The use of human fibroblasts grown on microcarriers for the formation of the connective tissue equivalent

Live equivalents of tissues, specifically those produced on the basis of fibroblasts and collagen gel, are widely used for repair of organ and tissues defects. In clinical practice, it is more convenient to use the fibroblasts grown on microcarriers or such a connective tissue equivalent when the fibroblasts on microcarriers are embedded in collagen gel. We studied the properties of a connective tissue equivalent produced by embedding the fibroblasts grown on microcarriers in collagen gel for its prospective use in clinical practice. According to our results, the optimal time of use of the live tissue equivalent amounts to three--four days after embedding of fibroblasts on microcarriers in gel. At that time, contraction only begins, which facilitates manipulations with the gel.     

Use of Human Fibroblasts Grown on Microcarriers for Formation of Connective Tissue Equivalent

Living equivalents of tissues, specifically those produced on the basis of fibroblasts and collagen gel, are widely used for repair of organ and tissues defects. In clinical practice, it is more convenient to use the fibroblasts grown on microcarriers or such a connective tissue equivalent when the fibroblasts on microcarriers are embedded in collagen gel. We studied the properties of a connective tissue equivalent produced by embedding the fibroblasts grown on microcarriers in collagen gel for its prospective use in clinical practice. According to our results, the optimal time of use of the living tissue equivalent amounts to three–four days after embedding of fibroblasts on microcarriers in gel. At that time, contraction only begins, which facilitates manipulations with the gel.     

Effects of lactoferrin on collagen gel contractile activity and myosin light chain phosphorylation in human fibroblasts.

When fibroblasts are plated on a type I collagen gel they reduce the size of the gel and the extent of collagen gel contraction reflects the motile activity of the fibroblasts. We found that both bovine and human lactoferrin (Lf) enhanced the collagen gel contractile activity of WI-38 human fibroblasts. Rho inhibitor (exoenzyme C3), Rho kinase inhibitor (Y-27632), myosin light chain kinase inhibitor (ML-7), MEK inhibitor (PD98059) and Src family tyrosine kinase inhibitor inhibited the Lf-enhanced collagen gel contraction. Treatment of fibroblasts with Lf induced the phosphorylation of myosin light chain (MLC) within 30 min. Lf-enhanced MLC phosphorylation was inhibited by Y-27632 and ML-7. These results suggest that Lf promotes the motility of fibroblasts by regulating MLC phosphorylation.     

The interaction of human papillary and reticular fibroblasts and human keratinocytes in the contraction of three-dimensional floating collagen lattices

Fibroblasts derived from the papillary and reticular dermis of human skin and human keratinocytes show differences in their abilities to contract floating three-dimensional gels constructed from type I collagen. Reticular fibroblasts produce greater gel contraction than papillary fibroblasts. When equal numbers of papillary and reticular fibroblasts are mixed in the gels, papillary fibroblasts consistently inhibit gel contraction by reticular fibroblasts indicating interaction between these cell types in the contraction process. Surprisingly, keratinocytes alone produce greater gel contraction than that produced by either fibroblast type. Cooperativity in the gel contraction process is observed when fibroblasts are incorporated into the collagen matrix and keratinocytes are seeded onto the gel surface. Keratinocytes and dermal fibroblasts adhere to the collagen fibril to induce gel contraction by different mechanisms. Fibroblast contraction of collagen gels does not require fibronectin but is a serum-dependent reaction. In contrast, keratinocyte contraction of collagen gels occurs in a serum-free environment. Polyclonal, affinity-purified antibodies to human plasma fibronectin at high concentrations do not inhibit gel contraction by keratinocytes, making unlikely the possibility that fibronectin synthesized by the keratinocyte is a significant factor in the gel contraction process. We are currently examining the possibilities either that keratinocytes are synthesizing other adhesion proteins or that receptors on the cell surface can interact directly with the collagen fiber.     

