Fibroblast morphogenesis on 3D collagen matrices: The balance between cell clustering and cell migration

Fibroblast clusters have been observed in tissues under a variety of circumstances: in fibrosis and scar, in the formation of hair follicle dermal papilla, and as part of the general process of mesenchymal condensation that takes place during development. Cell clustering has been shown to depend on features of the extracellular matrix, growth factor environment, and mechanisms to stabilize cell–cell interactions. In vitro studies have shown that increasing the potential for cell–cell adhesion relative to cell–substrate adhesion promotes cell clustering. Experimental models to study fibroblast clustering have utilized centrifugation, hanging drops, and substrata with poorly adhesive, soft and mechanically unstable properties. In this review, we summarize work on a new, highly tractable, cell clustering research model in which human fibroblasts are incubated on the surfaces of collagen matrices. Fibroblast clustering occurs under procontractile growth factor conditions (e.g., serum or the serum lipid agonist lysophosphatidic acid) but not under promigratory growth factor conditions (e.g., platelet-derived growth factor) and can be reversed by switching growth factor environments. Cell contraction plays a dual role in clustering to bring cells closer together and to stimulate cells to organize fibronectin into a fibrillar matrix. Binding of fibroblasts to a shared fibronectin fibrillar matrix stabilizes clusters, and fragmentation of the fibrillar matrix occurs when growth factor conditions are switched to promote cell dispersal.     

Oncogenic Ras-transformed human fibroblasts exhibit differential changes in contraction and migration in 3D collagen matrices

Tractional force exerted by tissue cells in 3D collagen matrices can be utilized for matrix remodeling or cell migration. The interrelationship between these motile processes is not well understood. The current studies were carried out to test the consequences of oncogenic Ras (H-Ras^V12) transformation on human fibroblast contraction and migration in 3D collagen matrices. Beginning with hTERT-immortalized cells, we prepared fibroblasts stably transformed with E6/E7 and with the combination HPV16 E6/E7 and H-Ras^V12. Oncogenic Ras-transformed cells lost contact inhibition of cell growth, formed colonies in soft agar and were unable to make adherens junctions. We observed no changes in the extent or growth factor dependence of collagen matrix contraction (floating or stress-relaxation) by oncogenic Ras-transformed cells. On the other hand, transformed cells in nested collagen matrices lost not only growth factor selectivity, but also cell-matrix density-dependent inhibition of migration. These findings demonstrate differential regulation of collagen matrix contraction and cell migration in 3D collagen matrices.     

Fibronectin production by cultured human lung fibroblasts in three-dimensional collagen gel culture

Summary In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.     

Tumor cell locomotion: differential dynamics of spontaneous and induced migration in a 3D collagen matrix

Although great strides have recently been made in elucidating the factors initiating tumor cell migration and the relevant cellular pathways involved, the constituent components of migratory dynamics for individual tumor cell motion have still not been resolved. Utilizing a three-dimensional (3D) collagen assay and computer-assisted, continuous single cell tracking, we investigated the basic parameters for both the spontaneous and norepinephrine-induced migration of highly metastatic MBA-MB-468 breast, PC-3 prostate, and SW 480 colon carcinoma cells. We show that tumor cells do not migrate with uniform migrational structure and speed as previously thought, but rather, the induction of locomotion elicits significant increases in speed, break frequency, and total cell displacement, but decreases in break length and no change in the recruitment of nonlocomotory cells. We furthermore illustrate the corresponding morphological changes of induced tumor cell migration with emphasis on motion in a collagen matrix. These results demonstrate the complexity of tumor cell migration, and the compulsion for incorporating not only knowledge of intracellular pathways, but also fundamental parameters of migratory behavior into any expansive theory of tumor cell migration and metastasis formation. We furthermore establish the analytical methodology of investigating both the stimulation and potential pharmaceutical inhibition of tumor cell migration.     

Contraction of collagen by human fibroblasts and keratinocytes

Summary In the process of wound healing keratinocytes and fibroblasts play an important role, keratinocytes in the re-epithelization process and fibroblasts in the process of wound contraction. We have studied the role of human keratinocytes and fibroblasts in the rearrangement of collagen in a collagen lattice model system. Our results revealed that keratinocytes as well as fibroblasts rearrange the collagen lattice; this occurs in a cell number and collagen concentration dependent manner. The optimal gel contraction is obtained in the presence of keratinocytes on the top of and of fibroblasts in the collagen lattice, the situation most closely approaching the in vivo situation. Between the two types of cells, differences in morphologic behavior were observed: when incorporated into the gel the keratinocytes retained their spherical shape throughout the whole culture period, but fibroblasts became elongated and formed extensions. Our data suggest that not only fibroblasts but also keratinocytes may be involved in the process of wound contraction. This work was supported by the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation, grant 84-10).     

