Fibroblast-populated Collagen Lattice Model 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.

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.

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.

English

Povidone-iodine (PI) 10% solution is an effective antiseptic. However, it appears to be toxic to the cells involved in wound healing. The aim of this study is to evaluate the toxicity of PI on cultured human fibroblast using fibroblast populated collagen lattice (FPCL) model. The cultured human fibroblast was divided into 6 groups i.e. 5 groups were exposed by PI 1, 0.1, 0.01, 0.001 and 0.0001%, and 1 group was exposed by phosphate-buffered-saline (PBS). Twenty-four hours later, the media was washed using PBS. The size of the FPCL media on each group was observed over time by serial photographs, which then were measured by Image-J computer program. Exposure of 0.1, 0.01, 0.001 and 0.0001 PI caused an obvious reduction of fibroblast’s contraction capability on FPCL media, which described temporary fibroblast injury, that showing a concentration-dependent recovery phenomenon after 48th hour. Furthermore, 1% PI exposure leads to a permanent fibroblast injury. In conclusion, PI exposure in concentration more than 0.1% has a permanent toxic effect on fibroblast that clearly observed using a simple FPCL model.

ShRNA targeting SFRP2 promotes the apoptosis of hypertrophic scar fibroblast

Hypertrophic scars result from a dysregulated process in wound healing. Although the basic mechanism is unclear, increased proliferation and decreased cell apoptosis are noticed in the development of hypertrophic scar. In previous study, we found that secreted frizzled-related protein 2 (SFRP2), which was associated with cell proliferation, apoptosis, and differentiation, was dramatically upregulated in hypertrophic scar (HS) tissue. In this study short hairpin RNA (shRNA) targeting SFRP2 was employed to characterize SFRP2 function in hypertrophic scar-derived fibroblasts (HSFb). Cell proliferation was assessed by MTT, dynamic growth curves, and BRDU assays. Meanwhile, Cell apoptosis was detected using fluorescence-activated cell sorting (FACS). Caspase-3 activity was assayed by spectrophotometry. Fibroblast populated collagen lattice (FPCL) model was employed to evaluate the contractility of HSFb. Further, real-time PCR and western blot were used to measure the mRNA and protein expressions of α-SMA in HSFb. In addition, mRNA levels of type I and III procollagen were assayed by quantitative real-time PCR. The results revealed that shRNA targeting SFRP2 significantly promoted the apoptosis of HSFb, while it had no effect on the cell proliferation. Decreased synthesis of a-smooth muscle actin (α-SMA) in HSFb and reduced contraction of fibroblasts in the FPCL model were observed. Quantitative RT-PCR suggested that the mRNAs of type I and III procollagen were significantly downregulated. In conclusion, as a novel anti-apoptosis gene, SPRP2 was present in hypertrophic scars. Importantly, shRNA targeting SFRP2 may provide a new approach to preventing the formation of HS.

In vitro study of the effects of plaunotol on oral cell proliferation and wound healing

Plaunotol is an acyclic diterpene alcohol extracted from a medicinal plant called plau-noi, Croton stellatopilosus Ohba, and has been widely used for the treatment of gastric ulcers in Japan. The aim of this study was to examine the effects of plaunotol on human gingival fibroblasts (HGFs) and human oral keratinocytes (HOKs). To assess the cytotoxic effect, HGFs and HOKs were treated with plaunotol. Subsequently, the morphology of cells was recorded and cells were subjected to MTT assay. To investigate cell proliferation effect, cells were treated with plaunotol and counted with a haemocytometer. To determine wound healing effect, the number of cells repopulated into the wounded areas in monolayer culture and in fibroblast-populated collagen lattice (FPCL) was measured. The results showed that 10 and 1 μg/ml (33 and 3.3 μmol/l) plaunotol induced toxicity in HGFs and HOKs, respectively. However, 0.1 μg/ml (0.33 μmol/l) plaunotol promoted HGF proliferation and wound healing in monolayer and FPCL models. In contrast, 0.1 μg/ml plaunotol could not induce HOK proliferation nor in vitro wound healing using monolayer culture, but it induced wound healing in a modified FPCL model. Our data suggested that plaunotol could promote oral cell proliferation and wound healing in vitro and may have an implication on oral wound healing.

