chronic wounds, whereas endothelial cells stain exclusively for the β-receptor.Pierce et al., 1994Pierce G.F. Tarpley J.E. Allman R.A. et al.Tissue repair processes in healing chronic pressure ulcers treated with recombinant platelet-derived growth factor BB.Am J Pathol. 1994; 145: 1399-1410PubMed Google Scholar observed that the number of fibroblasts increased in healing chronic wounds but not in non-healing chronic wounds treated with PDGF-BB, whereas in placebo-treated chronic wounds no difference in fibroblast number was seen between healing and non-healing wounds. This observation indicates that there are fibroblasts in some chronic wounds that do not respond to PDGF-BB, possibly due to deficient numbers of PDGF receptors or dysfunctional intracellular signal transduction. In order to test this hypothesis, we have compared fibroblasts cultured from chronic as well as from acute wounds, and also from normal skin with respect to their growth and mitogenic response to growth factors, especially to PDGF-BB. Furthermore, the levels of PDGF α-receptor and β-receptor on the different fibroblast strains were estimated. Fibroblasts from wounds were obtained from eight patients [aged 68 ± 5 y (mean ± SEM), four females] with chronic wounds (venous leg ulcers) and 10 patients (59 ± 5 y, six females) with acute wounds (traumatic), as well as from normal whole skin of the medial upper arm of five healthy volunteers (45 ± 4 y, three females). The chronic wounds had been present for more than 6 mo (6 mo, 1, 1.5, 3, 6, 10, 10, and 20 y), were larger than 50 cm2 in size, and were excised for subsequent autologous skin transplantation due to the non-healing state of the ulcers. Punch biopsy specimens (4 mm) were also taken from adjacent, clinically diagnosed lipodermatosclerotic skin and normal skin in five of the eight patients (aged 64 ± 6 y) with chronic wounds (1, 1.5, 6, 10, and 20 y old ulcers). The 10 acute wounds comprised seven with 14–45 d old exuberant granulation tissue, two expanded polytetrafluoroethylene tubes with 10 d old subcutaneous granulation tissue (Jorgensen et al., 1996Jorgensen L.N. Kallehave F. Karlsmark T. Gottrup F. Reduced collagen accumulation after major surgery.Br J Surg. 1996; 83: 1591-1594Crossref PubMed Scopus (32) Google Scholar), and one 4 d old 6 mm punch biopsy wound. No sites showed any clinical signs of infection. Furthermore, none of the patients had diabetes mellitus or were given systemic antibiotics, glucocorticoids, or chemotherapeutic agents. Informed consent was obtained from all patients participating in the study, which was approved by the local ethical committee. Granulation tissue was obtained from central nonepithelialized parts of the open wounds or from the expanded polytetrafluoroethylene tubes and used as explants. From the normal skin 6 mm whole dermis punch biopsies were used as explants after excision of the epidermis. The tissue specimens were immersed in ice-cold phosphate-buffered saline (PBS), kept at 5°C and washed three times in 50 ml ice-cold PBS before explanting, which occurred within 4 h after harvesting. Tissues were minced (1 mm3), explanted on 60 mm Petri dishes (Greiner, Frickenhausen, Germany) and fibroblast outgrowth supported with fibroblast growth medium (FGM, Clonetics, Walkersville, MD) supplemented with 10% heat-inactivated mycoplasma-screened fetal bovine serum (FBS, Gibco BRL, Grand Island, NY). FGM is composed of fibroblast basal medium (FBM) supplemented with basic fibroblast growth factor (bFGF, 1 ng per ml, final concentration), insulin (5 μg per ml), gentamicin (50 μg per ml), and amphotericin-B (10 ng per ml). The same batch of serum was used throughout the study. The explant procedure was compared with enzymatic digestion of granulation tissue obtained from the two polytetrafluoroethylene tubes. Fibroblasts were cultured either from explanted minced tissue as described above or from minced tissue