TGF-β1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels

Apoptosis of fibroblasts may be key for the removal of cells following repair processes. Contraction of three-dimensional collagen gels is a model of wound healing and remodeling. Here two potent inducers of contraction, TGF-β1 and fetal calf serum (FCS) were evaluated for their effect on fibroblast apoptosis in contracting collagen gels. Human fetal lung fibroblasts were cultured in floating type I collagen gels, exposed to TGF-β1 or FCS, and allowed to contract for 5 days. Apoptosis was evaluated using TUNEL and confirmed by DNA content profiling. Both TGF-β1 and serum significantly augmented collagen gel contraction. TGF-β1 also increased apoptosis assessed by TUNEL positivity and DNA content analysis. In contrast, serum did not affect apoptosis. TGF-β1 induction of apoptosis was associated with augmented expression of Bax, a pro-apoptotic member of the Bax/Bcl-2 family, inhibition of Bcl-2, an anti-apoptotic member of the same family, and inhibition of both cIAP-1 and XIAP, two inhibitors of the caspase cascade. Serum was associated with an increase in cIAP-1 and Bcl-2, anti-apoptotic proteins. Interestingly, serum was also associated with an apparent increase in Bax, a pro-apoptotic protein. Blockade of Smad3 with either siRNA or by using murine fibroblasts deficient in Smad3 resulted in a lack of TGF-β induction of augmented contraction and apoptosis. Contraction induced by different factors, therefore, may be differentially associated with apoptosis, which may be related to the persistence or resolution of the fibroblasts that accumulate following injury.     

Transforming growth factor beta stimulates fibroblast-collagen matrix contraction by different mechanisms in mechanically loaded and unloaded matrices

Studies were carried out to test the idea that transforming growth factor beta (TGFbeta) stimulated fibroblast contraction of collagen matrices by different mechanisms depending on mechanical loading on the cells and matrix. Under mechanically unloaded conditions (floating matrices), TGFbeta stimulated contraction directly as an agonist and indirectly by preactivating cells to express the myofibroblast phenotype. Increased contraction of floating matrices by preactivated cells appeared to result in part from an autocrine mechanism. Under mechanically loaded conditions (stressed matrices), TGFbeta had no direct agonist effect on contraction. Fibroblasts preactivated to become myofibroblasts showed increased ability to transfer tension to stressed matrices, and tension persisted even after the cells' actin cytoskeleton was disrupted. Our findings are consistent with the idea that fibroblasts activated to become myofibroblasts in vitro have increased contractile activity and indicate that multiple mechanisms that differ depending on mechanical loading on the cells and matrix are involved.     

ARHGEF4-mediates the actin cytoskeleton reorganization of hepatic stellate cells in 3-dimensional collagen matrices

The actin cytoskeleton of hepatic stellate cells (HSCs) is reorganized when they are cultured in 3D collagen matrices. Here, we investigated the molecular mechanism of actin cytoskeleton reorganization in HSCs cultured in 3D floating collagen matrices (FCM) compared to those on 2D polystyrene surfaces (PS). First, we found that the generation of dendritic cellular processes was controlled by Rac1. Next, we examined the differential gene expression of HSCs cultured on 2D PS and in 3D FCM by RNA-Seq and focused on the changes of actin cytoskeleton reorganization-related molecular components and guanine nucleotide exchange factors (GEFs). The results showed that the expression of genes associated with actin cytoskeleton reorganization-related cellular components, filopodia and lamellipodia, were significantly decreased, but podosome-related genes was significantly increased in 3D FCM. Furthermore, we found that a Rac1-specific GEF, ARHGEF4, played roles in morphological changes, migration and podosome-related gene expression in HSCs cultured in 3D FCM.

Abbreviations: 2D PS: 2-dimensional polystyrene surface; 3D FCM: 3-dimensional floating collagen matrices; ARHGEF4: Rho guanine nucleotide exchange factor 4; ARHGEF6: Rho guanine nucleotide exchange factor 6; GEF: guanine nucleotide exchange factor; HSC: hepatic stellate cell     

Differences in the mechanism of collagen lattice contraction by myofibroblasts and smooth muscle cells

