Influence of serum on adult and fetal dermal fibroblast migration adhesion, and collagen expression

Summary The wound healing response to injury can be affected by many factors such as cell migration and extracellular matrix elaboration. The objective of this study was to examine the serum- and age-dependent effects on cell migration, adhesion, and collagen expression by skin fibroblasts. Dermal fibroblasts were isolated and plated with and without serum for up to 7 d. Cell migration was determined by quantitative image analysis, adhesion was quantified using a centrifugation assay, and collagen expression was assessed by PCR and immunohistochemical staining. Both adult and fetal fibroblasts migrated significantly faster in serum-containing medium compared to serum-free medium. There was no significant difference in migration between the two cell types in either serum-containing or serum-free medium. There was no significant difference in adhesion in the presence of serum, although there was a greater faction of adherent fetal skin fibroblasts than adult fibroblasts in serum-free medium. Moreover, the adherent fraction of fetal fibroblasts in serum-free medium was not significantly different from that in serum-containing medium, suggesting that fetal skin fibroblasts possess serum-independent adhesion properties. Collagen mRNA expression was significantly up-regulated in serum-free compared to serum-containing medium for both cell types. With respect to collagen immunohistochemistry, both dermal fibroblast populations exhibited greater type I collagen compared to type III collagen staining. Quantitative assessment of collagen staining indicated significantly enhanced type I collagen secretion in the presence of serum by fetal skin fibroblasts. These findings suggest that intrinsic cellular characteristics may govern the observed differences in adult and fetal wound healing.     

SPARC-thrombospondin-2-double-null mice exhibit enhanced cutaneous wound healing and increased fibrovascular invasion of subcutaneous polyvinyl alcohol sponges.

Secreted protein acidic and rich in cysteine (SPARC) and thrombospondin-2 (TSP-2) are structurally unrelated matricellular proteins that have important roles in cell-extracellular matrix (ECM) interactions and tissue repair. SPARC-null mice exhibit accelerated wound closure, and TSP-2-null mice show an overall enhancement in wound healing. To assess potential compensation of one protein for the other, we examined cutaneous wound healing and fibrovascular invasion of subcutaneous sponges in SPARC-TSP-2 (ST) double-null and wild-type (WT) mice. Epidermal closure of cutaneous wounds was found to occur significantly faster in ST-double-null mice, compared with WT animals: histological analysis of dermal wound repair revealed significantly more mature phases of healing at 1, 4, 7, 10, and 14 days after wounding, and electron microscopy showed disrupted ECM at 14 days in these mice. ST-double-null dermal fibroblasts displayed accelerated migration, relative to WT fibroblasts, in a wounding assay in vitro, as well as enhanced contraction of native collagen gels. Zymography indicated that fibroblasts from ST-double-null mice also produced higher levels of matrix metalloproteinase (MMP)-2. These data are consistent with the increased fibrovascular invasion of subcutaneous sponge implants seen in the double-null mice. The generally accelerated wound healing of ST-double-null mice reflects that described for the single-null animals. Importantly, the absence of both proteins results in elevated MMP-2 levels. SPARC and TSP-2 therefore perform similar functions in the regulation of cutaneous wound healing, but fine-tuning with respect to ECM production and remodeling could account for the enhanced response seen in ST-double-null mice.     

A CXCL5- and bFGF-dependent effect of PDGF-B-activated fibroblasts in promoting trafficking and differentiation of bone marrow-derived mesenchymal stem cells

