Comparative growth dynamics and morphology between cultured myofibroblasts from granulating wounds and dermal fibroblasts

Rat myofibroblasts from granulating wound biopsies (RGW) were successfully cultured and compared in terms of growth and morphology with fibroblasts from uninjured rat dermis (RD). Populations of early passage (P-3) RGW myofibroblasts grew significantly more slowly than RD fibroblasts. Logarithmic growth was nearly the same in late passage (P-30) populations of both cell types. Early passage RGW myofibroblasts were similar to those in vivo, as shown by well-defined microfilament bundles. RD fibroblasts contained less well defined microfilaments. Both late passage RGW myofibroblasts and RD fibroblasts displayed evidence of morphologic dedifferentiation. These data show that morphologic features of myofibroblasts and fibroblasts in vivo are maintained in vitro. Evidence is presented that cultured animal myofibroblasts maintain differentiation in early passage, whereas late passage cells suggest that these differences disappear with time.     

Heparin induces alpha-smooth muscle actin expression in cultured fibroblasts and in granulation tissue myofibroblasts.

BACKGROUND: Heparin increases alpha-smooth muscle actin expression in smooth muscle cells in vivo and in vitro. It has been recently suggested that alpha-smooth muscle actin expression in fibroblasts is a marker of myofibroblastic differentiation. We have examined the effect of heparin and of four nonanticoagulant heparin derivatives on alpha-smooth muscle actin expression by fibroblasts in vitro and in vivo. EXPERIMENTAL DESIGN: For in vitro experiments, heparin was added for 7 days to different fibroblastic cultures. We studied cell proliferation and alpha-smooth muscle actin protein and mRNA expression. For in vivo studies, osmotic minipumps filled with NaCl or tumor necrosis factor-alpha without or with nonanticoagulant heparin were implanted subcutaneously. After 14 days, newly accumulated connective tissues around the pumps were processed for immunofluorescence and electron microscopic and biochemical studies. RESULTS: In vitro, heparin inhibited proliferation and increased the expression of alpha-smooth muscle actin protein and mRNA. Analysis of [3H]thymidine incorporation in synchronized cells suggested that heparin produces a selection of alpha-smooth muscle actin expressing cells. In vivo, the local application of tumor necrosis factor-alpha resulted in formation of a typical granulation tissue: immunofluorescence showed that accumulated fibroblastic cells express alpha-smooth muscle actin only in the presence of heparin derivatives. In tumor necrosis factor-alpha treated animals, electron microscopic examination established the presence of myofibroblasts, but alpha-smooth muscle actin was expressed in microfilament bundles only in the presence of heparin derivatives. CONCLUSIONS: These results show that heparin and its nonanticoagulant derivatives influence the expression of alpha-smooth muscle actin in fibroblastic cells both in vitro and in vivo and that this effect is probably related to the selection of a particular cell subpopulation. They suggest a possible role for heparin during the formation and evolution of granulation tissue.     

Sphingomyelinase in cultured skin fibroblasts from normal and Niemann-Pick type C patients

The spingomyelinase activities of extracts of normal cultured skin fibroblasts were compared with those obtained from Niemann-Pick disease Type A and B patients, and from a number of patients with a provisional clinical diagnosis of Niemann-Pick disease Type C. Even though fibroblasts from Type A and B patients were shown to be clearly deficient (<5% residual activity) the activity in Type C fibroblasts varied from approximately 50% of the lowest control value to normal activity.
Isoelectric focussing of extracts of normal cultured skin fibroblasts on polyacrylamide gels revealed the presence of sphingomyelinase heterogeneity. However, no characteristic change in the behaviour of the enzyme in corresponding extracts from Niemann-Pick Type C cells was observed, suggesting that, if the defect in this condition is expressed in fibroblasts, it does not manifest biochemically in the appearance or disappearance of a specific sphingomyelinase isoenzyme.
Our data suggest that the heterogeneity observed in fibroblast extracts may simply reflect an interaction of the enzyme either with itself or with other hydrophobic components present in the cellular extracts.     

