Fibroblast-populated Collagen Lattice Research Articles

Collagen Lattice Model, Populated with Heterogeneous Cancer-Associated Fibroblasts, Facilitates Advanced Reconstruction of Pancreatic Cancer Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a solid-tumor malignancy. To enhance the treatment landscape of PDAC, a 3D model optimized for rigorous drug screening is essential. Within the PDAC tumor microenvironment, a dense stroma comprising a large extracellular matrix and cancer-associated fibroblasts (CAFs) is well-known for its vital role in modulating tumor growth, cellular heterogeneity, bidirectional paracrine signaling, and chemoresistance. In this study, we employed a fibroblast-populated collagen lattice (FPCL) modeling approach that has the ability to replicate fibroblast contractility in the collagenous matrix to build dense stroma. This FPCL model allows CAF differentiation by facilitating multifaceted cell-cell interactions between cancer cells and CAFs, with the differentiation further influenced by mechanical forces and hypoxia carried within the 3D structure. Our FPCL models displayed hallmark features, including ductal gland structures and differentiated CAFs with spindle shapes. Through morphological explorations alongside in-depth transcriptomic and metabolomic profiling, we identified substantial molecular shifts from the nascent to mature model stages and potential metabolic biomarkers, such as proline. The initial pharmacological assays highlighted the effectiveness of our FPCL model in screening for improved therapeutic strategies. In conclusion, our PDAC modeling platform mirrors complex tumor microenvironmental dynamics and offers an unparalleled perspective for therapeutic exploration.

Fibrin Contributes to an Improvement of an in vitro Wound Repair Model using Fibroblast-populated Collagen Lattices

Incisional acute wounds of the skin are characterized by a rapid biomechanical response by stromal cell contraction that joins the wound lips through the fibrin cloth. In this work, we have performed an in vitro model using Fibroblast-Populated Collagen Lattices (FPCLs) that partially mimic that physiological process. Injured FPCLs under relaxed or stressed conditions were evaluated over time, and cross-sections of the lattices were stained with picrosirius red. Wounds filled with fibrin in relaxed FPCLs were closed earlier than controls, the fibrillar pattern of the collagen lattice was different between the wound and the edges of the lattice. On the other hand, stressed FPCLs did not close wounds, even those filled with fibrin, because the tension generated from the lattice borders maintained high tension towards the wound. Controls or fibrin-treated stressed FPCLs, showed high tension in the wound matrix, characterized by the high packing of collagen observed like yellow-red birefringent fibers when stained by picrosirius red. Despite wounds that remain open, fibrin-treated FPCLs exhibited less wound area than controls. With this work, we have demonstrated that FPCL models can be used to study wound closure, mainly when they are improved with other elements of the wound environment that allow us to analyze the biological process.

Abstract B061: Development of a pancreatic ductal adenocarcinoma 3D tumor model for high-throughput drug screening

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a persistent malignancy that is difficult to diagnose in its early stage and is one of the leading cancer-related deaths worldwide. Currently, limited therapeutic options are available for PDAC, resulting in an extremely low 5-year survival rate. To speed up the discovery of novel therapeutics, a reliable 3D model that highly simulates the PDAC tumor microenvironment is needed for large-scale drug screening. In this study, we propose a novel 3D model for restoring the PDAC tumor microenvironment by targeting cancer-associated fibroblasts (CAFs), which serve as the key component of the PDAC, providing its heterogeneity and contributing to reciprocal signaling and drug resistance. Our previous studies have demonstrated that Adipose-derived mesenchymal stem cells can differentiate into varying subtypes CAFs when co-cultured with PDAC cells both in vitro and in vivo. Following this strategy, we utilize the fibroblast-populated collagen lattice (FPCL) modeling approach, which relies on the contractility of fibroblasts in a collagen gel matrix, to provide the mechanical stress and hypoxic conditions for the 3D tumor models. Human Adipose-derived mesenchymal stem cells as CAF precursors and PDAC cell lines were mixed with collagen solution to make the collagen discs, resulting in the formation of 3D PDAC tumor models within a week. The morphological analyses, along with global transcriptome and metabolomic analyses, were performed to confirm the clinical-like mature ductal glands of PDAC cells and CAFs-like spindle-shaped of mesenchymal stem cells in the interior of PDAC-FPCL (PDAC-F). Moreover, the PDAC-F has shown a high tumorigenic potential in vivo animal model. Additionally, PDAC-F was shown to allow combination therapies screening of anticancer and antifibrotic drugs in the drug screening test, demonstrating that the combination therapeutics have better efficacy. In conclusion, our PDAC-F is built with distinct features of PDAC, reproducing the heterogeneous and high stiffness with a clinical-like PDAC morphology. In addition, the short modeling time and the high reproducibility of this PDAC-F open the possibilities for large-scale drug screening, which is expected to accelerate new drug development and provide effective treatment for pancreatic cancer patients. Citation Format: Xiaoyu Song, Yuma Nihashi, Yasuyuki S. Kida. Development of a pancreatic ductal adenocarcinoma 3D tumor model for high-throughput drug screening [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B061.