Adult bone marrow-derived mesenchymal stem cells (MSCs) are able to differentiate into myofibroblasts and be recruited into wound lesions and contribute to wound healing. The cellular and molecular mechanisms responsible for MSC trafficking and differentiation, however, are poorly understood. Local resting resident fibroblasts are activated after injury and play a critical role in recruiting MSCs. We investigated the role of platelet-derived growth factor-B-activated fibroblasts (PDGF-B-aFBs) in regulating recruitment, migration and differentiation of MSCs from GFP transgenic mice in an in vitro wound healing assay and a novel three-dimensional (3D) model. PDGF-B-aFBs caused significant increases in MSC migration velocity compared to control as demonstrated by time-lapse photography in an in vitro wound healing assay. Consistently, invasion/migration of MSCs into 3D collagen gels was enhanced in the presence of PDGF-B-aFBs. In addition, PDGF-B-aFBs induced differentiation of MSCs into myofibroblast. The regulatory effects of PDGF-B-aFBs are likely to be mediated by basic fibroblast growth factor (bFGF) and epithelial neutrophil activating peptide-78 (ENA-78 or CXCL5) as protein array analysis indicated elevated levels of these two soluble factors in culture supernatant of PDGF-B-aFBs. Blocking antibodies against bFGF and CXCL5 were able to inhibit both trafficking and differentiation of MSCs into 3D collagen gels while supplement of exogenous bFGF and/or CXCL5 promoted invasion/migration of MSCs into 3D collagen gels. Our results reveal that PDGF-B-aFBs play a key role in the recruitment/migration and differentiation of MSCs and implicate a bFGF- and CXCL5-dependent mechanism in mediating these effects.     

Antimicrobial peptide KSL-W promotes gingival fibroblast healing properties in vitro

We investigated the effect of synthetic antimicrobial decapeptide KSL-W (KKVVFWVKFK) on normal human gingival fibroblast growth, migration, collagen gel contraction, and α-smooth muscle actin protein expression. Results show that in addition to promoting fibroblast adhesion by increasing F-actin production, peptide KSL-W promoted cell growth by increasing the S and G2/M cell cycle phases, and enhanced the secretion of metalloproteinase (MMP)-1 and MMP-2 by upregulating MMP inhibitors, such as tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in fibroblasts. An in vitro wound healing assay confirmed that peptide KSL-W promoted fibroblast migration and contraction of a collagen gel matrix. We also demonstrated a high expression of α-smooth muscle actin by gingival fibroblasts being exposed to KSL-W. This work shows that peptide KSL-W enhances gingival fibroblast growth, migration, and metalloproteinase secretion, and the expression of α-smooth muscle actin, thus promoting wound healing.     

CD44 is critically involved in infarct healing by regulating the inflammatory and fibrotic response

Infarct healing is dependent on an inflammatory reaction that results in leukocyte infiltration and clearance of the wound from dead cells and matrix debris. However, optimal infarct healing requires timely activation of "stop signals" that suppress inflammatory mediator synthesis and mediate resolution of the inflammatory infiltrate, promoting formation of a scar. A growing body of evidence suggests that interactions involving the transmembrane receptor CD44 may play an important role in resolution of inflammation and migration of fibroblasts in injured tissues. We examined the role of CD44 signaling in infarct healing and cardiac remodeling using a mouse model of reperfused infarction. CD44 expression was markedly induced in the infarcted myocardium and was localized on infiltrating leukocytes, wound myofibroblasts, and vascular cells. In comparison with wild-type mice, CD44(-/-) animals showed enhanced and prolonged neutrophil and macrophage infiltration and increased expression of proinflammatory cytokines following myocardial infarction. In CD44(null) infarcts, the enhanced inflammatory phase was followed by decreased fibroblast infiltration, reduced collagen deposition, and diminished proliferative activity. Isolated CD44(null) cardiac fibroblasts had reduced proliferation upon stimulation with serum and decreased collagen synthesis in response to TGF-beta in comparison to wild-type fibroblasts. The healing defects in CD44(-/-) mice were associated with enhanced dilative remodeling of the infarcted ventricle, without affecting the size of the infarct. Our findings suggest that CD44-mediated interactions are critically involved in infarct healing. CD44 signaling is important for resolution of the postinfarction inflammatory reaction and regulates fibroblast function.     

Amyloid precursor-like protein 2 promotes cell migration toward fibronectin and collagen IV.