Cytogenetically marked clones in human fibroblasts cultured from normal subjects

Clones of cytogenetically abnormal cells have been recognized in fibroblasts cultured from normal human adult skin. No such clones have been observed in human embryo skin fibroblasts cultured in the same way. Although the culture conditions may have played some part in the emergence of these clones, it is possible that the abnormal cells from which the clones were derived were present in vivo.     

In vitro characterization of natural and synthetic dermal matrices cultured with human dermal fibroblasts

The ideal dermal matrix should be able to provide the right biological and physical environment to ensure homogenous cell and extracellular matrix (ECM) distribution, as well as the right size and morphology of the neo-tissue required. Four natural and synthetic 3D matrices were evaluated in vitro as dermal matrices, namely (1) equine collagen foam, TissuFleece, (2) acellular dermal replacement, Alloderm, (3) knitted poly(lactic-co-glycolic acid) (10:90)-poly(-caprolactone) (PLGA-PCL) mesh, (4) chitosan scaffold. Human dermal fibroblasts were cultured on the specimens over 3 weeks. Cell morphology, distribution and viability were assessed by electron microscopy, histology and confocal laser microscopy. Metabolic activity and DNA synthesis were analysed via MTS metabolic assay and [(3)H]-thymidine uptake, while ECM protein expression was determined by immunohistochemistry. TissuFleece, Alloderm and PLGA-PCL mesh supported cell attachment, proliferation and neo-tissue formation. However, TissuFleece contracted to 10% of the original size while Alloderm supported cell proliferation predominantly on the surface of the material. PLGA-PCL mesh promoted more homogenous cell distribution and tissue formation. Chitosan scaffolds did not support cell attachment and proliferation. These results demonstrated that physical characteristics including porosity and mechanical stability to withstand cell contraction forces are important in determining the success of a dermal matrix material.     

Enhancement of gap junctional intercellular communication of normal human dermal fibroblasts cultured on polystyrene dishes grafted with poly-N-isopropylacrylamide

Technology developed to allow recovery of cells without enzyme treatment, involving a dish grafted with a thermoreactive polymer gel of poly-N-isopropylacrylamide (PIPAAm), was found to significantly enhance gap junctional intercellular communication (GJIC) in normal human dermal fibroblasts (NHDF cells). NHDF cells were cultured for 4 days on PIPAAm-grafted dishes irradiated with various doses of electron beams, and GJIC was assayed by the scrape-loading dye transfer method. The area of dye transfer was greater in the PIPAAm-grafted dishes than in the control culture dishes, indicating that the PIPAAm-grafted dishes enhanced the GJIC of NHDF cells. Connexin-43 (Cx43) expression was analyzed because Cx43 is considered to be a main component of the gap junctional channel. PIPAAm-grafted dishes irradiated with 100, 250, or 500 kGy of electron beams showed significantly enhanced expression of Cx43-NP, Cx43-P1, and especially Cx43-P2. Enhanced expression of Cx43-P2, a functional transmembrane protein, may be related to the promotion of GJIC. These results suggest that the PIPAAm-grafted dish not only enables the enzyme-free recovery of a cell monolayer for use in the construction of a three-dimensional artificial tissue, but also significantly contributes to the enhancement of GJIC, which may partly promote tissue strength on the surface of the PIPAAm-grafted dish.     