that had been digested with 200 U bacterial collagenase type IA (Sigma, St Louis, MO) per ml FBM for 18 h at 37°C in a humidified atmosphere of 5% CO2/air essentially as described byRegan et al., 1991Regan M.C. Kirk S.J. Wasserkrug H.L. Barbul A. The wound environment as a regulator of fibroblast phenotype.J Surg Res. 1991; 50: 442-448Abstract Full Text PDF PubMed Scopus (52) Google Scholar. Cell growth was similar for the two procedures with a mean saturation density on post-plating day 10 (see Growth kinetics below) of 6.9 × 104 for the fibroblasts grown from explants compared with 7.3 × 104 for collagenase-treated tissue. Initial experiments also showed that the growth of human dermal fibroblasts, obtained from explanted skin from the posterior lower leg of a healthy 30 y old male, increased significantly when using FGM supplemented with 10% FBS compared with using Dulbecco's modified Eagle's medium (DMEM) with Glutamax, 4.5 g glucose per liter and supplemented with 10% FBS. The saturation density, achieved with DMEM with 10% FBS, was 2.1 ± 0.1 × 104 compared with 1.2 ± 0.1 × 105 for fibroblasts cultured in FGM supplemented with 10% FBS. Furthermore, FGM with 10% FBS supported outgrowth of fibroblasts from chronic wound tissue. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2/air. Culture medium was changed twice weekly. Subcultivation was performed using mycoplasma-tested trypsin/ethylenediaminetetraacetic acid (EDTA) (0.05%/0.02% in PBS pH 7.4; Biological Industries, Kibbutz Beit Haemek, Israel) and cells were split at a ratio of 3:1 at confluence (Hayflick and Moorhead, 1961Hayflick L. Moorhead P.S. The serial cultivation of human diploid cell strains.Exp Cell Res. 1961; 25: 585-621Crossref PubMed Scopus (5003) Google Scholar). Cells of the third passage were used for the assays. Phase-contrast microphotography was performed using an inverted microscope (Nikon TMS, 10 × objective) on all fibroblast strains. Selective cell strains were checked immunocytochemically for vimentin (Boehringer Mannheim, Germany), cytokeratins 5, 6, 8, and 17 (M 821; DAKO, Glostrup, Denmark), and von Willebrand factor (A 082; DAKO). All fibroblast strains examined were found positive for vimentin, and negative for cytokeratins and von Willebrand factor. The cell strains checked for mycoplasma by polymerase chain reaction were found negative. Day 0, 3.5 × 103 cells per cm2 were seeded in 24 well plates (Nunc, Roskilde, Denmark) in 1 ml complete medium per well, and enumerated 3, 4, 5, 6, 7, 8, 9, and 10 d post-plating in triplicates using a Bürker hemocytometer. Cells were re-fed with new medium days 4 and 7 during the 10 d growth period. Growth curves were generated for each individual fibroblast strain, and the population doubling time for the individual fibroblast strains was determined graphically during their logarithmic growth and the fibroblast density determined at saturation (Freshney, 1991Freshney R.I. Culture of animal cells.A Manual of Basic Technique. 2nd edn. Wiley-Liss, New York1991Google Scholar). All wells were inspected using inverted phase-contrast microscopy for aberrant cell behavior and for detached cells throughout the 10 d growth period. The effect of conditioned medium from the three groups of fibroblast strains on cell proliferation was studied in a complementary experiment. Fifteen milliliters of conditioned complete medium (FGM with 10% FBS) was obtained from confluent fibroblasts, in 75 cm2 plastic flasks grown for 72 h, from chronic and acute wounds, and dermis, respectively, from the same patients. The target cells (dermal fibroblasts) were seeded into 96 well plates (Nunc) at 3.5 × 103 cells per cm2 and 3.5 × 104 cells per cm2 and grown for 24 h, and then exposed to the conditioned media for 24 h. Fibroblasts were also treated with unconditioned complete media. All media were sterile-filtered (0.22 μm) before being added to the cells. Cell proliferation