The interaction of epidermal keratinocytes and fibroblasts during the formation of a live skin equivalent]

The effect of human fetal fibroblasts and adult keratinocytes on collagen contraction was studied. Keratinocytes embedded in collagen lattices did not spread and produced only a slight contraction. When keratinocytes were seeded on the surface of tht gel, the contraction began within 24 h and correlated with the formation of epithelial colonies. Transplantation of multilayered epithelial sheets on the gel significantly accelerated the onset of contraction. Keratinocytes seeded on and fibroblasts grown in collagen lattices cooperatively contracted the gel, and keratinocytes were able to stimulate gel contraction even when they had no contact with the collagen roughly populated with fibroblasts. Swiss 3T3 cells remained spherical in collagen lattices and did not contract the gel but when cultivated with keratinocytes they stimulated gel contraction. In their turn, keratinocytes influenced the behaviour of Swiss 3T3 cells which elongated and produced processes. We suggest that both keratinocytes and mesenchymal cells can affect gel contraction 1) by a direct contact with collagen lattices, and 2) through potentiation of the ability of another cell type to contract the gel.     

Blood plasma fibronectin does not influence fibroblast-induced collagen gel contraction

Human embryo fibroblasts were cultured within three-dimensional collagen gel in the Eagle medium supplemented with 10% calf serum. Addition of fibronectin (Fn) to the nutrient medium neither stimulated nor inhibited gel contraction by fibroblasts. Gel contraction also proceeded in the medium containing ultroser G or Fn exhausted calf serum. Addition of 10 or 50 micrograms/ml of Fn to the medium with ultroser G exerted no effect on the process of gel contraction. Addition of antibodies prepared against Fn (70 micrograms/ml) and tetrapeptide RGDS (100 and 500 micrograms/ml) to the medium supplemented with Fn exhausted calf serum did not change the rate of collagen gel contraction. The results obtained suggest that the rate of collagen gel contraction by fibroblasts is not dependent on the presence of exogenous fibronectin.     

Interactions of extracellular collagen and corneal fibroblasts: Morphologic and biochemical changes of rabbit corneal cells cultured in a collagen matrix

Summary Corneal fibroblasts, also known as keratocytes are surrounded by an extracellular matrix of collagen in vivo. To understand the physiology and pathology of these corneal fibroblasts, it is important to study their interactions with this extracellular matrix. We cultured rabbit corneal fibroblasts on tissue culture plastic dishes or in a hydrated collagen gel and compared the changes in morphology and mitotic activity. Corneal fibroblasts on plastic dishes were flattened and widely spread, whereas those in collagen gel became spindle-shaped with long processes. Examination with an electron microscope revealed that the corneal fibroblasts in collagen gel formed gap junctions with neighboring cells. Gap junctions were hardly ever observed between corneal fibroblasts cultured on plastic dishes. Corneal fibroblasts cultured in a collagen matrix showed much less incorporation of [³H]thymidine than did corneal fibroblasts cultured on plastic, and this incorporation decreased with increasing concentration of collagen. Our present results suggest that the morphologic and biochemical characteristics of corneal fibroblasts cultured in collagen gel are different from those cultured on plastic. This research was supported in part by grants from the Ministry of Education, Science and Culture of Japan, by a grant from Osaka Eye Bank, Osaka, Japan, and by an intramural research fund of Kinki University. Part of this research was presented at the annual meeting of the Japanese Ophthalmological Society (May 1985) at Kyoto, Japan, and at the annual meeting of the Association for Research in Vision and Ophthalmology (May 1987) at Sarasota, FL.     

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.     