Studying Monocyte-Myofibroblast Immunomodulation in a 3D Co-culture System.

Simple and reproducible 3D cell culture systems that mimic biological interactions within physiological tissues (biomimetics) can provide unique insight for scientific inquiries compared to 2D cell cultures. Fibroblast-populated collagen lattices (FPCLs) are commonly used for mimicking physiological collagen matrices, potentiating biomechanical stresses on embedded fibroblasts. Here, we describe a novel 3D co-culture model that incorporates human Tenon's capsule fibroblasts embedded in FPCLs co-cultured with THP-1 monocytes suspended in culture media. This method can be used for the assessment of cell-cell interactions in various stages of the wound healing process and can facilitate various types of immune cells in co-culture. This system can also be used to study pharmacological agents that may eventually improve clinical outcomes in patients affected by inflammatory disorders.

N-acetyl-L-hydroxyproline - A potent skin anti-ageing active preventing advanced glycation end-product formation invitro and exvivo.

Advanced glycation end-products (AGEs) represent a large group of compounds generated by a non-enzymatic reaction between reducing sugars and amino groups. The formation and accumulation of AGEs in the skin lead to protein crosslinking, dermal stiffening and yellowing, which ultimately contribute to cutaneous ageing. Amino acids have been described to exhibit anti-glycation effects. The objective of this study was to understand the inhibitory role of the amino acid derivative N-acetyl-L-hydroxyproline (NAHP) as an anti-glycation active for human skin. A cell-free assay investigating the inhibition of glycation of serum albumin by NAHP was used to determine the capability of NAHP to decrease AGE formation. Also, by assessing the amount of the AGE N-(carboxymethyl)lysine (CML) the anti-glycation abilities of NAHP were investigated utilizing dot blot analysis. The improvement of cell-matrix interaction by NAHP was determined invitro using a glycated fibroblast-populated collagen lattice (FPCL) dermis model. In skin biopsies, AGE autofluorescence was determined after treatment with NAHP and/or glucose exvivo. NAHP significantly and dose-dependently inhibited levels of AGEs, which were induced by the glycation of a protein solution. This decrease could be visualized by showing that the brownish appearance as well as the AGE-specific fluorescence of glucose-treated samples were reduced after the application of increasing amounts of NAHP. Also, CML formation was dose-dependently inhibited by NAHP. In FPCLs, the contractile capacity of fibroblasts was significantly disturbed after glycation. This could be prevented by the addition of NAHP. Compared to glyoxal-treated samples, the co-application of NAHP significantly decreased the diameter as well as the weight of glycated FPCLs. Exvivo application of glucose to skin explants showed a higher AGE fluorescence signal compared to control explants. Co-treatment with NAHP and glucose decreased the level of AGE fluorescence in comparison to glucose-treated explants. These data provide clear evidence that under glycation stress conditions treatment with NAHP inhibited AGE formation invitro and exvivo and prevented the loss of cellular contractile forces in a glycated dermis model. Thus, NAHP obviously provides a beneficial treatment option to counteract AGE-related changes in human skin such as dermal stiffening and yellowish skin appearance.

Roles of bovine sialoglycoproteins for anti-skin aging and accelerating skin wound healing.