was measured as incorporation of the thymidine analogue, 5-bromo-2′-deoxyuridine (BrdU, 10 μM, final concentration) into the dermal fibroblasts during the last hour of incubation. The incorporation of BrdU was determined using an ELISA kit (Boehringer Mannheim) and ΔOD (OD370 nm– OD492 nm) read by an ELISA reader (Labsystems, Helsinki, Finland). To study further the proliferative ability, one representative fibroblast strain from each group was pulse-labelled with BrdU. Cells were seeded to about 50% confluence (104 cells per cm2) on Chamber Glass Slides (Lab-Tek, Nunc) in complete culture medium and incubated for 24 h. BrdU (10 μM, final concentration) was added during the last hour of incubation. Cells were then washed three times with PBS, fixed with glycine buffer (50 mM glycine in 70% ethanol, pH 2.0). Cells synthesizing DNA were detected immunocytochemically using a kit from Boehringer Mannheim. Cells were seeded into 96 well plates (Nunc) at 1 × 104 cells per well in complete medium and incubated for 72 h. The medium was then replaced with 100 μl FBM containing 2.5% FBS per well and the cells were incubated for another 24 h. Growth factors in 25 μl FBM containing 1% (wt/vol) bovine serum albumin (BSA, Sigma) were added to a final volume of 125 μl and 25 μl FBM containing 1% BSA alone to the six control wells. Human recombinant PDGF-BB, PDGF-AA, and transforming growth factor (TGF)-β1 were obtained from Gibco BRL. Human recombinant bFGF and epidermal growth factor (EGF) were purchased from Sigma. Two-fold dilutions of each growth factor were used at six final concentrations in duplicate (PDGF, 0.6–20 ng per ml; bFGF, 0.15–5 ng per ml; EGF, 1.5–50 ng per ml; TGF-β1, 0.3–10 ng per ml). The cells were then incubated with or without growth factors for 18 h. BrdU in FBM medium (10 μl per well) was added to all wells to a final concentration of 10 μM except for the background control (unspecific binding of the anti-BrdU conjugate to the cells in the absence of BrdU), which received 10 μl of FBM medium alone. The cells were incubated for an additional 2 h and then washed three times with PBS. The effect of the growth factors on fibroblast proliferation was expressed as the percentage of BrdU incorporated, measured with the ELISA described above under Growth kinetics, into control-treated cells after subtraction of blank values for respective growth factor. The number of α-receptors and β-receptors were quantified in detergent-solubilized cell membranes using a membrane solid phase immunoassay (slot-blot technique) with monoclonal antibodies. The immunoreactions in the slot-blots were then quantified with densitometry, which is an established quantitative method (Towbin and Gordon, 1984Towbin H. Gordon J. Immunoblotting and dot immunobinding—current status and outlook.J Immunol Methods. 1984; 72: 313-340Crossref PubMed Scopus (781) Google Scholar). To improve the precision of the assay, cell membrane proteins at three 2-fold dilutions (each dilution assayed in duplicate) from the three groups were included in the same blot. Probing with the same concentration of the two primary antibodies against the α-receptors and β-receptors and color development were carried out simultaneously to decrease variability between the immunoreactions of α-receptor and β-receptor. Membrane extract was obtained after lysing confluent cells grown in FGM with 10% FBS with 1% (vol/vol) Triton X-100 with 3 mM phenylmethylsulfonyl fluoride for 30 min at 4°C (Ågren et al., 1996Ågren M.S. Haisa M. Grotendorst G.R. Differential expression of platelet-derived growth factor receptors in porcine fibroblasts cultured from skin and granulation tissue.Wound Repair Regeneration. 