Cultures of ligament fibroblasts in fibrin matrix gel.

The cellular properties of anterior cruciate ligament (ACL) and medial collateral ligament (MCL) fibroblasts have been analyzed in a three-dimensional fibrin matrix gel (FMG) system. The MCL fibroblasts proliferated significantly faster than ACL fibroblasts in 10% fetal bovine serum (FBS). FMG contraction resembles soft-tissue wound contraction. Transforming growth factor-beta1 (TGF-beta1) (5 ng/ml) caused a significantly faster rate of FMG contraction than control (0.5% FBS) in both ACL and MCL fibroblasts. Unlike the cells in 10% FBS, this faster rate of FMG contraction was achieved without increasing the initial cell number. In the FMG, the MCL fibroblasts demonstrated significantly higher collagen synthesis per cell than ACL fibroblasts between the days 2 and 6 of culture. These differences in cellular properties of the ACL and MCL fibroblasts that were observed in vitro may explain the differences in the healing potential of these ligaments in vivo.

Transforming growth factor-beta - and tumor necrosis factor-alpha -mediated induction and proteolytic activation of MMP-9 in human skin.

Both cytokines and matrix metalloproteinases (MMPs) are active during physiologic and pathologic processes such as cancer metastasis and wound repair. We have systematically studied cytokine-mediated MMP regulation. Cytokine-mediated proteinase induction and activation were initially investigated in organ-cultured human skin followed by determination of underlying cellular and molecular mechanisms using isolated skin cells. In this report we demonstrate that tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) synergistically induce pro-MMP-9 in human skin as well as isolated dermal fibroblasts and epidermal keratinocytes. Furthermore, TNF-alpha promotes proteolytic activation of pro-MMP-9 by conversion of the 92-kDa pro-MMP-9 to the 82-kDa active enzyme. This activation occurred only in skin organ culture and not by either isolated fibroblasts or keratinocyte, although the pro-MMP-9 activation could be measured in a cell-free system derived from TNF-alpha-activated skin. The cytokine-mediated induction of pro-MMP-9 in dermal fibroblasts was evident by increased mRNA. At the transcription level, we examined the cytokine-mediated transactivation of the 5'-region promoter of the human MMP-9 in dermal fibroblasts. The results demonstrated that TNF-alpha and TGF-beta could independently stimulate the 5'-flanking 670-base pair promoter. A TGF-beta-response element (-474) and an NF-kappaB-binding site (-601) were identified to be the cis-elements for TGF-beta or TNF-alpha activation, respectively. Taken together, these findings suggest a specific mechanism whereby multiple cytokines can regulate MMP-9 expression/activation in the cells of human skin. These results imply roles for these cytokines in the regulation of MMP-9 in physiologic and pathologic tissue remodeling.

TNF-alpha stimulates activation of pro-MMP2 in human skin through NF-(kappa)B mediated induction of MT1-MMP.