Tissue-engineered skin using aggregates of normal human skin fibroblasts and biodegradable material

Higher-density inoculation of fibroblasts into a three-dimensional scaffold should accelerate wound healing after skin implantation. This study attempted to develop tissue-engineered skin with a higher density of fibroblasts. We first attempted to fabricate three-dimensional high-cell-density aggregates (spheroids) of normal human fibroblasts for application to tissue-engineered skin. Our method consisted of rotational shaking with nontreated dishes, decreasing fibroblast-meterial interactions, and augmenting cell-cell interaction. To prompt aggregate formation, the medium was supplemented with insulin, dexamethasone, ascorbic acid, and basic fibroblast growth factors that potentiate secretion of extracellular matrices. Under such improved conditions, fibroblasts were able to form spheroidal aggregates within 24 to 36h of rotational culture. Although the formed aggregates were irregular in shape and were composed of only several cells after 12h, they became almost spheroidal after 24h. The aggregates grew even more round after 36h, and their surfaces became smooth. After 36h of rotational culture, the fibroblast aggregates were collected and reinoculated onto a biodegradable mesh composed of polyglycolic acid coated with collagen. The aggregates were trapped in the material and became attached after 24h. Finally, because transforming growth factor-β₃ (TGF-β₃) is known to accelerate wound healing, we conducted a semiquantitative analysis of TGF-β₃ mRNA in both the fibroblast monolayers (two-dimensional culture) and the aggregates (three-dimensional culture). Analysis of TGF-β₃ mRNA expression showed that mRNA expression was greater in the fibroblasts of aggregates than in a monolayer. Therefore, our newly developed dermal graft is expected to accelerate wound healing faster than conventional grafts.     

Sucralfate induces proliferation of dermal fibroblasts and keratinocytes in culture and granulation tissue formation in full-thickness skin wounds

Sucralfate is used to induce healing of gastrointestinal tract ulcers. We evaluated its potential utility in the healing of skin wounds. Initial experiments examined the effects of the sucralfate on proliferation of cultured dermal fibroblasts and keratinocytes. Sucralfate induced proliferation in quiescent cultures of both cell types. Additionally, sucralfate enhanced prostaglandin E₂ synthesis in basal keratinocytes and in interleukin-1-stimulated keratinocytes and dermal fibroblasts. Basal interleukin-1 and 6 release were not affected by sucralfate, but the agent enhanced interleukin-1-stimulated interleukin-6 release from fibroblasts. When applied daily to full-thickness wounds in rats, sucralfate increased the thickness of granulation tissue when assessed at day 12.     

Comparative study on motility of the cultured fetal and neonatal dermal fibroblasts in extracellular matrix.

One of the differences between fetal and adult skin healing is the ability of fetal wounds heal without contraction and scar formation. Extracellular matrix (ECM) provides a substratum for cells adhesion, migration, and proliferation and can directly influence the form and function of cells. As motility is essential for many important biological events, including wound healing, inflammatory response, embryonic development, and tumor metastasis, this study was designed to compare the motilities cultured dermal fetal and neonatal fibroblasts in the extracellular matrix. The motility of cultured fetal and neonatal fibroblasts was compared using a video-microscopy system that was developed in combination with a self-designed CO2 mini-incubator. To determine migration speed, cells were viewed with a 4X phase-contrast lens and video recorded. Images were captured using a color CCD camera and saved in 8-bit full-color mode. We found that cultured fetal fibroblasts move faster than neonatal fibroblast on type I collagen (fetal fibroblast, 15.1 micrometer/hr; neonatal fibroblast, 13.7 micrometer/hr), and in fibronectin (fetal fibroblast, 13.2 micrometer/hr; neonatal fibroblast, 13.0 micrometer/hr) and hyaluronic acid (fetal fibroblast, 11 micrometer/hr; neonatal fibroblast, 9.8 micrometer/hr).     