1996; 4: 288-296Crossref Scopus (2) Google Scholar). Extracts were diluted to lie within the linear range of the assay in Tris(hydroxymethyl)-aminomethane (Tris)–HCl-buffered-saline (pH 7.4), added to a 48 well slot-blot apparatus (Bio-Rad, Hercules, CA) and filtered through nitrocellulose membrane (Trans-Blot, 0.45 μm, Bio-Rad). Bound PDGF receptors were immunoreacted for 18 h at ambient temperature with mouse monoclonal antibodies (10 ng per ml, Genzyme, Cambridge, MA) against the human PDGF α-receptor and the human PDGF β-receptor (10 ng per ml, Genzyme) (Hart et al., 1987Hart C.E. Seifert R.A. Ross R. Bowen-Pope D.F. Synthesis, phosphorylation, and degradation of multiple forms of the platelet-derived growth factor receptor studied using a monoclonal antibody.J Biol Chem. 1987; 262: 10780-10785Abstract Full Text PDF PubMed Google Scholar). The primary antibodies were immunoreacted for 90 min with biotinylated swine anti-mouse IgG diluted 1:2500 (E 0453; DAKO) followed by treatment for 30 min with streptavidin complexed with biotinylated alkaline phosphatase (K 0391; DAKO) according to the manufacturer's instructions. Nitro blue tetrazolium/ 5-bromo-4-chloro-3-indolyl phosphate was applied to visualize the receptor-bound complexes. The blots were scanned (Hewlett Packard ScanJet 4). The intensities of the bands were quantified using a software from Jandel (SigmaGel), normalized to the protein content (DC Protein Assay Kit with bovine serum albumin as standard, Bio-Rad) of the cell extracts and expressed as arbitrary units per μg cell protein. Kruskal–Wallis one-way analysis of variance was applied to the population doubling time, cell density, and PDGF-receptor data. Wilcoxon matched-pairs signed-ranks test was applied when comparing levels of α- and β-receptor within the three fibroblast groups. In case of statistical significance (p < 0.05), Bonferroni's correction for multiple hypotheses was used to ensure that type I errors were not multiplied. The effect of ulcer age on fibroblast growth was tested using the Student's t-test for unpaired observations. Numerical data are given as mean ± SEM. Fibroblasts from acute wounds did not show any morphologic difference at a light microscopic level compared with those from normal dermis. They all displayed the characteristic spindle-shaped appearance and swirl growth pattern. Chronic wound fibroblasts commonly exhibited an altered morphology with irregular cell shape, multinucleation, larger and more variable cell size, and accumulation of debris (Figure 1a–c) .Figure 1Different morphology and growth kinetics for fibroblasts cultured from chronic wounds, acute wounds, and normal dermis. Representative phase-contrast microphotographs of fibroblasts from chronic wounds (a), acute wounds (b), and dermis (c) 10 d post-plating. Note the irregular shape, large size, and sparsely growth of the chronic wound fibroblasts (a) compared with spindle-shaped and tightly grown acute wound (b) and normal dermal fibroblasts (c). Scale bar: 200 μm. Each growth curve depicted on a logarithmic y-axis in (d)represents the mean of eight different fibroblast strains from chronic wounds (○), 10 different fibroblast strains from acute wounds (U25CF;), and five different fibroblast strains from normal dermis (□). Error bars: SEM.View Large Image Figure ViewerDownload (PPT) Although plating efficiency was not determined per se, a negligible number of detached fibroblasts was observed in any well prior to enumeration on the third post-plating day. Overall, chronic wound fibroblasts grew at a slower rate than those obtained from acute wounds and normal dermis (Figure 1d). The mean population doubling time, as determined from the slope of the individual growth curves, was almost four times longer for chronic wound fibroblasts than for acute wound and dermal fibroblasts (Table I). Chronic wound fibroblasts reached a density 10 d post-plating of about a fourth of the cell density of fibroblasts from acute wounds and dermis (Table I). Five of the eight chronic wound fibroblast strains approached or reached a replicative senescent growth behavior. Those fibroblast strains were