Both rat derived vascular smooth muscle cells (SMC) and human myofibroblasts contain α smooth muscle actin (SMA), but they utilize different mechanisms to contract populated collagen lattices (PCLs). The difference is in how the cells generate the force that contracts the lattices. Human dermal fibroblasts transform into myofibroblasts, expressing α‐SMA within stress fibers, when cultured in lattices that remain attached to the surface of a tissue culture dish. When attached lattices are populated with rat derived vascular SMC, the cells retain their vascular SMC phenotype. Comparing the contraction of attached PCLs when they are released from the culture dish on day 4 shows that lattices populated with rat vascular SMC contract less than those populated with human myofibroblast. PCL contraction was evaluated in the presence of vanadate and genistein, which modify protein tyrosine phosphorylation, and ML‐7 and Y‐27632, which modify myosin ATPase activity. Genistein and ML‐7 had no affect upon either myofibroblast or vascular SMC‐PCL contraction, demonstrating that neither protein tyrosine kinase nor myosin light chain kinase was involved. Vanadate inhibited myofibroblast‐PCL contraction, consistent with a role for protein tyrosine phosphatase activity with myofibroblast‐generated forces. Y‐27632 inhibited both SMC and myofibroblast PCL contraction, consistent with a central role of myosin light chain phosphatase. J. Cell. Biochem. 111: 362–369, 2010. © 2010 Wiley‐Liss, Inc.     

The D2 period of collagen II contains a specific binding site for the human discoidin domain receptor, DDR2

The human discoidin domain receptors (DDRs), DDR1 and DDR2, are expressed widely and, uniquely among receptor tyrosine kinases, activated by the extracellular matrix protein collagen. This activation is due to a direct interaction of collagen with the DDR discoidin domain. Here, we localised a specific DDR2 binding site on the triple-helical region of collagen II. Collagen II was found to be a much better ligand for DDR2 than for DDR1. As expected, DDR2 binding to collagen II was dependent on triple-helical collagen and was mediated by the DDR2 discoidin domain. Collagen II served as a potent stimulator of DDR2 autophosphorylation, the first step in transmembrane signalling. To map the DDR2 binding site(s) on collagen II, we used recombinant collagen II variants with specific deletions of one of the four repeating D periods. We found that the D2 period of collagen II was essential for DDR2 binding and receptor autophosphorylation, whereas the D3 and D4 periods were dispensable. The DDR2 binding site on collagen II was further defined by recombinant collagen II-like proteins consisting predominantly of tandem repeats of the D2 or D4 period. The D2 construct, but not the D4 construct, mediated DDR2 binding and receptor autophosphorylation, demonstrating that the D2 period of collagen II harbours a specific DDR2 recognition site. The discovery of a site-specific interaction of DDR2 with collagen II gives novel insight into the nature of the interaction of collagen II with matrix receptors.     

Nested collagen matrices: a new model to study migration of human fibroblast populations in three dimensions

Fibroblast-3D collagen matrix culture provides a model system to analyze cell physiology under conditions that more closely resemble tissue than conventional 2D cell culture. Previous work has focused primarily on remodeling and contraction of collagen matrices by fibroblasts, and there has been little research on migration of cell populations within the matrix. Here, we introduce a nested collagen matrix model to analyze migration of fibroblasts in 3D collagen matrices. Nested collagen matrices were prepared by embedding contracted cell-containing matrices (also called dermal equivalents) inside cell-free matrices; migration occurred from the former to the latter. Control experiments with human dermal fragments in place of dermal equivalents confirmed the reliability of the model. Human fibroblast migration in nested collagen matrices occurred after a lag phase of 8-16 h, and cells migrating out of the inner matrices were bipolar with leading dendritic extensions. Migration was myosin II, Rho kinase and metalloproteinase-dependent but did not require plasma fibronectin. Platelet-derived growth factor but not lysophosphatidic acid or serum stimulated cell migration, although all three of these physiological agonists promote matrix remodeling and contraction. The nested collagen matrix model is a relatively easy, rapid and quantitative method to measure migration of cell populations. Our studies using this model demonstrate important differences between regulation of fibroblast migration and remodeling in collagen matrices.     

Adherence,proliferation and collagen turnover by human fibroblasts seeded into different types of collagen sponges