Low density lipoprotein receptor-related protein (LRP) is required for lactoferrin-enhanced collagen gel contractile activity of human fibroblasts

Fibroblasts plated on a type I collagen gel can reduce the size of the gel in a way that mimics the reorganization of the collagen matrix that accompanies the wound healing process. We demonstrated previously that lactoferrin (Lf) specifically binds to WI-38 human fibroblasts and enhances their collagen gel contractile activity. The effect of Lf correlated with the phosphorylation of myosin light chain (MLC), suggesting that Lf promotes fibroblast contractile activity by regulating MLC phosphorylation. We found here that the binding of Lf to WI-38 cells was inhibited by recombinant receptor-associated protein (RAP), a universal competitor for ligand binding to LRP (LDL receptor-related protein), and RAP can also promote the collagen gel contractile activity. These observations suggest that LRP is a receptor that mediates the Lf-induced enhancement of collagen gel contractile activity in WI-38 fibroblasts. To confirm the hypothesis, we utilized LRP antisense oligonucleotide, which was modified by morpholino linkage. Suppression of LRP expression abrogated the Lf-induced enhancement the contractile activity in fibroblasts. Treatment of fibroblasts with Lf enhanced the phosphorylation of ERK1/2 and the activation of MLC kinase (MLCK). These effects were attenuated by suppression of LRP expression. These findings suggest that LRP is involved in the Lf-enhanced collagen gel contractile activity of WI-38 fibroblasts by converting the Lf binding signal into the activation of ERK1/2 and MLCK.     

Inhibition of transforming growth factor-β signaling potentiates tumor cell invasion into collagen matrix induced by fibroblast-derived hepatocyte growth factor

Interaction between tumor cells and stromal fibroblasts plays essential roles in tumor progression. However, its detailed molecular mechanism remains unclear. To understand the mechanism, we investigated molecules mediating this interaction using the three-dimensional (3D) co-culture system of Panc-1 pancreatic carcinoma cells with normal fibroblasts. When the two kinds of cells were placed on the top of collagen gel, the tumor cells scattered into the fibroblast layer, apparently undergoing epithelial‐mesenchymal transition. When fibroblasts were placed within collagen gel, Panc-1 cells actively invaded into the collagen gel, extending a microtubule-based long protrusion. Although transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF) individually stimulated the tumor cell invasion into collagen gel without fibroblasts, TGF-β signaling inhibitors (SB431542 and LY2157299) significantly enhanced the Panc-1 cell invasion in the 3D co-culture with fibroblasts. Experiments with HGF/Met signaling inhibitors or with the fibroblast conditioned medium revealed that HGF was a major invasion-promoting factor secreted from fibroblasts and SB431542 increased the HGF secretion by blocking the HGF-suppressing activity of cancer cell-derived TGF-β. These results indicate that HGF and TGF-β are critical regulators for both tumor–stroma interaction and tumor invasion. The results also suggest that TGF-β signaling inhibitors may promote tumor progression under some pathological conditions.     

Collagen gel contraction by fibroblasts requires cellular fibronectin but not plasma fibronectin

Fibroblasts embedded in three-dimensional lattices of collagen fibrils have been known to require serum constituents to induce a cell-mediated contraction of collagen gels. The gel contraction was studied with human skin fibroblasts cultured in the presence of fetal bovine serum (FBS). Removal of bovine serum fibronectin (sFN) from FBS did not affect the extent of gel contraction. Gel contraction occurred in serum-free defined media. Therefore, it is concluded that sFN is not required for gel contraction. That cellular FN (cFN) synthesized and secreted by fibroblasts plays a crucial role in gel contraction was suggested by the following experiments: (1) We obtained monoclonal antibodies (mAb A3A5) against fibroblast surface antigens, which suppressed the fibroblast-mediated gel contraction. Immunoblot analyses showed that mAb A3A5 recognizes cFN secreted by human fibroblasts and human plasma FN (pFN), but not bovine sFN in FBS used for culture. (2) Addition of rabbit antisera, which recognize human cFN, to a serum-free gel culture inhibited contraction. Uninvolvement of human pFN in gel contraction was further confirmed by the fact that neither pretreatment of fibroblasts with excess amounts of human pFN nor the presence of excess amounts of human pFN in gels affected the extent of gel contraction. This study seems to be the first demonstration of functional difference between cFN and pFN (or sFN) and proposes a novel mode of binding of fibroblasts with collagen fibrils via cFN during cell-mediated collagen morphogenesis.     