The effect of interferon-alpha2b on an in vitro model Dupuytren's contracture.

The effects of interferon-alpha2b (IFN-alpha2b) on Dupuytren's and control palmar fibroblasts were evaluated using the fibroblast-populated collagen lattice model. Three paired strains of Dupuytren's and control fibroblasts were exposed to IFN-alpha2b for 96 hours before incorporation into triplicate collagen lattices. Contraction of the lattices was recorded and Northern blot analysis of cytoskeletal mRNA was performed. Comparisons of Dupuytren's and control fibroblasts revealed significantly increased contractility of the Dupuytren's fibroblasts in 2 of the 3 strains. Treatment with IFN-alpha2b significantly inhibited contraction in both Dupuytren's and control fibroblasts. In Dupuytren's fibroblasts, treatment with IFN-alpha2b significantly down-regulated mRNA expression for cytoplasmic beta-actin and gamma-actin.

Scar Contracture: Cellular and Connective Tissue Aspects in Peyronie's Disease

The understanding of the mechanism of how the plaque in Peyronie's disease causes functional distortions is presented in terms of scar formation and maturation. The repair process is important for survival. The expected biological response to tissue loss and disruption is scarring but in some situations, such as Peyronie's disease, the resultant scar creates major problems. The development of scar involves the deposition of connective tissue which histologically can be studied in patients and in animal models as well as with in vitro models. Abnormal scarring can involve excessive connective tissue deposition. Clinically examples of excess scarring are hypertrophic scars common in healed burn patients and keloid scars seen in patients with this inherited defect in healing. In addition to excessive connective tissue deposition, another aspect of abnormal scarring is scar contracture, when the compaction of the scar tissue distorts the anatomical appearance and functional capacity of adjacent tissues and organs. The fibroblast populated collagen lattice model demonstrates the organization of connective tissue in a controlled environment and the information gained can be related to scarring. The compaction of scar tissue involves the reorganization of collagen fibers by resident fibroblasts using cell tractional forces rather than cell contraction forces. These findings were documented with the fibroblast populated collagen lattice model. In Peyronie's disease the distortion caused by the scar or plaque is neither related to scar contracture nor active compaction of scar tissue but, rather, the expansion of the surrounding unscarred tissue and the inflexibility of the scar tissue composing the plaque. Peyronie's disease plaques are rigid structures due to the organization of the deposited collagen fibers. Those fibers are independent in their connective tissue organization, which makes the plaque incompatable with adjacent structures.

In vitro and in vivo analysis of the inability of fetal rabbit wounds to contract

Fetal rabbit wounds that are sutured show excellent repair without obvious scarring. In contrast, an unsutured wound in a rabbit fetus does not close, and it appears that the process of wound contraction does not occur. Experiments were carried out to illustrate the mechanisms responsible for the noncontraction of open fetal rabbit wounds. Results showed that the lack of wound contraction was not an artifact caused by rapid fetal growth. With regard to the ability of cultured fetal fibroblasts to show cytoplasmic muscle-induced cell contraction, we found that, in cultured fetal fibroblasts, cell contraction was induced by adenosine triphosphate. Contractile abilities of fetal-derived fibroblasts were equivalent to those of adult-derived fibroblasts. The fetal fibroblasts also demonstrated the generation of superior contractile activity when examined in a fibroblast-populated collagen lattice model. Finally, the ability of amniotic fluid to alter wound contraction was addressed by means of the fibroblast-populated collagen lattice in vitro model. Increasing concentrations of amniotic fluid inhibited fetal fibroblast lattice contraction. Therefore, rabbit amniotic fluid contains an inhibitor that may be partially responsible for the noncontraction of fetal rabbit wounds in utero.