Fibroblasts are the most predominant cell subpopulation in the dermal layer of human skin, they play an important role in maintaining skin architecture and function. The senescence of fibroblasts is one of major causes of skin aging and chronic wound in the elderly, which is accompanied with a reduction of α2,6-sialylation on the cell surface. In this study, we investigated the effects of the bovine sialoglycoproteins on normal human dermal fibroblasts (NHDF). The results showed that bovine sialoglycoproteins could promote the proliferation and migration of NHDF cells, and accelerate the contraction of fibroblast-populated collagen lattice (FPCL). The average doubling time of NHDF cells treated with bovine sialoglycoproteins (0.5 mg/mL) was 31.1 ± 1.0 h whereas that was 37.9 ± 2.7 h for the control (p ˂ 0.05). Moreover, the expression of basic fibroblast growth factor (FGF-2) was upregulated, while that of transforming growth factor-beta 1 (TGF-β1) and human type I collagen (COL-I) were downregulated in treated NHDF cells. Furthermore, bovine sialoglycoproteins treatment significantly enhanced the α2,6-sialylation on the cell surfaces, which was consistent with the upregulation of α2,6-sialyltransferase I (ST6GAL1) expression. These results indicated that the bovine sialoglycoproteins might be developed as a reagent against skin aging in the cosmetic industry, or as a new candidate for accelerating skin wound healing and inhibiting scar formation.

Stretching Promotes Wound Contraction Through Enhanced Expression of Endothelin Receptor B and TRPC3 in Fibroblasts.

The conditioned medium from stretched keratinocyte was added to the fibroblast populated collagen lattice. Then, we analyzed the levels of endothelin receptor in the human hypertrophic scar tissue and stretched fibroblasts. To address the function of TRPC3, we have used an overexpression system with the collagen lattice. Finally, the TRPC3 overexpressing fibroblasts were transplanted to mouse dorsal skin, and the rate of skin wound contraction was assessed. Conditioned medium from stretched keratinocytes increased the rate of contraction of fibroblast populated collagen lattice. In human hypertrophic scar and stretched fibroblasts, endothelin receptor type B was increased. Cyclic stretching of TRPC3 overexpressing fibroblasts activated NFATc4, and stretched human fibroblasts showed more activation of NFATc4 in response to ET-1. The wound treated with TRPC3 overexpressing fibroblasts showed more contraction than control wound. These findings suggest that cyclical stretching of wounds have an effect on both keratinocytes and fibroblasts, where keratinocytes secret more ET-1, and fibroblasts develop more sensitivity to ET-1 by expressing more endothelin receptors and TRPC3.

Therapeutic Potential of Adipose Stem Cell-Derived Conditioned Medium on Scar Contraction Model.

Scars are composed of stiff collagen fibers, which contract strongly owing to the action of myofibroblasts. To explore the substances that modulate scar contracture, the fibroblast-populated collagen lattice (FPCL) model has been used. However, the molecular signature of the patient-derived FPCL model has not been verified. Here, we examined whether the patient-derived keloid FPCL model reflects scar contraction, analyzing detailed gene expression changes using comprehensive RNA sequencing and histological morphology, and revealed that these models are consistent with the changes during human scar contracture. Moreover, we examined whether conditioned media derived from adipose stem cells (ASC-CM) suppress the scar contracture of the collagen disc. Detailed time-series measurements of changes in disc area showed that the addition of ASC-CM significantly inhibited the shrinkage of collagen discs. In addition, a deep sequencing data analysis revealed that ASC-CM suppressed inflammation-related gene expression in the early phase of contraction; in the later phase, this suppression was gradually replaced by extracellular matrix (ECM)-related gene expression. These lines of data suggested the effectiveness of ASC-CM in suppressing scar contractures. Therefore, the molecular analysis of the ASC-CM actions found in this study will contribute to solving medical problems regarding pathological scarring in wound prognosis.

Down-Regulating Scar Formation by Microneedles Directly via a Mechanical Communication Pathway.