Tumor necrosis factor-alpha (TNF-(alpha)) is an important mediator during the inflammatory phase of wound healing. Excessive amounts of pro-inflammatory cytokines such as TNF-(alpha) are associated with inflammatory diseases including chronic wounds. Matrix metalloproteinases (MMPs) are involved in matrix re-modeling during wound healing, angiogenesis and tumor metastasis. As with pro-inflammatory cytokines, high levels of MMPs have been found in inflammatory states such as chronic wounds. In this report we relate these two phenomena. TNF-(alpha) stimulates secretion of active MMP-2, a type IV collagenase, in organ-cultured full-thickness human skin. This suggests a mechanism whereby excess inflammation affects normal wound healing. To investigate this observation at the cellular and molecular levels, we examined TNF-(alpha) mediated activation of pro-MMP-2, induction of MT1-MMP, and the intracellular signaling pathways that regulate the proteinase in isolated human dermal fibroblasts. We found that TNF-(alpha) substantially promoted activation of pro-MMP-2 in dermal fibroblasts embedded in type-I collagen. In marked contrast, collagen or TNF-(alpha) individually had little influence on the fibroblast-mediated pro-MMP-2 activation. One well-characterized mechanism for pro-MMP-2 activation is through a membrane type matrix metalloproteinase, such as MT1-MMP. We report that TNF-(alpha) significantly induced MT1-MMP at the mRNA and protein levels when the dermal fibroblasts were grown in collagen. Although the intracellular signaling pathway regulating mt1-mmp gene expression is still obscure, both TNF-(alpha) and collagen activate the NF-(kappa)B pathway. In this report we provide three sets of evidence to support a hypothesis that activation of NF-(kappa)B is essential to induce MT1-MMP expression in fibroblasts after TNF-(alpha) exposure. First, SN50, a peptide inhibitor for NF-(kappa)B nuclear translocation, simultaneously blocked the TNF-(alpha) and collagen mediated MT1-MMP induction and pro-MMP-2 activation. Secondly, TNF-(alpha) induced I(kappa)B to breakdown in fibroblasts within the collagen lattice, a critical step leading to NF-(kappa)B activation. Lastly, a consensus binding site for p65 NF-(kappa)B (TGGAGCTTCC) was found in the 5'-flanking region of human mt1-mmp gene. Based on these results and previous reports, we propose a model to explain TNF-(alpha) activation of MMP-2 in human skin. Activation of NF(kappa)B signaling in fibroblasts embedded in collagen induces mt1-mmp gene expression, which subsequently activates the pro-MMP-2. The findings provide a specific mechanism whereby TNF-(alpha) may affect matrix remodeling during wound healing and other physiological and pathological processes.

Temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor and gelatinase-B in chronic wound fluid switches from a chronic to acute wound profile with progression to healing.

The plasminogen activator/plasmin system is known to initiate a proteolytic cascade resulting in the activation of matrix metalloproteinases in vitro leading to the degradation of extracellular matrix. To investigate whether or not this cascade is present during delayed wound healing and contributes to the pathophysiological basis of impaired healing we examined the temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor-1 and gelatinase-B in fluid collected from chronic venous leg ulcers compared to acute surgical mastectomy wounds. Using a chromogenic substrate assay, levels of active urokinase plasminogen activator in chronic wounds were found to be about five fold higher compared to sera and two fold higher compared to mastectomy wounds. Levels of active plasminogen activator inhibitor-1 in chronic wounds were four times higher than those found in sera and two times higher than those found in mastectomy wound fluid. Using a fibrin overlay system and reverse zymography, we found that when the wound was not healing, the expression of urokinase plasminogen activator in chronic wound fluid was initially detected in the active forms (50 and 33 kDa), but that as the wound healed and decreased in size, was detected as an inhibitor- bound urokinase plasminogen activator-plasminogen activator inhibitor-1 complex ( congruent with 80-116 kDa). When the expression of active urokinase plasminogen activator was highest, no plasminogen activator inhibitor-1 was detectable. In contrast, urokinase plasminogen activator was always detected in the inhibitor bound form as a urokinase plasminogen activator-plasminogen activator inhibitor-1 complex in blood- and plasma-derived serum and mastectomy wound fluid. Plasminogen activator inhibitor-1 was detected in blood-derived serum and mastectomy wound fluid but not in plasma derived serum. Expression of matrix metalloproteinase-9 in chronic wound fluids, analyzed by gelatin zymography, showed that when urokinase plasminogen activator was detected in the active forms, matrix metalloproteinase-9 was overexpressed by approximately twice that found in mastectomy wounds and approximately 30 times that detected in blood-derived sera. When urokinase plasminogen activator appeared almost entirely as an enzyme- inhibitor complex, the level of expression of matrix metalloproteinase-9 was similar to that seen in mastectomy wound fluid. We conclude that the switch in urokinase plasminogen activator expression from an active to inhibitor bound form correlates with the decrease seen in matrix metalloproteinase-9 expression suggesting the presence of a proteolytic cascade initiated by the plasminogen activator/plasmin system during wound healing leading to the activation of matrix metalloproteinase-9. In addition, expression of urokinase plasminogen activator and matrix metalloproteinase-9 appear to be useful biomarkers to determine clinical wound healing status.

Developmental differences in the expression and modulation of extracellular matrix proteases and inhibitors in mouse skin fibroblasts.