Expansion and delivery of human fibroblasts on micronized acellular dermal matrix for skin regeneration

In order to obtain an abundant supply of autologous dermal fibroblasts for the manufacture of engineered autologous dermal substitutes, we fabricated the micronized acellular dermal matrix (MADM) microcarriers and expanded human fibroblasts on them. This novel approach eliminated the need for the repeated trypsinizations that may disrupt cell–extracellular matrix interactions and impair cell viability. This cell expansion protocol simultaneously formed an engineered particulate dermal substitute (EPDS) avoiding cell reseeded on the scaffolds process. We further tested its feasibility and effectiveness in athymic murine subcutaneous injection and full-thickness cutaneous wound model. Our results showed that MADM microcarriers retained the ultrastructure of the acellular dermal matrix, had good biocompatibility, and supported human fibroblast expansion either as a direct culture substrate or through culturing cells in conditioned medium prepared from them. In the animal study, EPDS formed a thick layer of tissue below the subcutaneous muscle tissue at 3 weeks following EPDS injection into subcutaneous tissue. In full-thickness cutaneous wound, the degree of wound healing with EPDS implantation was better than that without EPDS although healing rates were not significantly different between wounds implanted with or without EPDS. This demonstrates the potential utility of MADM not only as a cell culture substrate to expand fibroblasts but also as a cell transplantation vehicle for skin regeneration, with several advantages over current expansion–transplantation protocols for skin regeneration. In addition, EPDS may be used for cosmetic or reconstructive soft tissue augmentation in a minimally invasive fashion.     

Secreted collagen ratios in normal human and osteogenesis imperfecta skin fibroblasts

We examined the effects of several variables on the ratio of type I:type III collagen secreted by human caucasian skin fibroblasts in normal and osteogenesis imperfecta (OI) phenotypes. Isotopically labelled collagen extracted from fibroblast medium was analyzed by DEAE-cellulose chromatography and identified by appropriate methods. Type I procollagen was the major form of collagen secreted into the medium by normal cells cultured from one mid-term fetus, infants (n = 3), children (n = 3), adolescents (n = 2), and adults (n = 3). Interstrain differences in collagen production under standardized conditions were significantly greater than intrastrain variation (anova, p = 0.0051). There was no significant alteration in the type I:type III collagen ratio due to variation in: phase of cell growth, doublings (between 13th and 22nd), rate of isotope incorporation, labelling time (24-72 hrs) in the presence of ascorbic acid (50 micrograms/ml), age of donor (with the possible exception of adolescence), and site of biopsy (genital and non-genital sites). Variable conversion of type I procollagen to collagen did not perturb the type I:type III collagen ratio. Cell strains from OI patients (Sillence classification): type I (one strain); type II, III and IV (3 strains each) had greater interstrain than intrastrain variation in the collagen ratio (p = 0.0149). Interstrain differences were greater in OI cell strains relative to normal cell strains (p less than 0.01). In the aggregate, OI cells had significantly lower type I collagen production relative to type III (I/III ratio = 1.18) when compared with normal cells (I/III ratio = 2.90; t test, p less than 0.0001). These findings imply abnormal synthesis, secretion or stability of type I procollagen and greater phenotypic heterogeneity in OI skin fibroblasts relative to normal cells.     

Impaired migration, integrin function, and actin cytoskeletal organization in dermal fibroblasts from a subset of aged human donors

Deficits in the motility of fibroblasts contribute to age-related impairment of wound healing. We analyzed 'young' fibroblasts from four healthy donors 22-30 years old and 'aged' fibroblasts from six healthy donors 81-92 years old for migratory ability on type I collagen, secretion of matrix metalloproteases (MMPs), attachment to matrices and, expression and function of integrin alpha2beta1. Cells from each donor were analyzed separately in each experiment. Whereas migration of young fibroblasts was uniformly robust, three aged lines migrated well and three migrated poorly. Synthesis of MMP1 and TIMP1, but not MMP2 or MMP9, was increased in the aged fibroblasts relative to the young fibroblast lines irrespective of their motility. All lines of young and aged fibroblasts attached to plastic or collagen with similar efficiency. Although young and aged fibroblasts expressed comparable levels of the alpha2 integrin; the lines of aged fibroblasts that were poor migrators exhibited a significant reduction in alpha2beta1 function relative to fibroblasts with normal migratory capacities. Moreover, the lines of aged fibroblasts that exhibited poor migration demonstrated a disordered actin cytoskeleton and a reduced ability to contract collagen gels. In conclusion, aged fibroblasts, unlike young fibroblasts, displayed variable migratory capacities. Deficient migration by specific lines of aged fibroblasts was not related to the capacity to attach, express alpha2 integrin, or secrete MMPs and TIMP1, but was characterized by disorganized cytoskeletal actin and reduced alpha2beta1 function.     

