Response of fibroblasts to transforming growth factor-?1 on two-dimensional and in three-dimensional hyaluronan hydrogels.
ABSTRACT: Transforming growth factor-?1 (TGF-?1), an important cytokine with multiple functions, is secreted during wound healing. Previous studies have utilized two-dimensional (2D) cell culture to elucidate the functions of TGF-?1; however, 2D culture does not represent the complex three-dimensional (3D) in vivo environment. Using a synthetic hyaluronan (HA) extracellular matrix (ECM) hydrogel, we investigated the effect of TGF-?1 on fibroblasts cultured in three conditions--on tissue culture polystyrene (TCP), on HA (2D), and in HA (3D). After TGF-?1 treatment (0.1 to 20?ng/mL), morphological features and ECM regulation were analyzed by immunocytochemistry, Western blot, quantitative polymerase chain reaction, and zymogram assays. On TCP, cells showed the typical spindle shape with strong alpha smooth muscle actin (?-SMA) staining of cytoplasmic myofilaments along the cell axes after TGF-?1 treatment; on HA (2D), spindle-shape cells showed little ?-SMA staining; in HA (3D), cells were smaller and rounded with less ?-SMA deposition. The ?-SMA gene and protein expression on TCP were significantly upregulated by TGF-?1, but TGF-?1 did not induce ?-SMA expression in the presence of HA (both 2D and 3D). 3D HA culture significantly downregulated collagen I, III, and fibronectin expression, increased matrix metalloproteinase 1 and 2 (MMP1/MMP2) activity, upregulated MMP1 mRNA and downregulated TIMP3 mRNA expression. This study suggested that exogenous HA, particularly in 3D culture, appears to suppress ECM production, enhances ECM degradation and remodeling, and inhibits myofibroblast differentiation without decreasing TGF-? receptor expression.
Project description:Chemical modification of engineered microenvironments surrounding living cells represents a means for directing cellular behaviors through cell-matrix interactions. Presented here is a temporally controlled method for modulating the properties of biomimetic, synthetic extracellular matrices (ECM) during live cell culture employing the rapid, bioorthogonal tetrazine ligation with trans-cyclooctene (TCO) dienophiles. This approach is diffusion-controlled, cytocompatible, and does not rely on light, catalysts, or other external triggers. Human bone-marrow-derived mesenchymal stem cells (hMSCs) were initially entrapped in a hydrogel prepared using hyaluronic acid carrying sulfhydryl groups (HA-SH) and a hydrophilic polymer bearing both acrylate and tetrazine groups (POM-AT). Inclusion of a matrix metalloprotease (MMP)-degradable peptidic cross-linker enabled hMSC-mediated remodeling of the synthetic environment. The resultant network displayed dangling tetrazine groups for subsequent conjugation with TCO derivatives. Two days later, the stiffness of the matrix was increased by adding chemically modified HA carrying multiple copies of TCO (HA-TCO) to the hMSC growth media surrounding the cell-laden gel construct. In response, cells developed small processes radially around the cell body without a significant alteration of the overall shape. By contrast, modification of the 3D matrix with a TCO-tagged cell-adhesive motif caused the resident cells to undergo significant actin polymerization, changing from a rounded shape to spindle morphology with long cellular processes. After additional 7 days of culture in the growth media, quantitative analysis showed that, at the mRNA level, RGD tagging upregulated cellular expression of MMP1, but downregulated the expression of collagen I/III and tenascin C. RGD tagging, however, was not sufficient to induce the classic osteoblastic, chondrogenic, adipogenic, or fibroblastic/myofibroblastic differentiation. The modular approach allows facile manipulation of synthetic ECM to modulate cell behavior, thus potentially applicable to the engineering of functional tissues or tissue models.
Project description:Glioblastoma multiforme (GBM) tumors, which arise from glia in the central nervous system (CNS), are one of the most deadly forms of human cancer with a median survival time of ?1 year. Their high infiltrative capacity makes them extremely difficult to treat, and even with aggressive multimodal clinical therapies, outcomes are dismal. To improve understanding of cell migration in these tumors, three-dimensional (3D) multicomponent composite hydrogels consisting of collagen and hyaluronic acid, or hyaluronan (HA), were developed. Collagen is a component of blood vessels known to be associated with GBM migration; whereas, HA is one of the major components of the native brain extracellular matrix (ECM). We characterized hydrogel microstructural features and utilized these materials to investigate patient tumor-derived, single cell morphology, spreading, and migration in 3D culture. GBM morphology was influenced by collagen type with cells adopting a rounded morphology in collagen-IV versus a spindle-shaped morphology in collagen-I/III. GBM spreading and migration were inversely dependent on HA concentration; with higher concentrations promoting little or no migration. Further, noncancerous astrocytes primarily displayed rounded morphologies at lower concentrations of HA; in contrast to the spindle-shaped (spread) morphologies of GBMs. These results suggest that GBM behaviors are sensitive to ECM mimetic materials in 3D and that these composite hydrogels could be used to develop 3D brain mimetic models for studying migration processes.