Use of shark collagen for cell culture and zymography

The uses of shark collagen as a matrix for cell culture and as a substrate for zymography were investigated. Fibroblasts were cultured on a gel matrix of shark type I collagen at 30 degrees C. The collagen gel had contracted by 4 days of incubation. Individual fibroblasts were visible against the transparent background of the contracted collagen as long, lean star-shaped cells. The matrix metalloproteinases (MMPs) from fibroblasts secreted from the medium more easily digested shark gelatin than pig gelatin. MMP-2, -9, and that of potential form were recognizable in the zymographic gel of shark gelatin.     

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).     

The collagen recognition sequence for fibroblasts depends on collagen topography

We found that the peptide Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) inhibited spreading of human fibroblasts inside collagen gels and markedly decreased gel contraction, but this peptide had no effect on cell spreading on collagen-coated surfaces. On the other hand, the peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), which inhibited cell spreading on collagen-coated surfaces, did not inhibit cell spreading within collagen gels and was a less effective inhibitor of collagen gel contraction than GRGESP. Based on these findings, we conclude that human fibroblasts can interact with different collagen cell recognition sequences depending upon topographical organization of the collagen.     

Thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the proteolytically activated receptor-1 and a protein kinase C-dependent pathway

Myofibroblasts are ultrastructurally and metabolically distinctive fibroblasts that express smooth muscle (SM)-alpha actin and are associated with various fibrotic lesions. The present study was undertaken to investigate the myofibroblast phenotype that appears after activation of normal lung fibroblasts by thrombin. We demonstrate that thrombin induces smooth muscle-alpha actin expression and rapid collagen gel contraction by normal lung fibroblasts via the proteolytically activated receptor-1 and independent of transforming growth factor-beta pathway. Using antisense oligonucleotides we demonstrate that a decreased level of PKCepsilon abolishes SM-alpha actin expression and collagen gel contraction induced by thrombin in normal lung fibroblasts. Inhibition of PKCepsilon translocation also abolishes thrombin-induced collagen gel contraction, SM-alpha actin increase, and its organization by normal lung fibroblasts, suggesting that activation of PKCepsilon is required for these effects. In normal lung fibroblasts PKCepsilon binds to SM-alpha actin after thrombin treatment, but in activated fibroblasts derived from scleroderma lung they associate even in untreated cells. This suggests that SM-alpha actin may serve as a substrate for PKCepsilon in lung fibroblasts when activated by thrombin. We propose that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via a PKC-dependent pathway. Thrombin-induced differentiation of normal lung fibroblasts to a myofibroblast phenotype resembles the phenotype observed in scleroderma lung fibroblasts. Therefore, we conclude that chronic exposure to thrombin after microvascular injury leads to activation of normal lung fibroblasts and to the appearance of a myofibroblast phenotype in vivo. Our study provides novel, compelling evidence that thrombin is an important mediator of the interstitial lung fibrosis associated with scleroderma.     

Contractile capacity of fibroblasts from different sources in the model of living skin equivalent

Human fibroblasts contract collagen gel in vitro and produce a connective tissue-like structure termed the living skin equivalent. In this study, the contractile capacity of postnatal dermal fibroblasts, bone marrow mesenchymal cells and mesenchymal cells derived from the fat tissue has been compared to that of fetal dermal fibroblasts in the model of living skin equivalent. The results show that fetal fibroblasts contract the collagen gel approximately six times stronger than do all other fibroblast cell types, with the numbers of all these cells being equal. A deeper insight into the behavior of fibroblasts differing in their origin will help to develop new approaches to the treatment and regulation of wound healing and fibrosis formation.     