Excessive extracellular matrix deposition drives fibroblasts into a state of high mechanical stress, exacerbating pathological fibrosis and hypertrophic scar formation, leading to tissue dysfunction. This study reports a minimally invasive and convenient approach to obtaining scarless tissue using a silk fibroin microneedle patch (SF MNs). We found that by tuning the MN size and density only, the biocompatible MNs significantly decreased the scar elevation index in the rabbit ear hypertrophic scar model and increased ultimate tensile strength close to regular skin. To advance our understanding of this recent approach, we built a fibroblast-populated collagen lattice system and finite element model to study MN-mediated cellular behavior of fibroblasts. We found that the MNs reduced the fibroblasts generated contraction and mechanical stress, as indicated by decreased expression of the mechanical sensitive gene ANKRD1. Specifically, SF MNs attenuated the integrin-FAK signaling and consequently down-regulated the expression of TGF-β1, α-SMA, collagen I, and fibronectin. It resulted in a low-stress microenvironment that helps to reduce scar formation significantly. Microneedles’ physical intervention via the mechanotherapeutic strategy is promising for scar-free wound healing.

Collagen denaturation in the infarcted myocardium involves temporally distinct effects of MT1-MMP-dependent proteolysis and mechanical tension

Tissue injury results in profound alterations in the collagen network, associated with unfolding of the collagen triple helix, proteolytic degradation and generation of fragments. In the infarcted myocardium, changes in the collagen network are critically involved in the pathogenesis of left ventricular rupture, adverse remodeling and chronic dysfunction. We hypothesized that myocardial infarction is associated with temporally and spatially restricted patterns of collagen denaturation that may reflect distinct molecular mechanisms of collagen unfolding. We used a mouse model of non-reperfused myocardial infarction, and in vitro assays in fibroblast-populated collagen lattices. In healing infarcts, labeling with collagen hybridizing peptide (CHP) revealed two distinct patterns of collagen denaturation. During the inflammatory and proliferative phases of infarct healing, collagen denaturation was pericellular, localized in close proximity to macrophages and myofibroblasts. qPCR array analysis of genes associated with matrix remodeling showed that Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) is markedly upregulated in infarct macrophages and fibroblasts, suggesting its involvement in pericellular collagen denaturation. In vitro, MT1-MMP-mediated pericellular collagen denaturation is involved in cardiac fibroblast migration. The effects of MT1-MMP on collagen denaturation and fibroblast migration involve the catalytic site, and require hemopexin domain-mediated actions. In contrast, during the maturation phase of infarct healing, extensive collagen denaturation was noted in the hypocellular infarct, in the infarct border zone and in the mitral valve annulus, in the absence of MT1-MMP. In vitro, mechanical tension in attached collagen lattices was sufficient to induce peripheral collagen denaturation. Our study suggests that in healing infarcts, early pericellular collagen denaturation may be important for migration of macrophages and reparative myofibroblasts in the infarct. Extensive denaturation of collagen fibers is noted in mature scars, likely reflecting mechanical tension. Chronic collagen denaturation may increase susceptibility of the matrix to proteolysis, thus contributing to progressive cardiac dilation and post-infarction heart failure.

IL-10 alleviates lipopolysaccharide-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts.

Hypertrophic scar (HS) is a severe fibrotic skin disease. It has always been a major problem in clinical treatment, mainly because its pathogenesis has not been well understood. The roles of bacterial contamination and prolonged wound inflammation were considered significant. IL‐10 is a potent anti‐inflammatory cytokine and plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL‐10 alleviates lipopolysaccharide (LPS)‐induced inflammatory response and skin scarring and explore the possible mechanism of scar formation. Our results showed that the expression of TLR4 and pp65 was higher in HS and HS‐derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS‐derived fibroblasts (NSFs). LPS could up‐regulate the expression of TLR4, pp65, Col I, Col III and α‐SMA in NSFs, but IL‐10 could down‐regulate their expression in both HSFs and LPS‐induced NSFs. Blocking IL‐10 receptor (IL‐10R) or the phosphorylation of STAT3, their expression was up‐regulated. In addition, in vitro and in vivo models results showed that IL‐10 could alleviate LPS‐induced fibroblast‐populated collagen lattice (FPCL) contraction and scar formation. Therefore, IL‐10 alleviates LPS‐induced skin scarring via IL‐10R/STAT3 axis regulating TLR4/NF‐κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of fibroblasts to myofibroblasts. Our results indicate that IL‐10 can alleviate the LPS‐induced harmful effect on wound healing, reduce scar contracture, scar formation and skin fibrosis. Therefore, the down‐regulation of inflammation may lead to a suitable scar outcome and be a better option for improving scar quality.