Project description:Aberrant remodeling of trabecular meshwork (TM) extracellular matrix (ECM) may induce ocular hypertensive phenotypes in human TM (hTM) cells to cause ocular hypertension, via a yet unknown mechanism. Here, we show that, in the absence of exogenous transforming growth factor-beta2 (TGF?2), compared with control matrices (VehMs), glucocorticoid-induced cell-derived matrices (GIMs) trigger non-Smad TGF?2 signaling in hTM cells, correlated with overexpression/activity of structural ECM genes (fibronectin, collagen IV, collagen VI, myocilin), matricellular genes (connective tissue growth factor [CTGF], secreted protein, acidic and rich in cysteine), crosslinking genes/enzymes (lysyl oxidase, lysyl oxidase-like 2-4, tissue transglutaminase-2), and ECM turnover genes/enzymes (matrix metalloproteinases-MMP2,14 and their inhibitors-TIMP2). However, in the presence of exogenous TGF?2, VehMs and GIMs activate Smad and non-Smad TGF?2 signaling in hTM cells, associated with overexpression of ?-smooth muscle actin (?-SMA), and differential upregulation of aforementioned ECM genes/proteins with new ones emerging (collagen-I, thrombospondin-I, plasminogen activator inhibitor, MMP1, 9, ADAMTS4, TIMP1); with GIM-TGF?2-induced changes being mostly more pronounced. This suggests dual glaucomatous insults potentiate profibrotic signaling/phenotypes. Lastly, we demonstrate type I TGF? receptor kinase inhibition abrogates VehM-/GIM- and/or TGF?2-induced upregulation of ?-SMA and CTGF. Collectively, pathological TM microenvironments are sufficient to elicit adverse cellular responses that may be ameliorated by targeting TGF?2 pathway.
Project description:To elucidate molecular pharmacology of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-i, Ripasudil and Y27632) on their efficiency for aqueous outflow, 2D or 3D cultures of a human trabecular meshwork (HTM) were prepared in the presence of TGF?2. Those were examined by transendothelial electrical resistance (TEER, 2D), electronic microscopy (EM, 2D and 3D), expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, and fibronectin (FN) by immunolabeling and/or quantitative PCR (3D), and solidity of 3D organoids by a micro-squeezer. TGF?2 significantly increased the TEER values in 2D cultures, and the ECM expression indicated that the 3D organoids assumed a more densely packed shape. ROCK-i greatly reduced the TGF?2-induced enhancement of TEER and the immunolabeled ECM expression of the 3D organoids. In contrast, the mRNA expression of COL1 was increased, and those of COL4 and FN were unchanged. EM revealed that TGF?2 caused the HTM cells to become more compact and abundant ECM deposits within the 3D organoids were observed. These were significantly inhibited by ROCK-i. The dense solids caused by the presence of TGF?2 were significantly suppressed by ROCK-i. Current study indicates that ROCK-i cause beneficial effects toward the spatial configuration of TGF?2-induced HTM 3D organoids.
