Dermal Fibroblasts from Different Layers of Pig Skin Exhibit Different Profibrotic and Morphological Characteristics

In vitro studies of human dermal fibroblast (DF) heterogeneity have long been reported, yet in vivo studies and related research on animals are rare. The objectives of the study were to determine whether the DFs of pigs exhibit heterogeneity and to identify an animal model for the in vivo study of DF heterogeneity. The skin of three female red Duroc pigs (FRDPs) was separated into six layers, and the second and fifth layers (i.e., the superficial and deep dermis) were used in the establishment of wound models and cell cultures. To create the wound models, 54 tongue‐shaped flaps were created on one side of the dorsum, and the underlying dermis was then fully replaced with the superficial or deep dermis (the superficial and deep groups, respectively). Skin samples were harvested at postoperative weeks 1, 2, and 3 for measurements of the normal and wounded skin thicknesses. Cells cultured from the superficial and deep dermis (i.e., superficial and deep DFs) were subjected to quantitative estimation of collagen and electron microscopy. The wounded skin thickness in the deep group was significantly greater than that in the superficial group. In contrast with the long deep DFs, the superficial DFs were short and exhibited microvilli‐like cell surface projections. Compared with the superficial DFs, the deep DFs exhibited a greater density of rough endoplasmic reticulum and produced significantly more collagen. Similar to humans, FRDPs exhibit DF heterogeneity and should thus be a good animal model for in vivo studies of DF heterogeneity. Anat Rec, 299:1585–1599, 2016. © 2016 Wiley Periodicals, Inc.     

Chondrogenic Differentiation of Adult Dermal Fibroblasts

Cell sources for generation of articular cartilage ex vivo are limited. To explore options other than stem cells, dermal fibroblasts were tested for their developmental potential when cultured on the cartilage matrix proteoglycan, aggrecan. A previous study suggested such an effort would be successful (M. M. French et al., Journal of Cell Biology 145:1103-1115, 1999). The adult dermal fibroblast cell line, RAB-9, was used in these assays. While initial attempts to differentiate the cells were unsuccessful, after pretreatment with insulin growth factor one (IGF-I), the cells were able to differentiate in culture on aggrecan. After 24 h in culture on aggrecan, the majority of the cells formed dense aggregates reminiscent of condensing mesenchymal cells in development. At 1 week, these aggregates stained positively with both Safranin O and antibodies against collagen type II. This staining was maintained through the conclusion of the experiment at week 4. RT-PCR for collagen II supports the hypothesis that dermal fibroblasts can be triggered to differentiate by culture on cartilage matrix proteoglycans. A three-fold increase in collagen type II mRNA expression is seen when cells are cultured on aggrecan in comparison to controls. These results provide an initial step towards a cell source that may prove equally successful for the generation of cartilage in the laboratory.     

Electrical Stimulation Increases Random Migration of Human Dermal Fibroblasts

Exogenous electrical stimulation (ES) has been investigated as a therapy for chronic wounds, as the skin produces currents and electrical fields (EFs) during wound healing. ES therapies operate by applying small EFs to the skin to mimic the transepithelial potentials that occur during the granulation phase of wound healing. Here, we investigated the effect of short duration (10 min) ES on the migration of HDFs using various magnitudes of physiologically relevant EFs. We modeled cutaneous injury by culturing HDFs in custom chambers that allowed the application of ES and then performed timelapse microscopy on a standard wound model. Using MATLAB to process cell coordinate data, we determined that the cells were migrating randomly and fit mean squared displacement data to the persistent random walk equation using nonlinear least squares regression analysis. Results indicated that application of 25–100 mV/mm DC EFs to HDFs on either uncoated or FN-coated surfaces demonstrated no significant changes in viability or proliferation. Of significance is that the HDFs increased random migration behavior under some ES conditions even after 10 min, providing a mechanism to enhance wound healing.     

Staphylococcus aureus Induces Hypoxia and Cellular Damage in Porcine Dermal Explants