Mathematical Modeling Can Advance Wound Healing Research.

Significance: For over 30 years, there has been sustained interest in the development of mathematical models for investigating the complex mechanisms underlying each stage of the wound healing process. Despite the immense associated challenges, such models have helped usher in a paradigm shift in wound healing research. Recent Advances: In this article, we review contributions in the field that span epidermal, dermal, and corneal wound healing, and treatments of nonhealing wounds. The recent influence of mathematical models on biological experiments is detailed, with a focus on wound healing assays and fibroblast-populated collagen lattices. Critical Issues: We provide an overview of the field of mathematical modeling of wound healing, highlighting key advances made in recent decades, and discuss how such models have contributed to the development of improved treatment strategies and/or an enhanced understanding of the tightly regulated steps that comprise the healing process. Future Directions: We detail some of the open problems in the field that could be addressed through a combination of theoretical and/or experimental approaches. To move the field forward, we need to have a common language between scientists to facilitate cross-collaboration, which we hope this review can support by highlighting progress to date.

IL-10 Alleviates Lipopolysaccharide-Induced Skin Scarring <i>via</i> IL-10R/STAT3 Axis Regulating TLR4/NF-κB Pathway in Dermal Fibroblasts

Background: Hypertrophic scar (HS) is a serious fibrotic skin disease. The roles of bacterial contamination and prolonged inflammation in wound were considered to be significant. IL-10 plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL-10 alleviates lipopolysaccharide (LPS)-induced inflammatory response and skin scarring, explore the possible mechanism in scar formation. Methods: RT-qPCR, Western blotting, histochemistry, immunostaining, ELISA array, electron microscope, fibroblast-populated collagen lattice (FPCL) and a rabbit ear scar model were used to investigate and validate the effect of IL-10 on LPS-stimulated scar formation. Findings: Our results showed that the expression of TLR4 and pp65 was higher in HS and HS-derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS-derived fibroblasts (NSFs). LPS could upregulate the expression of TLR4, pp65, Col I, Col III and α-SMA in NSFs, but IL-10 could downregulate their expression in both HSFs and LPS-induced NSFs. Blocking IL-10 receptor (IL-10R) or the phosphorylation of STAT3, their expression was upregulated. In addition, in vitro and in vivo models results showed that IL-10 could alleviate LPS-induced fibroblast-populated collagen lattice (FPCL) contraction and scar formation. Interpretation: Our study suggests that IL-10 may improve LPS-induced harmful to wound healing, reduce scar contracture and scar formation via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of HSFs to NSFs. Therefore, the downregulation of inflammation may be a better option for improving scar quality, and become potential therapeutic targets for scarring. Funding Statement: This research was supported by grants from the National Natural Science Foundation of China (81571914, and 81741105), and the Natural Science Foundation of Shaanxi Province (2018JQ8048). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Written consent was obtained from all participants before surgery. All protocols used in this study were approved by the Ethics Committee of Xijing Hospital, affiliated to the Fourth Military Medical University of China. Animal experiments were approved by the Experimental Animal Committee of the Fourth Military Medical University (Xi’an, China).

Wnt4 negatively regulates the TGF-β1-induced human dermal fibroblast-to-myofibroblast transition via targeting Smad3 and ERK.