Project description:Biological scaffolds composed of tissue-derived extracellular matrix (ECM) can promote homologous (i.e., tissue-specific) cell differentiation through preservation of biophysical and biochemical motifs found in native tissues. Solubilized ECMs derived from decellularized tendon and cartilage have recently been promoted as tissue-specific biomaterials, but whether tissue-specific bioactivity is preserved following solubilization is unknown. This study explored the tissue-specific bioactivity of soluble decellularized tendon and cartilage ECMs on human bone marrow-derived mesenchymal stem cells (MSCs) presented across different culture microenvironments, including two-dimensional (2D) tissue culture plastic, aligned electrospun nanofibers, cell pellets, and cell-seeded photocrosslinkable hydrogels.Tendon and cartilage ECMs were decellularized using established methods and solubilized either via pepsin digestion or urea extraction. The effect of soluble ECMs on cell proliferation and differentiation was initially explored by supplementing basal medium of human MSCs cultured on 2D tissue culture plastic. In subsequent experiments, MSCs were cultured on aligned electrospun nanofibers, ascell pellets, or encapsulated within photocrosslinkable methacrylated gelatin (GelMA) hydrogels. Urea-extracted tendon and cartilage ECMs were added as supplements.Pepsin-digested ECMs did not promote homologous differentiation in human MSCs, whether provided as a medium supplement or three-dimensional (3D) hydrogels. In contrast, urea-extracted ECMs tended to promote tissue-specific differentiation of MSCs cultured in 2D and 3D microenvironments. The application of the small molecule TGF-? signaling inhibitor SB-431542 largely negated the tissue-specific gene expression patterns mediated by tendon and cartilage ECMs. This suggests that the action of endogenous TGF-? was required, but was not sufficient, to impart tissue-specific bioactivity of urea-extracted ECMs. When urea-extracted cartilage ECM was incorporated within a photocurable GelMA hydrogel it independently enhanced chondrogenesis in encapsulated MSCs, and showed additive prochondrogenesis upon TGF-? supplementation in the medium.Urea-extracted ECM fractions of decellularized tendon and cartilage are soluble supplements capable of enhancing tissue-specific differentiation of adult stem cells.
Project description:This study evaluated the feasibility of a novel three-dimensional (3D) porous composite of uncalcined and unsintered hydroxyapatite (u-HA) and poly-d/l-lactide (PDLLA) (3D-HA/PDLLA) for the bony regenerative biomaterial in maxillofacial surgery, focusing on cellular activities and osteoconductivity properties in vitro and in vivo. In the in vitro study, we assessed the proliferation and ingrowth of preosteoblastic cells (MC3T3-E1 cells) in 3D-HA/PDLLA biomaterials using 3D cell culture, and the results indicated enhanced bioactive proliferation. After osteogenic differentiation of those cells on 3D-HA/PDLLA, the osteogenesis marker genes runt-related transcription factor-2 (Runx2), and Sp7 (Osterix) were upregulated. For the in vivo study, we evaluated the utility of 3D-HA/PDLLA biomaterials compared to the conventional bone substitute of beta-tricalcium phosphate (?-TCP) in rats with critical mandibular bony defects. The implantation of 3D-HA/PDLLA biomaterials resulted in enhanced bone regeneration, by inducing high osteoconductivity as well as higher ?-TCP levels. Our study thus showed that the novel composite, 3D-HA/PDLLA, is an excellent bioactive/bioresorbable biomaterial for use as a cellular scaffold, both in vitro and in vivo, and has utility in bone regenerative therapy, such as for patients with irregular maxillofacial bone defects.
Project description:The extracellular matrix (ECM), one of the key components of tumor microenvironment, has a tremendous impact on cancer development and highly influences tumor cell features. ECM affects vital cellular functions such as cell differentiation, migration, survival and proliferation. Gene and protein expression levels are regulated in cell-ECM interaction dependent manner as well. The rate of unsuccessful clinical trials, based on cell culture research models lacking the ECM microenvironment, indicates the need for alternative models and determines the shift to three-dimensional (3D) laminin rich ECM models, better simulating tissue organization. Recognized advantages of 3D models suggest the development of new anticancer treatment strategies. This is among the most promising directions of 3D cell cultures application. However, detailed analysis at the molecular level of 2D/3D cell cultures and tumors in vivo is still needed to elucidate cellular pathways most promising for the development of targeted therapies. In order to elucidate which biological pathways are altered during microenvironmental shift we have analyzed whole genome mRNA and miRNA expression differences in LLC1 cells cultured in 2D or 3D culture conditions.In our study we used DNA microarrays for whole genome analysis of mRNA and miRNA expression differences in LLC1 cells cultivated in 2D or 3D culture conditions. Next, we indicated the most common enriched functional categories using KEGG pathway enrichment analysis. Finally, we validated the microarray data by quantitative PCR in LLC1 cells cultured under 2D or 3D conditions or LLC1 tumors implanted in experimental animals.Microarray gene expression analysis revealed that 1884 genes and 77 miRNAs were significantly altered in LLC1 cells after 48 h cell growth under 2D and ECM based 3D cell growth conditions. Pathway enrichment results indicated metabolic pathway, MAP kinase, cell adhesion and immune response as the most significantly altered functional categories in LLC1 cells due to the microenvironmental shift from 2D to 3D. Comparison of the expression levels of selected genes and miRNA between LLC1 cells grown in 3D cell culture and LLC1 tumors implanted in the mouse model indicated correspondence between both model systems.Global gene and miRNA expression analysis in LLC1 cells under ECM microenvironment indicated altered immune response, adhesion and MAP kinase pathways. All these processes are related to tumor development, progression and treatment response, suggesting the most promising directions for the development of targeted therapies using the 3D cell culture models.