We describe an in vitro model that we have used to evaluate dermal substitutes and to obtain data on cell proliferation, the rate of degradation of the dermal equivalent, contractibility and de novo synthesis of collagen. We tested three classes of collagenous materials: (1) reconstituted non-crosslinked collagen, (2) reconstituted collagen that was chemically crosslinked with either glutaraldehyde, aluminium alginate or acetate, and (3) native collagen fibres, with or without other extracellular matrix molecules (elastin hydrolysate, hyaluronic acid or fibronectin). The non-crosslinked reconstituted collagen was degraded rapidly by human fibroblasts. Teh chemically crosslinked materials proved to be cytotoxic. Native collagen fibres were stable. In the absence of ascorbic acid, the addition of elastin hydrolysate to this type of matrix reduced the rate of collagen degradation. Both elastin hydrolysate and fibronectin partially prevented fibroblast-mediated contraction. Hyaluronic acid was only slightly effective in reducing the collagen degradation rate and more fibroblast-mediated contraction of the material was found than for the native collagen fibres with elastin hydrolysate and fibronectin. In the presence of ascorbate, collagen synthesis was enhanced in the native collagen matrix without additions and in the material containing elastin hydrolysate, but not in the material with hyaluronic acid. These results are indicative of the suitability of tissue substitutes for in vivo application.     

Up-regulated type I collagen expression by the inhibition of Rac1 signaling pathway in human dermal fibroblasts

Tissue remodeling is known to play important roles in wound healing. Although Rac1 is reported to be one of the key signaling molecules in cutaneous wound healing process, the exact mechanisms of Rac1-mediated tissue remodeling is still unknown. This study investigated the role of Rac1 in the regulation of extracellular matrix in cultured human dermal fibroblasts obtained by skin biopsy from three healthy donors. Protein levels of type I collagen in cultured human fibroblasts were increased by the treatment with Rac1 inhibitor NSC23766 in a dose-dependent manner. However, the mRNA levels of α2(I) collagen was not altered by the inhibitor. On the other hand, by the addition of inhibitor, half-lives of type I collagen protein were increased and MMP1 levels were reduced. These data suggest that blockade of Rac1 signaling results in accumulation of type I collagen due to decreased collagenase activity. This study also suggests that controlling Rac1 signaling is a new therapeutic approach to chronic/untreatable ulcer.     

A 3D in situ cell counter reveals that breast tumor cell (MDA‐MB‐231) proliferation rate is reduced by the collagen matrix density

Many cell types require the biophysical and biochemical cues within the 3D extracellular matrix (ECM) to exhibit their true physiologically relevant behavior. As a result, cell culture platforms have been evolving from traditional 2D petridish plates into 3D biomatrices, and there is a need for developing analytic tools to characterize 3D cell culture. The existing cell counting method, using a hemocytometer or coulter counter, requires that cells are suspended in fluids prior to counting. This poses a challenge for 3D cell culture as cells are embedded in a 3D biomatrix. We use a facile 3D cell counting method that overcomes this limitation and allows for in situ cell counting in a 3D cell culture using equipment that is commonly available in a biology lab. Using a breast tumor cell line, MDA‐MB‐231, as a model system, we demonstrated that MDA‐MB‐231 cells (1) grow slower within a 3D collagen matrix than on a 2D substrate for an extended growth time (a week) with a comparable, initial cell‐to‐cell distance, (2) their cell growth rate decreases with the increase of collagen concentration, showing a linear growth rate rather than an exponential growth rate. Further work using flow cytometry showed that the observed growth rate reduction was consistent with the retardation of the transition to S (synthesis) phase in the cell cycle. This work demonstrates the validity of the 3D cell counting method and the importance of cell–ECM interactions in cell proliferation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:990–996, 2015     

Increased strength in the Col-Tgel induces apoptosis in the human dental pulp stem cells: 3D culturing of human dental pulp stem cells at different strengths of collagen

Human dental pulp stem cells (HDPSCs) have great potential to be used in regenerative medicine. To use these stem cells effectively for this purpose, they should be grown in a 3D cell culture that mimics their natural niches instead of a 2D conventional cell culture. The aim of this study was to grow the HDPSCs in the 3D cell culture created by Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) in two different strengths to find a suitable 3D cell culture environment for these stem cells. Two stiffness of the 3D Col-Tgel were used to grow the HDPSCs: soft and medium matrix with strength of 0.9–1.5 kPa and 14–20 kPa, respectively. HDPSCs express markers similar to MSCs, therefore seven such markers were analyzed in the HDPSCs during their growth in the 2D and in the 3D soft and medium Col-Tgel. The CD105 and CD90 markers were significantly (p < 0.05) downregulated in HDPSCs cultured in both 3D cell culture conditions compared with HDPSCs in 2D cell culture. Furthermore, CD34 marker, a negative marker, expressed by a few cells in HDPSCs culture was upregulated (p < 0.05) in HDPSCs cultured in medium 3D Col-Tgel, indicating cells that expressing the marker grow better in medium 3D Col-Tgel. The apoptosis results revealed that HDPSCs in medium 3D Col-Tgel had the least number of live cells and a significantly (p < 0.05) higher early apoptosis rate compared to HDPSCs in 2D and 3D Col-Tgel medium. MTT analysis also showed a significant difference among the three cell culture conditions. We conclude that HDPSCs cultured on 3D soft Col-Tgel showed better proliferation than cells cultured in 3D medium gel. These results demonstrate that the ideal environment to grow HDPSCs in 3D is the soft Col-Tgel not medium Col-Tgel.     