Abnormal activation of Wnt signaling has been demonstrated in the wound healing process and the pathogenesis of fibrotic disorders, with Wnt4 specifically identified as having a key role in the pathogenesis of renal, pulmonary and liver fibrosis. Wnt4 also was found to be upregulated by transforming growth factor-β1 (TGF-β1) in fetal and postnatal murine fibroblasts and bone marrow mesenchymal cells, suggesting an underlying cooperation between Wnt4 and TGF-β1 in fibrosis. However, the specific roles of Wnt4 in TGF-β1-induced skin myofibroblast transition and hypertrophic scar formation remain unclear. In the present study, we first observed reduced Wnt4 expression in hypertrophic scar tissue compared with that in normal skin tissue. Following upregulation by TGF-β1, Wnt4 inhibited the TGF-β1-induced transdifferentiation of fibroblasts into myofibroblasts. Using fibroblast-populated collagen lattice contraction assays, we showed that the increased contractility induced by TGF-β1 was significantly blocked by exogenous Wnt4 and the α-smooth muscle actin (α-SMA) expression was decreased in fibroblasts in the collagen lattices. In addition, knockdown of Wnt4 resulted in further increases in α-SMA and collagen I expressions. Further investigation showed that Wnt4 could inhibit the autocrine effect of TGF-β1 as well as block the phosphorylation of Smad3 and ERK but not of AKT or JNK. Lastly, using hypertrophic scar-derived fibroblasts, we showed that the elevated α-SMA and collagen I levels were markedly reduced after treatment with Wnt4. Taken together, our results suggest that Wnt4 negatively regulates TGF-β1-induced fibroblast activation, which may represent a novel therapeutic strategy for the treatment and prevention of hypertrophic scars.

KGF-1 accelerates wound contraction through the TGF-β1/Smad signaling pathway in a double-paracrine manner

KGF-1 plays an important role in the wound healing process. Loss of the KGF-1 gene in diabetic mice attenuated the process of wound contraction, suggesting that KGF-1 contributes to wound contraction. However, the mechanism remains unclear. To investigate the role of KGF-1 in diabetic wound contraction, we established a keratinocyte-fibroblast co-culture system. Concentrations of transforming growth factor β1 (TGF-β1) in conditioned supernatant treated with KGF-1 (KGF-1 group), tk;4KGF-1-neutralizing antibody (anti-KGF-1 group), TGF-β1 (TGF-β1tk;1 group), KGF-1 and TGF-β1-neutralizing antibody (KGF-1 + anti-TGF-β1 group) were tested by ELISA. Conditioned medium was added to fibroblast-populated collagen lattice (FPCL) to investigate the effect of KGF-1 on fibroblastqj contraction. TGF-β1, Col-I, p-Smad2, p-Smad3, and α-smooth muscle actin (α-SMA) were examined by Western blotting. A diabetic rat wound model was utilized to evaluate wound morphology, histology, immunohistochemistry, and protein expression in wound tissue after treatment with KGF-1. ELISA assays revealed that the concentration of TGF-β1 in the conditioned supernatant in the KGF-1 group was significantly higher. The contractile capacity of FPCL stimulated by conditioned medium derived from the KGF-1 group was significantly elevated; however, the contractile activity of FPCL induced by KGF-1 was attenuated by TGF-β1-neutralizing antibody. The Western blot results suggest that KGF-1 is able to stimulate TGF-β1 activation with increased Col-I, p-Smad2, p-Smad3, and α-SMA expression. Diabetic wounds treated with KGF-1 had a higher degree of contraction with significantly higher expression of TGF-β1, Col-I, p-Smad2, p-Smad3, and α-SMA. Our findings demonstrate that KGF-1 promotes fibroblast contraction and accelerates wound contraction via the TGF-β1/Smad signaling pathway in a double-paracrine manner.

A novel FPCL model producing directional contraction through induction of fibroblast alignment by biphasic pulse direct current electric field