Project description:Cystogenesis and tubulogenesis are basic building blocks for many epithelial organs, including the kidney. Most researchers have used two-dimensional (2D) cell culture to investigate signaling pathways downstream of hepatocyte growth factor (HGF). We hypothesize that three-dimensional (3D) collagen-grown Madin-Darby canine kidney (MDCK) cells, which form cysts and then tubulate in response to HGF, are a much more in vivo-like system for the identification of novel tubulogenes. With the use of a canine microarray containing over 20,000 genes, 2,417 genes were identified as potential tubulogenes that were differentially regulated, exclusively in 3D-grown MDCK cells. Among these, 840 were dependent on MAPK signaling. Importantly, this work shows that many putative tubulogenes, previously identified via microarray analysis of 2D cultures, including by us, do not change in 3D culture and vice versa. The use of a 3D-culture system allowed for the identification of novel MAPK-dependent and -independent genes that regulate early renal tubulogenesis in vitro, e.g., matrix metalloproteinase 1 (MMP1). Knockdown of MMP1 led to defects in cystogenesis and tubulogenesis in 3D-grown MDCK cells, most likely due to problems establishing normal polarity. We suggest that data obtained from 2D cultures, even those using MDCK cells treated with HGF, should not be automatically extrapolated to factors important for cystogenesis and tubulogenesis. Instead, 3D culture, which more closely replicates the biological environment and is therefore a more accurate model for identifying tubulogenes, is preferred. Results from the present analysis will be used to build a more accurate model of the signaling pathways that control cystogenesis and tubulogenesis.
Project description:The mechanism of pathological osteogenesis in Ankylosing spondylitis (AS) is largely unknown. Our previous studies demonstrated that the imbalance between BMP-2 and Noggin secretion induces abnormal osteogenic differentiation of marrow-derived mesenchymal stem cells (MSCs) from AS patients in a two-dimensional culture environment. In this study, HA/β-TCP scaffolds were further used as a three-dimensional (3D) biomimetic culture system to mimic the bone microenvironment in vivo to determine the abnormal osteogenic differentiation of AS-MSCs. We demonstrated that when cultured in HA/β-TCP scaffolds, AS-MSCs had a stronger osteogenic differentiation capacity than that of MSCs from healthy donors (HD-MSCs) in vitro and in vivo. This dysfunction resulted from BMP2 overexpression in AS-MSCs, which excessively activated the Smad1/5/8 and ERK signalling pathways and finally led to enhanced osteogenic differentiation. Both the signalling pathway inhibitors and siRNAs inhibiting BMP2 expression could rectify the enhanced osteogenic differentiation of AS-MSCs. Furthermore, BMP2 expression in ossifying entheses was significantly higher in AS patients. In summary, our study demonstrated that AS-MSCs possess enhanced osteogenic differentiation in HA/β-TCP scaffolds as a 3D biomimetic microenvironment because of BMP2 overexpression, but not Noggin. These results provide insights into the mechanism of pathological osteogenesis, which can aid in the development of niche-targeting medications for AS.
Project description:Tumor cell response to irradiation also depends on their microenvironment. Therefore ongoing investigation of three-dimensional (3D) cell culture models provide researchers with essential data studying and remodeling radiotherapeutic implications in cancer treatment. 3D culture models were shown to mimic in vivo cell microenvironment more accurately than the standard two-dimensional cell monolayer (2D) cultures. Growing evidence suggests that 2D and 3D cultured cell gene expression pattern discrepancies following irradiation is highly dependent on cell-ECM interactions. It has been shown that laminin-rich-extracellular matrix (lr-ECM) used in 3D cultures not only alters cancer cell phenotype and response to external stimuli but also affects their differentiation, migration and survivability. Thus, a change in these fundamental cell properties demands us to reconsider data previously collected using 2D in vitro models. RNA was harvested from two colorectal cancer cell lines cultivated under 3D cell culture conditions, 4h after treatment of single (2 Gy or 10 Gy) or fractionated (5x2 Gy) ionizing radiation dose.