To investigate developmental differences in the wound repair process between fetal and adult skin fibroblasts, we studied the expression of plasminogen activator, plasminogen activator inhibitor, matrix metalloproteinase, and tissue inhibitor of metalloproteinase in E-15, E-17, newborn and adult mouse skin fibroblasts cultured within three dimensional matrices of either collagen or fibrin. Fibrin overlay and reverse overlay analyses revealed that mouse skin fibroblasts secreted tissue plasminogen activator and type1 plasminogen activator inhibitor. However, only E-15 and E-17 fibroblasts secreted the active form of tissue plasminogen activator, while in newborn and adult fibroblasts tissue plasminogen activator was conjugated to type1 plasminogen activator inhibitor. Only adult fibroblasts expressed a high level of active type1 plasminogen activator inhibitor. Gelatin zymography revealed that the predominant matrix metalloproteinase secreted by all the mouse fibroblasts was gelatinase A (matrix metalloproteinase -2). Matrix metalloproteinase -2 was partially activated in the adult fibroblasts cultured within a collagen matrix. The tissue inhibitor of metalloproteinase-2 was expressed by all fibroblasts, but levels were highest in the newborn and adult fibroblasts. When E-15 fibroblasts were cultured within a fibrin matrix, tissue plasminogen activator was downregulated. Transforming growth factor-betadownregulated tissue plasminogen activator while upregulating type1 plasminogen activator inhibitor, and platelet-derived growth factor enhanced tissue plasminogen activator expression in E-15 fibroblasts. Therefore, plasminogen activator and its inhibitor, and matrix metalloproteinase and its associated tissue inhibitor are differentially expressed in fetal and adult fibroblasts, and their expression is controlled by extracellular matrix components and growth factors present in wounds.

Contractility, transforming growth factor-beta, and plasmin in fetal skin fibroblasts: role in scarless wound healing.

The early fetus responds to cutaneous wounds in a fundamentally different way from the adult; fetal wounds heal without scars. Wound contraction is a vital component of wound healing. The cytokine transforming growth factor (TGF)-beta promotes wound contraction and can be activated by the serine protease plasmin. Herein, we explored whether murine skin fibroblast contractile properties, TGF-beta, and plasmin formation are developmentally regulated. Our results showed that early fetal mouse embryonic day 15 skin fibroblasts contracted a collagen gel less, secreted less active and total TGF-beta, and generated less plasmin than either late fetal (embryonic day 17) or adult skin fibroblasts. Furthermore, there was a slight positive correlation between the formation of plasmin and the level of activation of TGF-beta. We conclude that early fetal mouse skin fibroblasts contract a collagen gel and secrete and activate TGF-beta to a lesser extent than do late fetal and adult skin fibroblasts. We speculate that the fetal skin fibroblast undergoes a developmental transition that causes wounds in mouse to contract at or after embryonic day 17. Further, this developmental transition is influenced by growth factor-fibroblast interactions and coincides with the emergence of the skin fibroblast's ability to generate plasmin and activate TGF-beta.

The molecular basis of keloid and hypertrophic scar formation.

Excess scar formation secondary to traumatic or surgical injuries can have devastating consequences, ranging from body disfigurement to organ dysfunction. Hypertrophic scars and keloids are skin fibrotic conditions that can be caused by minor insults to skin, such as acne or ear piercing, or by severe injuries such as burns. Differences between keloids, hypertrophic scars and normal scars include distinct scar appearance, histologic morphology and cellular function in response to growth factors. Recent advances in our understanding of the wound healing process reveal possible causes for hypertrophic scars and keloids. This information might assist in the development of efficacious treatment for hypertrophic scar and keloid formation.

Osteopontin is produced by rat cardiac fibroblasts and mediates A(II)-induced DNA synthesis and collagen gel contraction.