Although parallel alignment of fibroblasts to the tension lines of scar has been evidenced in vivo, how scar contracture generates directional contraction remains largely unclear due to the lack of effective in vitro model. Fibroblast populated collagen lattice (FPCL), a widely used in vitro model, fails to mimic scar contracture since it produces concentric contraction with the random orientation of fibroblast. We hypothesized that a novel FPCL model with fibroblast alignment might produce directional contraction and then simulate scar contracture better. Here, we showed that although direct current electric fields (DCEFs) enabled fibroblasts aligned perpendicularly to the field vector, it also promoted electrotactic migration of fibroblast in FPCL. By contrast, biphasic pulse direct current electric fields (BPDCEFs), featured by reversal of the EF direction periodically, abolished the electrotactic migration, but induced fibroblast alignment in a pulse frequency dependent manner. Specifically, BPDCEF at a pulse frequency of 0.0002 Hz induced fibroblast alignment comparable to that induced by DCEF under the same field strength (300 mV/mm), leading to an enhanced contraction of FPCL along the direction of cell alignment. FPCL pretreated by BPDCEF showed an elliptical contraction whereas it was concentric in control FPCL. Further study revealed that F-actin redistributions acted as a key mechanism for the induction of fibroblasts alignment by BPDCEF. Cytochalasin D, an inhibitor of actin dynamics, abolished F-actins redistribution, and significantly suppressed the fibroblasts alignment and the directional contraction of FPCL. Importantly, BPDCEF significantly increased RhoA activity in fibroblasts, while this response was attenuated by C3 transferase pre-treatment, a potent inhibitor of RhoA, caused F-actin depolymerization and actin filament bundle randomly distributed. Taken together, our study suggests a crucial role for fibroblast orientation in scar contracture, and provides a novel FPCL model that may be feasible and effective for investigating scar contracture in vitro.

Fibroblast populated collagen lattices exhibit opposite biophysical conditions by fibrin or hyaluronic acid supplementation.

Fibrin and hyaluronic acid are important components of the provisional wound matrix. Through interactions with fibroblasts, they provide biophysical cues that regulate the viscoelastic properties of the extracellular matrix. To understand the roles of fibrin and hyaluronic acid in a collagenous environment, we used fibroblast populated collagen lattices (collagen, collagen-fibrin, and collagen-hyaluronic acid). Compared with collagen and collagen-hyaluronic acid cultures, collagen-fibrin cultures showed less contraction, which is correlated with increased elastic (G') and complex (|G*|) moduli, and reduced proportions of dendritic fibroblasts, despite increased αv integrin expression. Stiffness decreased during culture in collagen-fibrin environment, meanwhile phase shift (δ) values increased, clearly associated with the rise in fibrinolytic and gelatinolytic activities. These processes changed the viscoelastic properties of the system toward G' and |G*| values observed on day 5 in collagen cultures. Although less collagen turnover was observed in collagen-fibrin cultures than in collagen and collagen-hyaluronic acid cultures, collagen neosynthesis was apparently insufficient to contribute to the overall viscoelastic properties of the system. Collagen-hyaluronic acid cultures showed very limited changes during time. Firstly, they exhibited the highest δ values, suggesting an increase in the viscous behavior due to the hygroscopic properties of hyaluronic acid. These results showed that fibrin and hyaluronic acid not only affect differently the viscoelastic properties of the culture, they can tune fibroblastic activity by regulating cell attachment and extracellular matrix remodeling.

Inhibition of Pathological Phenotype of Hypertrophic Scar Fibroblasts Via Coculture with Adipose-Derived Stem Cells.

Hypertrophic scar (HS) is a dermal fibroproliferative disease characterized by fibroblast over-proliferation, overproduction, and deposition of the extracellular matrix. Growing evidence demonstrated that adipose-derived stem cells (ASCs) secrete a plethora of trophic and antifibrotic factors, which suppress inflammation and ameliorate fibrosis of different tissues. However, few studies investigate their effect on repressing HS activity. This study evaluated the suppressing effect of ASCs on HS fibroblast bioactivity and the possible mechanism via a coculture model. HS-derived fibroblasts (HSFs) and ASCs were isolated from individual patients. HSFs or HSFs treated with transforming growth factor-β1 (TGF-β1) were cocultured with ASCs and the change of HSF cellular behaviors, such as cell proliferation, migration, contractility, and gene/protein expression of scar-related molecules, were evaluated by cell counting assay, cell cycle analysis, scratch wound assay, fibroblast-populated collagen lattice (FPCL) contractility assay, real-time quantitative polymerase chain reaction, ELISA, and western blotting assay. After 5 days of ASC coculture treatment, the expression levels of collagen I (Col 1), collagen III (Col 3), fibronectin (FN), TGF-β1, interleukin-6 (IL-6), interleukin-8 (IL-8), connective tissue growth factor (CTGF), and alpha-smooth muscle actin (α-SMA) in HSFs decreased significantly while the expression levels of decorin (DCN) and MMP-1/TIMP-1 (matrix metalloproteinase/tissue inhibitor of MMP) ratio increased significantly. Besides, after 5 days of exogenous TGF-β1 stimulation, the expression levels of Col 1, FN, TGF-β1, IL-6, CTGF, and α-SMA in HSFs increased significantly. Impressively, all these increased gene expression levels were reversed by 5 days of ASCs coculture treatment. Additionally, the proliferation, migration, and contractility of HSFs were all significantly reduced by ASC coculture treatment. Furthermore, the protein levels of TGF-β1 and intracellular signal pathway-related molecules, such as p-smad2, p-smad3, p-Stat3, and p-ERK, were downregulated significantly in HSFs after 5 days of ASCs coculture treatment. This study demonstrated that coculture of HSFs with ASCs not only inhibited proliferation, migration, and contractility of HSFs but also decreased the expression levels of HSF-related or TGF-β1-induced molecules. Additionally, the antifibrotic effect on HSFs was likely mediated by the inhibition of multiple intracellular signaling. The results of this study suggest the therapeutic potential of ASCs for HS treatment, which is worth of further investigation.