Previous studies have established that in response to wounding, the expression of amyloid precursor-like protein 2 (APLP2) in the basal cells of migrating corneal epithelium is greatly up-regulated. To further our understanding of the functional significance of APLP2 in wound healing, we have measured the migratory response of transfected Chinese hamster ovary (CHO) cells expressing APLP2 isoforms to a variety of extracellular matrix components including laminin, collagen types I, IV, and VII, fibronectin, and heparan sulfate proteoglycans (HSPGs). CHO cells overexpressing either of two APLP2 variants, differing in chondroitin sulfate (CS) attachment, exhibit a marked increase in chemotaxis toward type IV collagen and fibronectin but not to laminin, collagen types I and VII, and HSPGs. Cells overexpressing APLP2-751 (CS-modified) exhibited a greater migratory response to fibronectin and type IV collagen than their non-CS-attached counterparts (APLP2-763), suggesting that CS modification enhanced APLP2 effects on cell migration. Moreover, in the presence of chondroitin sulfate, transfectants overexpressing APLP2-751 failed to exhibit this enhanced migration toward fibronectin. The APLP2-ECM interactions were also explored by solid phase adhesion assays. While overexpression of APLP2 isoforms moderately enhanced CHO adhesion to laminin, collagen types I and VII, and HSPGs lines, especially those overexpressing APLP2-751, exhibited greatly increased adhesion to type IV collagen and fibronectin. These observations suggest that APLP2 contributes to re-epithelialization during wound healing by supporting epithelial cell adhesion to fibronectin and collagen IV, thus influencing their capacity to migrate over the wound bed. Furthermore, APLP2 interactions with fibronectin and collagen IV appear to be potentiated by the addition of a CS chain to the core proteins.     

Internal fluid flow increases cellular interconnects between Medial Collateral Ligament fibroblasts and cellular extensions within three-dimensional collagen matrixes

The interconnectivity of fibroblasts within the ligamentous extracellular matrix has been largely overlooked. Studies on the cell-to-cell contacts with their neighbors via gap junctions in ligament fibroblasts, and works on the ability of fibroblasts to generate interconnected networks in vivo, suggest interfibroblastic interactions play an important role in fundamental biological processes, including homeostasis and wound healing. The current study examines how fluidic shear stresses imposed by internal flow can be used to mediate the formation of three-dimensional, interconnected fibroblast networks within collagen solutions. Several fibroblast-collagen solutions were exposed to shear stresses via Poiselle Flow. The consequent changes in cell networking, interconnections, and cell morphology within collagen matrixes exhibited by cells derived from Bovine Medial Collateral Ligaments were analyzed. Results illustrate that higher imposed stresses generate cells with more dendritic and/or branched morphologies, which form more visible three-dimensional networks within collagen matrixes than fibroblast-collagen solutions that were unexposed to shear stress.     

Endothelial cells enhance adipose mesenchymal stromal cell‐mediated matrix contraction via ALK receptors and reduced follistatin: Potential role of endothelial cells in skin fibrosis

Abnormal cutaneous wound healing can lead to formation of fibrotic hypertrophic scars. Although several clinical risk factors have been described, the cross‐talk between different cell types resulting in hypertrophic scar formation is still poorly understood. The aim of this in vitro study was to investigate whether endothelial cells (EC) may play a role in skin fibrosis, for example, hypertrophic scar formation after full‐thickness skin trauma. Using a collagen/elastin matrix, we developed an in vitro fibrosis model to study the interaction between EC and dermal fibroblasts or adipose tissue‐derived mesenchymal stromal cells (ASC). Tissue equivalents containing dermal fibroblasts and EC displayed a normal phenotype. In contrast, tissue equivalents containing ASC and EC displayed a fibrotic phenotype indicated by contraction of the matrix, higher gene expression of ACTA2, COL1A, COL3A, and less secretion of follistatin. The contraction was in part mediated via the TGF‐β pathway, as both inhibition of the ALK4/5/7 receptors and the addition of recombinant follistatin resulted in decreased matrix contraction (75 ± 11% and 24 ± 8%, respectively). In conclusion, our study shows that EC may play a critical role in fibrotic events, as seen in hypertrophic scars, by stimulating ASC‐mediated matrix contraction via regulation of fibrosis‐related proteins.     