derived from venous leg ulcers that had been present for more than 3 y. Statistical analyses revealed that those fibroblasts (n = 5) grew to a statistically significantly (p < 0.01) lower saturation cell density (6.6 ± 2.0 × 103 cells per cm2) than those from leg ulcers younger (n = 3) than 3 y (4.3 ± 0.4 × 104 cells per cm2). The mean population doubling time was also extended by the fibroblasts from the old ulcers (166.8 ± 53.5 h) compared with fibroblasts from ulcers younger than 3 y (38.6 ± 6.6 h), although this difference did not reach statistical significance (p = 0.074). Acute wound fibroblasts reached a significantly (p < 0.05) lower saturation cell density than dermal fibroblasts 10 d post-plating (Table I).Table IPopulation doubling time was increased and saturation density decreased for chronic wound fibroblastsChronic woundspAcute woundspNormal dermisNumber of strains8105Population doubling time (h)118.7 ± 39.7aMean ± SEM.<0.0532.6 ± 5.5NSbA statistically non-significant (p > 0.05) difference.27.9 ± 3.3Saturation density (cells per cm2 × 10–4)2.0 ± 0.7<0.058.8 ± 1.7<0.0515.5 ± 1.9a Mean ± SEM.b A statistically non-significant (p > 0.05) difference. Open table in a new tab In five of the chronic wounds, fibroblasts of peri-wound skin were also expanded in vitro to see if they differed from those in the chronic wounds in terms of growth. The growth of fibroblasts from the chronic venous leg ulcers was severely diminished compared with those from adjacent uninjured tissues. One biopsy from adjacent normal skin in one patient and one biopsy from adjacent lipodermatosclerotic skin in another patient were excluded due to interfering epithelial outgrowth from the biopsies. In the remaining three patients, the growth of fibroblasts from peri-ulcer lipodermatosclerotic skin appeared to be similar to fibroblasts from normal skin of the same lower limb, which again was lower in the chronic wounds (Table II). Thus, it appears that the fibroblasts in the chronic wounds are different regarding cellular growth from those of adjacent tissue.Table IIPopulation doubling time was increased and saturation density decreased for chronic wound fibroblasts compared with adjacent lipodermatosclerotic and normal skin in three patientsChronic woundsLipodermatosclerotic dermisNormal dermisPopulation doubling time (h)111.0 ± 67.7aMean ± SEM.45.9 ± 6.740.4 ± 4.9Saturation density (cells per cm2 × 10–4)2.7 ± 1.29.1 ± 1.99.6 ± 0.4a Mean ± SEM. Open table in a new tab The possibility of the chronic wound fibroblasts producing growth inhibitory molecules that might decrease cell growth was investigated. BrdU incorporation, expressed as ΔOD, into dermal fibroblasts from normal skin (3.5 × 103 cells per cm2) exposed to conditioned media for 24 h from fibroblasts derived from one chronic wound and from adjacent normal skin was 0.449 ± 0.020 (n = 3) and 0.406 ± 0.020 (n = 2), respectively. The corresponding results for the acute wound and adjacent skin were 0.413 ± 0.026 (n = 5) and 0.492 ± 0.031 (n = 3), respectively. Unconditioned complete media resulted in a ΔOD of 0.780 ± 0.032 (n = 6). Similar results were obtained when the target fibroblasts were seeded at an initial higher density (3.5 × 104 cells per cm2). These results thus indicate that the observed decreased growth of chronic wound fibroblasts was not due to an autocrine suppressive mechanism. Further proof of a diminished proliferation of chronic wound fibroblasts was an abolished or decreased DNA synthesis as evidenced by no or reduced incorporation of the thymidine analog BrdU (Figure 2). Human recombinant mitogens added to growth-arrested cells elicited DNA synthesis, indicating the presence of functional receptors on the various cell strains. BrdU-incorporation in the fibroblast strains, however, from the four chronic wounds of the longest duration (6, 10, 10, and 20 y) was below the limit of detection. PDGF-BB was the most mitoge