A model for one-dimensional morphoelasticity and its application to fibroblast-populated collagen lattices.

The mechanical behaviour of solid biological tissues has long been described using models based on classical continuum mechanics. However, the classical continuum theories of elasticity and viscoelasticity cannot easily capture the continual remodelling and associated structural changes in biological tissues. Furthermore, models drawn from plasticity theory are difficult to apply and interpret in this context, where there is no equivalent of a yield stress or flow rule. In this work, we describe a novel one-dimensional mathematical model of tissue remodelling based on the multiplicative decomposition of the deformation gradient. We express the mechanical effects of remodelling as an evolution equation for the effective strain, a measure of the difference between the current state and a hypothetical mechanically relaxed state of the tissue. This morphoelastic model combines the simplicity and interpretability of classical viscoelastic models with the versatility of plasticity theory. A novel feature of our model is that while most models describe growth as a continuous quantity, here we begin with discrete cells and develop a continuum representation of lattice remodelling based on an appropriate limit of the behaviour of discrete cells. To demonstrate the utility of our approach, we use this framework to capture qualitative aspects of the continual remodelling observed in fibroblast-populated collagen lattices, in particular its contraction and its subsequent sudden re-expansion when remodelling is interrupted.

Gallic acid attenuates TGF-β1-stimulated collagen gel contraction via suppression of RhoA/Rho-kinase pathway in hypertrophic scar fibroblasts

AimsTo examine the effect and molecular mechanism of gallic acid (GA) on transforming growth factor-β1 (TGF-β1)-stimulated hypertrophic scar fibroblast (HSF) contraction. Materials and methodsA fibroblast-populated collagen lattice (FPCL) was developed to examine the effect of GA on TGF-β1-enhanced HSF contraction. The changes in crucial factors related to cell contraction including α-smooth muscle actin (α-SMA), F-actin, and the phosphorylation level of myosin light chain (MLC) were evaluated using western blot and immunostaining. The activation and expression of RhoA/ROCK after the TGF-β1 challenge and GA insult were evaluated using RhoA-G-LISA and RhoA-ELISA kit while the phosphorylation level of MYPT1 and the expression of ROCK1 and ROCK2 were examined by western blot, respectively. Key findingsGA significantly suppressed TGF-β1-stimulated HSF contraction in a dose- and time-dependent manner. Moreover, the TGF-β1-enhanced α-SMA expression, F-actin formation, and MLC phosphorylation were obviously attenuated by GA. TGF-β1 significantly stimulated RhoA activation but did not alter the expression of RhoA in the HSFs. However, both the activation and expression of RhoA decreased obviously with GA pretreatment followed by TGF-β1 stimulation. Furthermore, GA inhibited ROCK activity but did not affect its expression after TGF-β1 stimulation. SignificanceThese results suggest that GA exhibited the potential to prevent HSF contraction after TGF-β1 stimulation by down regulating the RhoA/ROCK signal cascade, followed by the inhibition of the expression of α-SMA, F-actin formation, and phosphorylation of MLC.