Role of plasma-derived fibrin on keratinocyte and fibroblast wound healing

Fibrin has excellent biocompatibility and biological properties to support tissue regeneration and promote wound healing. However, the role of diluted fibrin in wound healing has yet to be elucidated as it is commonly used in high concentration. This study was aimed to examine the effects of diluted plasma-derived fibrin (PDF) on keratinocyte and fibroblast wound healing in term of cell proliferation, migration, extracellular matrix (ECM) production and soluble factor secretion. Two PDF concentrations, 10 and 20% (v/v) were tested on keratinocytes and fibroblasts indirectly co-cultured in the transwell system. The control group was cultured with 5% FBS. Results showed that PDF reduced the keratinocyte growth rate and fibroblast migration, and increased the fibroblast ECM gene expression whereby significant differences were found between the 20% PDF group and the 5% FBS group. Similar trend was seen for the 10% PDF group but the differences were not significant. Comparison of the soluble factors between the PDF groups demonstrated that the level of growth-related oncogene alpha, interleukin-8 and epithelial neutrophil-activating peptide-78 were significantly higher in the 10% PDF group, whilst interleukin-1 alpha and granulocyte–macrophage colony stimulating factor were significantly more concentrated in the 20% PDF group. Our results suggested that PDF selectively elevated the expression of collagen type 1 and collagen type 3 in fibroblasts but slowed down the migration in concentration-dependent manner. These novel findings provide new insight into the role of PDF in wound healing and may have important implications for the use of fibrin in skin tissue engineering.     

Microporous Dermal-Mimetic Electrospun Scaffolds Pre-Seeded with Fibroblasts Promote Tissue Regeneration in Full-Thickness Skin Wounds

Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone) electrospun scaffold (70:30 col/PCL) containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM), and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344) rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14%) over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold). Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration. Collectively these results suggest that microporous electrospun scaffolds pre-seeded with fibroblasts promote greater wound-healing than acellular scaffolds.     

Evidence that PI3K, Rac, Rho, and Rho kinase are involved in basic fibroblast growth factor-stimulated fibroblast-Collagen matrix contraction

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase while platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to determine the signaling mechanisms of basic fibroblast growth factor (bFGF)-stimulated fibroblast-collagen matrix contraction. Both bFGF and LPA promoted equally collagen matrix contraction well. Three different inhibitors, LY294002 for phosphatidylinositol-3-kinase (PI3K), C3 exotransferase for Rho and Y27632 for Rho kinase, suppressed the bFGF-stimulated fibroblast-collagen matrix contraction. With bFGF stimulation, fibroblasts spread with prominent stress fiber network formation and focal adhesions. In the presence of Rho kinase inhibitor, focal adhesions and stress fibers were mostly lost. We demonstrated that bFGF stimulation for fibroblast caused transient Rac and Rho activation but did not activate Cdc42. In addition, bFGF enhanced fibroblast migration in wound healing assay. The present study implicates PI3K, Rac, Rho, and Rho kinase as being involved in bFGF-stimulated collagen matrix contraction. The elucidation of bFGF-triggered signal transduction may be an important clue to understand the roles of bFGF in wound healing.     

Modification of collagen by 3-deoxyglucosone alters wound healing through differential regulation of p38 MAP kinase

Background

Wound healing is a highly dynamic process that requires signaling from the extracellular matrix to the fibroblasts for migration and proliferation, and closure of the wound. This rate of wound closure is impaired in diabetes, which may be due to the increased levels of the precursor for advanced glycation end products, 3-deoxyglucosone (3DG). Previous studies suggest a differential role for p38 mitogen-activated kinase (MAPK) during wound healing; whereby, p38 MAPK acts as a growth kinase during normal wound healing, but acts as a stress kinase during diabetic wound repair. Therefore, we investigated the signaling cross-talk by which p38 MAPK mediates wound healing in fibroblasts cultured on native collagen and 3DG-collagen.

Methodology/Principal Findings

Using human dermal fibroblasts cultured on 3DG-collagen as a model of diabetic wounds, we demonstrated that p38 MAPK can promote either cell growth or cell death, and this was dependent on the activation of AKT and ERK1/2. Wound closure on native collagen was dependent on p38 MAPK phosphorylation of AKT and ERK1/2. Furthermore, proliferation and collagen production in fibroblasts cultured on native collagen was dependent on p38 MAPK regulation of AKT and ERK1/2. In contrast, 3DG-collagen decreased fibroblast migration, proliferation, and collagen expression through ERK1/2 and AKT downregulation via p38 MAPK.

Conclusions/Significance

Taken together, the present study shows that p38 MAPK is a key signaling molecule that plays a significantly opposite role during times of cellular growth and cellular stress, which may account for the differing rates of wound closure seen in diabetic populations.     