We developed a porcine dermal explant model to determine the extent to which Staphylococcus aureus biofilm communities deplete oxygen, change pH, and produce damage in underlying tissue. Microelectrode measurements demonstrated that dissolved oxygen (DO) in biofilm-free dermal tissue was 4.45 ± 1.17 mg/liter, while DO levels for biofilm-infected tissue declined sharply from the surface, with no measurable oxygen detectable in the underlying dermal tissue. Magnetic resonance imaging demonstrated that biofilm-free dermal tissue had a significantly lower relative effective diffusion coefficient (0.26 ± 0.09 to 0.30 ± 0.12) than biofilm-infected dermal tissue (0.40 ± 0.12 to 0.48 ± 0.12; P < 0.0001). Thus, the difference in DO level was attributable to biofilm-induced oxygen demand rather than changes in oxygen diffusivity. Microelectrode measures showed that pH within biofilm-infected explants was more alkaline than in biofilm-free explants (8.0 ± 0.17 versus 7.5 ± 0.15, respectively; P < 0.002). Cellular and nuclear details were lost in the infected explants, consistent with cell death. Quantitative label-free shotgun proteomics demonstrated that both proapoptotic programmed cell death protein 5 and antiapoptotic macrophage migration inhibitory factor accumulated in the infected-explant spent medium, compared with uninfected-explant spent media (1,351-fold and 58-fold, respectively), consistent with the cooccurrence of apoptosis and necrosis in the explants. Biofilm-origin proteins reflected an extracellular matrix-adapted lifestyle of S. aureus. S. aureus biofilms deplete oxygen, increase pH, and induce cell death, all factors that contribute to impede wound healing.     

Differential Expression of Wound Fibrotic Factors between Facial and Trunk Dermal Fibroblasts

Clinically, wounds on the face tend to heal with less scarring than those on the trunk, but the causes of this difference have not been clarified. Fibroblasts obtained from different parts of the body are known to show different properties. To investigate whether the characteristic properties of facial and trunk wound healing are caused by differences in local fibroblasts, we comparatively analyzed the functional properties of superficial and deep dermal fibroblasts obtained from the facial and trunk skin of seven individuals, with an emphasis on tendency for fibrosis. Proliferation kinetics and mRNA and protein expression of 11 fibrosis-associated factors were investigated. The proliferation kinetics of facial and trunk fibroblasts were identical, but the expression and production levels of profibrotic factors, such as extracellular matrix, transforming growth factor-β1, and connective tissue growth factor mRNA, were lower in facial fibroblasts when compared with trunk fibroblasts, while the expression of antifibrotic factors, such as collagenase, basic fibroblast growth factor, and hepatocyte growth factor, showed no clear trends. The differences in functional properties of facial and trunk dermal fibroblasts were consistent with the clinical tendencies of healing of facial and trunk wounds. Thus, the differences between facial and trunk scarring are at least partly related to the intrinsic nature of the local dermal fibroblasts.     

Altered Expression of Connective Tissue Genes in Postnatal Chondroinduced Human Dermal Fibroblasts

In a novel model for postnatal chondroinduction, normal human dermal fibroblasts (hDFs) cultured with demineralized bone powder (DBP) express chondrocyte features after 7 days. Representational difference analysis (RDA) prior to overt chondroblastogenesis (3 days) revealed altered expression of connective tissue genes (collagens, collagen receptors, and post-translational enzymes). Northern or RT-PCR analysis at 3, 7, 14, or 21 d showed different expression patterns for those genes. COL3A1 was transiently upregulated, whereas upregulation of COL11A1, integrin &#102 -11, lysyl oxidase, and lysyl hydroxylase 2 mRNAs persisted for 7 days. Downregulation of decorin was sustained for 21 d. The expression of the post-translational enzymes induced by DBP was unique when compared with human skin and human articular chondrocytes. Thus, the data suggest an "induced chondroblast" stage with a unique connective tissue gene expression profile that may result in a matrix supportive of chondrogenesis by postnatal cells.     

Differential Expression of Wound Fibrotic Factors between Facial and Trunk Dermal Fibroblasts

Clinically, wounds on the face tend to heal with less scarring than those on the trunk, but the causes of this difference have not been clarified. Fibroblasts obtained from different parts of the body are known to show different properties. To investigate whether the characteristic properties of facial and trunk wound healing are caused by differences in local fibroblasts, we comparatively analyzed the functional properties of superficial and deep dermal fibroblasts obtained from the facial and trunk skin of seven individuals, with an emphasis on tendency for fibrosis. Proliferation kinetics and mRNA and protein expression of 11 fibrosis-associated factors were investigated. The proliferation kinetics of facial and trunk fibroblasts were identical, but the expression and production levels of profibrotic factors, such as extracellular matrix, transforming growth factor-β1, and connective tissue growth factor mRNA, were lower in facial fibroblasts when compared with trunk fibroblasts, while the expression of antifibrotic factors, such as collagenase, basic fibroblast growth factor, and hepatocyte growth factor, showed no clear trends. The differences in functional properties of facial and trunk dermal fibroblasts were consistent with the clinical tendencies of healing of facial and trunk wounds. Thus, the differences between facial and trunk scarring are at least partly related to the intrinsic nature of the local dermal fibroblasts.     