The contraction of collagen matrices by dermal fibroblasts

Floating collagen gel cultures containing human foreskin fibroblasts have been observed to undergo a rapid contraction process. The initial rate of contraction (i.e., within the first 2 hr) was observed to be a linear function of cell number within the concentration range of 10(5)-10(6) cells/gel. Observation of thick, deresined sections of such contracting gels in the SEM, as well as observation of thin sections in the TEM, suggest that the fibroblasts exert a tension upon the surrounding collagen fibers. These observations further indicate that the fibroblasts migrate from the interior regions of the gel matrix and eventually form a monolayer of cells encapsulating the contracted collagen disc. These observations are discussed in terms of the possible mechanisms involved in gel contraction.     

Contraction of collagen gels seeded with tendon cells

Knowledge of the adaptation of the soft tissue to mechanical factors and biomolecules would be essential to better understand the mechanism of tendon injury and to improve the outcome of tendon repair. The responses to these factors could be different for the distinct types of cells in the tendon: cells from the tendon sheath, fibroblasts from the epitenon surface, or fibroblasts from the internal endotenon. In this study, we examined the mechanical and histological characteristics of the rate of contraction of the collagen gel seeded with epitenon and endotenon fibroblasts. The rate of contraction and the mechanical property of the contracted construct depend on the gel concentration and also the treatment of TGF-beta1.     

The 56 kDa protein of human genital skin fibroblasts is identical to that radiolabelled by [3H]dihydrotestosterone 17 beta-bromoacetate

Analysis of soluble proteins from human genital skin fibroblasts by two-dimensional polyacrylamide gel electrophoresis reveals an abundant protein doublet of mol. wt 56,000 with isoelectric points (pI) of 6.7 and 6.5. This protein is absent in non-genital skin fibroblasts as well as in genital skin fibroblasts of most patients with complete forms of androgen insensitivity. The protein specifically binds androgen. A protein of similar estimated molecular weight (58,000) from human genital skin fibroblasts has recently been found to be covalently radiolabelled by the affinity ligand dihydrotestosterone 17 beta-bromoacetate (DHT-BA). In the present study these proteins have been found to be indistinguishable on one- and two-dimensional gel electrophoresis. Antibodies raised against the 56 kDa pI 6.7/6.5 protein also recognized the protein covalently radiolabelled by DHT-BA. A third protein of estimated mol. wt 59,000 has been found to be associated with several steroid hormone receptor complexes but has no known ligand binding activity. This protein was found to be clearly separable from the 56/58 kDa protein on two-dimensional gel electrophoresis as it has a more acidic pI of approximately 5.4. Furthermore, antibodies against the 59 kDa protein do not recognize the 56 kDa species, and vice versa.     

Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro

Analyses were made of the requirements for the formation of a continuous basal lamina during myogenesis of quail muscle in vitro. A culture system was developed in which mass cultures of differentiating muscle cells were embedded in a native gel of rat tail collagen. Fibroblastic cells, which were also present in the cultures, migrated into the gel and within a few days surrounded the newly formed myotubes. In this environment, a continuous basal lamina was formed at the surface of the myotubes as demonstrated by immunofluorescent staining with monoclonal antibodies against type IV collagen, laminin, and heparan sulfate, as well as by electron microscopic immunolocalization. To distinguish between the role of the fibroblasts and the collagen gel in promoting basal lamina formation, clones of quail muscle cells lacking fibroblasts were subsequently embedded in a native rat tail collagen gel. Under these conditions, only very limited fluorescent staining for basement membrane components was observed associated with the myotubes. However, the introduction of chick muscle or skin fibroblasts into the clonal cultures just before gel formation resulted in the formation of an extensive basal lamina on the surface of the myotubes. Conditioned medium from fibroblast cultures by itself was not effective in promoting basal lamina formation. These results clearly show that during myogenesis in vitro fibroblasts must be in close proximity to the myotubes for a continuous basal lamina to form. These results probably relate closely to the interactions that must occur during myogenesis in vivo between the muscle cells and the surrounding connective tissue including the developing tendons.