Topical application of plasma fibronectin in full-thickness skin wound healing in rats

Fibronectin (Fn) has been shown to play an important role in wound healing because it appears to be the stimulus for migration of fibroblasts and epidermal cells. The purpose of this study was to investigate whether topical application of plasma Fn (pFn) improves healing of full-thickness skin wounds in rats. A round section of full-thickness skin (diameter of approximately 15 mm) was resected in rats. Animals were then divided into two groups, and wounds were treated topically with a single application of human plasma albumin (control group) or human pFn (FN group). Wound closure rate, hydroxyproline concentration, and histologic features (immunohistochemical staining) were evaluated. The FN group had a significantly higher wound closure rate and hydroxyproline level in the skin than the control group. Histologic analysis of macrophage and fibroblast migration, collagen regeneration, and epithelialization were significantly increased in the FN group compared with the control group. A single topical application of pFn increased the migration of macrophages, myofibroblasts, and fibroblasts. Moreover, further release of transforming growth factor-beta1 from activated fibroblasts, keratinocytes, and epithelial cells may also contribute to the beneficial effect of pFn on wound healing.     

Collagen type V enhances matrix contraction by human periodontal ligament fibroblasts seeded in three-dimensional collagen gels

Extracellular matrix components play an important role in modulating cellular activity. To study such capacities of the matrix, fibroblasts are frequently cultured in a three-dimensional gel and contraction is assessed as a measure of cellular activity. Since a connective tissue contains several types of collagen, we investigated the effect of gels composed of collagen I alone or in combination with 10% collagen III and/or 5% collagen V on contraction by human periodontal ligament fibroblasts. Gels containing collagen V contracted much faster than those without this type of collagen. Blocking of the integrin beta1-subunit with an activity-blocking antibody delayed (gels with collagen V) or almost completely blocked (gels without collagen V) contraction. Use of an antibody directed against integrin alpha2beta1 resulted in delay of gel contraction for gels both with and without collagen V. Anti-integrin alpha v beta3 or RGD peptides partially blocked contraction of gels containing collagen V, but had no effect on gels consisting of collagen I alone. The beta1-containing integrins are involved in the basal contraction by fibroblasts that bind to collagens I and III. The enhanced contraction, stimulated by collagen V, appears to be mediated by integrin alpha v beta3. We conclude that collagen V may play an important modulating role in connective tissue contraction. Such a modulation may occur during the initial stages of wound healing and/or tissue regeneration.     

Hepatocyte growth factor inhibits the formation of the basement membrane of alveolar epithelial cells in vitro

Hepatocyte growth factor (HGF) is a pulmotrophic factor for the regeneration of injured pulmonary tissue. We investigated the role of HGF in basement membrane formation during wound healing by immortalized alveolar type II epithelial cells that could form a continuous basement membrane when they were cultured on collagen fibrils in the presence of entactin-contaminated laminin-1. Cells cultured with 5.0 ng/ml HGF neither formed a continuous basement membrane on collagen fibrils nor maintained a continuous basement membrane architecture on a basement membrane substratum. The cells showed increased secretion of matrix metalloproteinase-9 and urokinase-type plasminogen activator, and the HGF-induced inhibition of basement membrane formation was attenuated by addition of 200 ng/ml tissue inhibitor of matrix metalloproteinase-1. Cells sequentially exposed to HGF and 1.0 ng/ml transforming growth factor-beta1 had enhanced basement membrane formation compared with those receiving these reagents in the reverse order or concurrently. HGF simultaneously stimulated proliferation and migration of the cells so that it advanced wound closure on the basement membrane substratum. The present results indicate that the role of HGF in wound healing is the stimulation of reepithelization, but this factor may also contribute to the degradation of the basement membrane.     

Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing

Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and β1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of β1 and β3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing.     

Role of the α1β1 integrin complex in collagen gel contraction in vitro by fibroblasts

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the β1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF-β, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the α2β1 and, to a lesser extent the α1β1 integrin complexes. The present studies were undertaken to determine the role of the α1 integrin chain, a collagen/laminin receptor, in collagen gel contration by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the α1β1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the α1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the α1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the α1β integrin complex plays a significant role in cellular interactions with interstital collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing. © 1995 Wiley-Liss, Inc.