Autologous Versus Allogeneic T Cell-Stimulated IL-6 Production by Dermal Fibroblasts

T cells adhere to human dermal fibroblasts (HDF). This cellular interaction leads to a pronounced secretion of the proinflammatory cytokines IL-6 and IL-8 via a juxtacrine stimulation induced by HDF-associated IL-1. Upon stimulation, fibroblasts express various surface proteins such as MCH-I molecules, which may interact with corresponding receptors on T cells. The present study was conducted to further investigate the mechanism of this complex interaction with regard to the secretion of IL-6 in cocultures of T cells and HDF. IL-6 was time- and dose-dependently upregulated in such cocultures. Spatial separation of the cells by microporous membranes resulted in a 90% reduction of IL-6 secretion, but when cells had limited cell contact IL-6 secretion was increased again. Allogeneic cocultures of T cells and HDF showed increased capacity of IL-6 stimulation as compared to autologous cultures. Our results suggest that MHC-I/T cell receptor interaction modulates IL-6 secretion in allogeneic and autologous cocultures.     

Senescent Cultures of Human Dermal Fibroblasts Modified Phenotype When Immobilized in Fibrin Polymer

One of the limitations in tissue engineering is the restricted ability to expand the number of cells, because somatic cells can duplicate a limited number of times before they lose the ability to divide, leading to a senescent state. Here we report that the interaction of senescent fibroblasts with fibrin polymer can modify the senescent phenotype and partially restore the ability of growth-arrested cells to continue replicating. Primary human dermal fibroblasts were grown to >90% SA/β-Gal (senescence associated β-galactosidase). The senescent cells were immobilized in fibrin-polymers by mixing fibrinogen and thrombin solutions. Immobilized senescent cell cultures grew, however, their growth arrested after 24 h of immobilization. The percentage of cells with a positive reaction at SA/β-Gal did not decrease significantly after immobilization, but the intensity of the stain decreased. The glycolytic activity in immobilized senescent fibroblast was re-established at pre-senescent levels. In conclusion, fibrin induces changes in the phenotype of senescent human fibroblasts. This simple procedure could complement available tissue-engineering techniques to increase the amount of biomass seeded on a fibrin scaffold, which could be beyond senescence.     

Lipopolysaccharide Induces Mucus Cell Metaplasia in Mouse Lung

A murine model of lipopolysaccharide (LPS)-induced airway inflammation and epithelial cell phenotypic change, and the time courses of these events are described. A single intratracheal instillation of Pseudomonas aeruginosa LPS in mice resulted in massive recruitment of neutrophils to the lung 2 d after treatment as assessed by differential cell counts of the inflammatory cells in bronchoalveolar lavage fluid and histologic assessment of hematoxylin and eosin (H&E)-stained lung sections. The LPS-induced neutrophilic inflammation subsided substantially on Day 4 and essentially vanished by Day 7. Airway epithelial mucus cells were not detected by Alcian blue periodic acid-Schiff staining until Day 4 after LPS treatment and became more abundant in number as well as in mucus content on Day 7. The expression of Muc5ac messenger RNA (mRNA) as well as glycoprotein was enhanced on Day 2, peaked on Day 4, and decreased on Day 7, whereas enhanced expression of mucin core 2 beta6 N-acetylglucosaminyltransferase (C2GnT)-M mRNA was not detected until Day 4 and peaked on Day 7. The expression of C2GnT-L mRNA in the lung, a marker for activated leukocytes as well as mucus cells, peaked on Day 2 and remained moderately high until Day 7. C2GnT-L mRNA expression in LPS-treated lung correlated with the presence of neutrophils and the appearance of mucus cells in the airway epithelium. We conclude that mucus cell metaplasia and hyperplasia can be generated in mouse lungs with a single intratracheal instillation of LPS. In addition, C2GnT-M may serve as a marker for mucus cells in mouse lung. This LPS-induced mucus cell metaplasia and hyperplasia model should be useful for the study of Pseudomonas-induced airway mucus hypersecretory diseases.     