The Rho family GEF Asef2 regulates cell migration in three dimensional (3D) collagen matrices through myosin II

Cell migration is fundamental to a variety of physiological processes, including tissue development, homeostasis, and regeneration. Migration has been extensively studied with cells on 2-dimensional (2D) substrates, but much less is known about cell migration in 3D environments. Tissues and organs are 3D, which is the native environment of cells in vivo, pointing to a need to understand migration and the mechanisms that regulate it in 3D environments. To investigate cell migration in 3D environments, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that the Rho family guanine nucleotide exchange factor (GEF) Asef2 inhibits cell migration in 3D type I collagen (collagen I) matrices. Treatment of cells with the myosin II (MyoII) inhibitor blebbistatin abolished the decrease in migration by Asef2. Moreover, Asef2 enhanced MyoII activity as shown by increased phosphorylation of serine 19 (S19). Furthermore, Asef2 increased activation of Rac, which is a Rho family small GTPase, in 3D collagen I matrices. Inhibition of Rac activity by treatment with the Rac-specific inhibitor NSC23766 abrogated the Asef2-promoted increase in S19 MyoII phosphorylation. Thus, our results indicate that Asef2 regulates cell migration in 3D collagen I matrices through a Rac-MyoII-dependent mechanism.     

Role of LRP-1 in cancer cell migration in 3-dimensional collagen matrix

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a member of Low Density Lipoprotein Receptor (LDLR) family, which is ubiquitously expressed and which is described as a multifunctional endocytic receptor which mediates the clearance of various extracellular matrix molecules including serine proteinases, proteinase-inhibitor complexes, and matricellular proteins. Several studies showed that high LRP-1 expression promotes breast cancer cell invasiveness, and LRP-1 invalidation leads to cell motility abrogation in both tumor and non-tumor cells. Furthermore, our group has reported that LRP-1 silencing prevents the invasion of a follicular thyroid carcinoma despite increased pericellular proteolytic activities from MMP2 and uPA using a 2D-cell culture model. As the use of 3D culture systems is becoming more and more popular due to their promise as enhanced models of tissue physiology, the aim of the present work is to characterize for the first time how the 3D collagen type I matrix may impact the ability of LRP-1 to regulate the migratory properties of thyroid carcinoma using as a model FTC-133 cells. Our results show that inhibition of LRP-1 activity or expression leads to morphological changes affecting cell-matrix interactions, reorganizations of the actin-cytoskeleton especially by inhibiting FAK activation and increasing RhoA activity and MLC-2 phosphorylation, thus preventing cell migration. Taken together, our results suggest that LRP-1 silencing leads to a decrease of cell migratory capacity in a 3D configuration.     

Actin cytoskeleton assembly regulates collagen production via TGF‐β type II receptor in human skin fibroblasts

The dermal compartment of skin is primarily composed of collagen‐rich extracellular matrix (ECM), which is produced by dermal fibroblasts. In Young skin, fibroblasts attach to the ECM through integrins. During ageing, fragmentation of the dermal ECM limits fibroblast attachment. This reduced attachment is associated with decreased collagen production, a major cause of skin thinning and fragility, in the elderly. Fibroblast attachment promotes assembly of the cellular actin cytoskeleton, which generates mechanical forces needed for structural support. The mechanism(s) linking reduced assembly of the actin cytoskeleton to decreased collagen production remains unclear. Here, we report that disassembly of the actin cytoskeleton results in impairment of TGF‐β pathway, which controls collagen production, in dermal fibroblasts. Cytoskeleton disassembly rapidly down‐regulates TGF‐β type II receptor (TβRII) levels. This down‐regulation leads to reduced activation of downstream effectors Smad2/Smad3 and CCN2, resulting in decreased collagen production. These responses are fully reversible; restoration of actin cytoskeleton assembly up‐regulates TβRII, Smad2/Smad3, CCN2 and collagen expression. Finally, actin cytoskeleton‐dependent reduction of TβRII is mediated by induction of microRNA 21, a potent inhibitor of TβRII protein expression. Our findings reveal a novel mechanism that links actin cytoskeleton assembly and collagen expression in dermal fibroblasts. This mechanism likely contributes to loss of TβRII and collagen production, which are observed in aged human skin.