In situ image analysis of interactions between normal human keratinocytes and fibroblasts cultured in three-dimensional fibrin gels

The non-invasive investigation of different cells to interact and become spatially organised in a three-dimensional (3D) environment or scaffold is an important challenge in tissue engineering and tissue physiology. The aim of the present study was to develop 3D cell culture systems using fibrin gels, which would allow for the single and co-culture of different cell types with in situ image analysis. Two chambers were constructed for mono-culture and co-culture of human dermal fibroblasts and keratinocytes. During cell culture, in situ imaging and morphological characterisation of cells was assessed using brightfield light and/or fluorescence microscopy, and later confirmed by staining of fixed cells using immunofluorescence microscopy. The results showed that it was possible to investigate fibroblast and keratinocyte interactions in a fibrin scaffold for at least 12 days. Using this model system it was found that when a co-culture of fibroblasts and keratinocytes were plated on top of the fibrin gels, fibroblasts were seen to migrate into the gels within 2-3 days in contrast to keratinocytes, which did not enter. However, keratinocytes were found to retard fibroblast migration into gels when compared to fibroblasts cultured on their own, illustrating the dependency of intracellular communication on cell position for reconstructive approaches.     

Comparative study of adult human skin fibroblasts and umbilical fibroblast-like cells

Expression of markers, collagens, and HLA-1 by human skin fibroblasts and fibroblast-like cells isolated from the umbilical Wharton’s jelly was compared. Skin fibroblasts express collagens (proteins characteristic of differentiated cells of this histogenetic series) and HLA-1, while umbilical cells express, in addition to collagens, juvenile surface markers and almost no HLA-1. This indicates that fibroblast-like cells isolated from different sources are different and can serve as sources for the creation of cell preparations with different characteristics in future. __________ Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 53–59, January, 2006     

Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds

Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-β₁ and TGF-β₂ precursor proteins, but an increase in expression of TGF-β₃ precursor protein and TGF-β type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.

Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

Background  

Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging.     

Behavior of human dermal fibroblasts in three-dimensional fibrin clots: dependence on fibrinogen and thrombin concentration

Fibrin sealant products are used in hemostasis and tissue sealing, and potentially as a cell delivery vehicle. In this study, fibrin sealant was evaluated as a delivery vehicle for human dermal fibroblasts. Fibroblast proliferation and migration were assessed in various dilutions of fibrin sealant by changing the fibrinogen and thrombin concentration. Fibroblasts proliferated well within three-dimensional (3-D) fibrin clots consisting of fibrinogen (5-17 mg/mL) and thrombin (1-167 U/mL). These fibroblasts also retained good morphology and growth characteristics after migrating out of the 3-D fibrin clots. Furthermore, using Western blot and fluorescence-activated cell-sorting analysis, we found that the expression of growth factors and interleukins in the entire fibroblast-fibrin construct was dependent on the fibrin sealant formulation. For example, in a formulation in which fibroblasts showed modest proliferation and migration, interleukin 8 was secreted to a lesser extent than in a formulation that supported robust proliferation and migration. To our knowledge, this is the first time that it has been shown that modifying the concentration of fibrinogen and thrombin affects fibroblast behavior within formed 3-D fibrin clots. In addition, some of these formulations present an ideal delivery vehicle for fibroblasts that could be used for the treatment of chronic wounds.