再生丝素膜对鼠胚真皮层成纤维细胞遗传特性的影响

目的 :研究再生丝素膜 (WL组 ,HY组 ,SD组 )对大鼠鼠胚成纤维细胞的遗传物质的影响。方法 :采用浸提液法在体外培养鼠胚成纤维细胞 ,计算细胞的微核率 (MNF)和染色体畸变率 (CAF) ,用单细胞电泳法 (SCGEassay)检测基因组DNA的损伤程度。结果 :WL组、SD组再生丝素膜和空白对照组的细胞微核率 (MNF)和染色体畸变率(CAF)无显著性差异 (P >0 0 5 ) ,无明显彗星现象 ;而HY组再生丝素膜与阴性对照比较有显著差异 (P <0 0 1)。结论 :WL组和SD组再生丝素膜对种子细胞的遗传学特性无明显影响 ,优于HY组。     

Bactericidal/Permeability-Increasing Protein Is Expressed by Human Dermal Fibroblasts and Upregulated by Interleukin 4

The bactericidal/permeability-increasing protein (BPI) is an antibiotic- and endotoxin-neutralizing protein of granulocytes and epithelial cells. Constitutive expression of BPI, which increases upon interleukin 4 stimulation, by human dermal fibroblast was demonstrated, suggesting an important role of BPI in gram-negative bacterial clearance and a dampened response to endotoxin in the skin.     

Chitosan/Pluronic Hydrogel Containing bFGF/Heparin for Encapsulation of Human Dermal Fibroblasts

Abstract

In order to act as a cell reservoir, a hydrogel has to have a suitable mechanical strength and should also enhance proliferation of cells. We have developed a hydrogel composed of chitosan and Pluronic containing a complex of basic fibroblast growth factor (bFGF) and heparin for cell encapsulation, and its ability as a scaffold for cells was evaluated. The hydrogel was composed of glycidyl methacrylated chitooligosaccharide (COS) and diacrylated Pluronic F127 (Pluronic) in various blend ratios. A mixture of COS and Pluronic was homogeneously mixed with cells, bFGF and heparin; then, the hydrogel precursor was photo-irradiated. With increasing amount of COS, the chemically cross-linked COS/Pluronic hydrogel showed an approx. 2-fold increase of its elastic modulus and a lower mass erosion rate than the hydrogel composed of only Pluronic after 28 days. The amount of bFGF was increased according to the presence of heparin within the hydrogel, but the amount of released bFGF was decreased by increasing the content of COS. The hydrogel containing bFGF with heparin showed higher proliferation of cells compared to the hydrogel without heparin at 20% COS. However, when the blend ratio of COS was 50%, the hydrogel showed less proliferation of cells than those with 20% COS. Therefore, the chemically cross-linked COS/Pluronic hydrogel be useful for a protein-delivery system and tissue-engineering scaffold.     

IL-4 and IL-13 Induce Chemotaxis of Human Foreskin Fibroblasts, But Not Human Fetal Lung Fibroblasts

Through shared receptors, IL-4 and IL-13 have been suggested to regulate not only inflammatory cells, but also to play a role in stimulating fibroblasts during fibrotic processes. Previous studies have shown that IL-4 is a chemoattractant for foreskin fibroblasts. The current study was designed to determine the effect of IL-4 and IL-13 on the migration of two types of fibroblasts: foreskin and human fetal lung fibroblasts (HFL-1). Using the Boyden blindwell chamber method, human foreskin or fetal lung fibroblasts (both 10(6)/mL) were placed in upper wells with various concentrations of IL-4 or IL-13 in the lower wells as chemoattractants. Both IL-4 (1 pg/mL) and IL-13 (100 pg/mL) induced foreskin fibroblast chemotaxis, up to 50 +/- 8 and 24 +/- 7 cells/5 high-power fields, respectively (both p < 0.05). In contrast, neither cytokine induced migration of the lung fibroblasts although both type of cells express IL-4 receptor and IL-13alpha1 receptor. These results suggest that fibroblasts are heterogeneous with regard to their ability to respond to cytokine-driven chemotaxis. Therefore, the role of specific cytokines in mediating fibrotic responses might vary depending on local mesenchymal cell responses.     

体外培养不同代次成纤维细胞胶原基因表达及成纤维细胞对EGF的反应性

目的　观察体外培养不同代次人成纤维细胞Ⅰ、Ⅲ型胶原基因的表达及对EGF刺激的反应性。方法　将正常人二倍体成纤维细胞进行连续传代培养 ,以不同代次的细胞为实验对象 ,应用RT PCR、3 H TdR掺入量测定等方法对不同代次细胞进行检测。结果　随细胞代次的增加 ,Ⅰ、Ⅲ型胶原mRNA含量逐渐降低 ,3 H TdR掺入量降低 ,EGF促进P10 培养的成纤维细胞增殖 ,对衰老成纤维细胞 (P60 )无生长增殖的调控能力。结论　为建立人成纤维细胞衰老模型提供部分实验依据。