Angiotensin II (AII) is a critical factor in cardiac remodeling which involves hypertrophy, fibroblast proliferation, and extracellular matrix production. However, little is known about the mechanism by which AII accelerates these responses. Osteopontin is an acidic phosphoprotein with RGD (arginine-glycine-aspartate) sequences that are involved in the vascular smooth muscle cell remodeling process. We identified the presence of osteopontin mRNA and protein in cultured rat cardiac fibroblasts and its prominent regulation by AII (10(-11) M). Osteopontin message levels were increased fourfold (P < 0.01) and protein fivefold (P < 0.05) at 24 h after addition of AII (10(-7) M). This response was inhibited by the AT1 receptor blocker, losartan. Osteopontin mRNA levels were increased in hypertrophied ventricles from animals with renovascular hypertension (1.6-fold, P < 0.05) and aortic banding (2.9-fold, P < 0.05). To examine the function of osteopontin, we determined its effects on (a) the ability of cardiac fibroblasts to contract three-dimensional collagen gels and (b) cardiac fibroblast growth. A monoclonal antibody against osteopontin partially blocked AII-induced three-dimensional collagen gel contraction by cardiac fibroblasts (64+/-4 vs. 86+/-5% in the presence of antibody, P < 0.05), while osteopontin itself promoted contraction of the gels by fibroblasts (71+/-5%, P < 0.05 compared with control). Either a monoclonal antibody against beta3 integrin which is a ligand for osteopontin or the RGD peptide blocked both AII and osteopontin-induced collagen gel contraction. Thus, the osteopontin RGD sequence binds to beta3 integrins on the fibroblast to promote fibroblast binding to collagen. All induced a threefold increase in DNA synthesis of cardiac fibroblasts, which was completely blocked by antibodies against osteopontin and beta3 integrin, or by RGD peptide, but not by controls. Thus, All-induced growth of cardiac fibroblasts also requires osteopontin engagement of the beta3 integrin. Taken together, these results provide the first evidence that osteopontin is a potentially important mediator of AII regulation of cardiac fibroblast behavior in the cardiac remodeling process.

Role of growth factors in scar contraction: an in vitro analysis.

Excessive scar contracture by wound fibroblasts can have devastating consequences, ranging from body disfigurement to joint immobility. The ability of fibroblasts isolated from lesions of hypertrophic scars, keloids, normal skin, or normal scars in contracting the provisional wound matrix (i.e., fibrin clot) was compared and analyzed. Hypertrophic scar fibroblasts showed a consistently higher basal level of fibrin matrix gel (FMG) contraction than other fibroblasts. This heightened basal level of contractility may be attributed partially to the autocrine effect of transforming growth factor-beta 1 (TGF-beta 1). Normal and keloid fibroblasts exhibited similar basal rates of FMG contraction, and both responded to platelet-derived growth factor (PDGF) and TGF-beta by increasing FMG contraction two- to threefold. However, 45% of the TGF-beta-induced increase in FMG contraction by keloid fibroblasts, but not normal fibroblasts, was mediated by the autocrine production of PDGF. Therefore, fibroblasts isolated from different scars exhibit varied degrees of FMG contraction. In addition, the mechanism underlying growth factor-mediated contraction differed vastly among fibroblasts of different scar origin. The significance of these differences in growth factor-mediated FMG contraction is discussed.

Elevated levels of plasminogen activator inhibitor-1 may account for the altered fibrinolysis by keloid fibroblasts.

Using a 3-dimensional fibrin gel model system simulating fibroplasia of wound repair, we investigated the interaction between keloid fibroblasts and fibrin matrix and compared it with that of normal fibroblasts. Normal skin fibroblasts caused fibrin gel degradation under serum-free conditions, whereas keloid fibroblasts did not cause microscopically detectable gel degradation. Fibrin gel degradation occurred through plasmin-mediated fibrinolysis, which was initiated by fibroblasts exhibited high uPA but low plasminogen activator inhibitor-1 (PAI-1) activities, and transforming growth factor-beta 1 prevented fibrinolysis of normal fibroblasts by upregulating PAI-1 while downregulating uPA activities. In contrast, keloid fibroblasts exhibited an intrinsically high level of PAI-1 and a low level of uPA. This change in the ratio of activator and inhibitor activities was attributed to altered fibrin degradation by keloid fibroblasts. The PAI-1 increase was also demonstrated at the RNA level by Northern analysis. In terms of the pivotal role of the plasmin/plasminogen activator system in matrix remodeling, the elevated PAI-1 level exhibited by keloid fibroblasts may have significant consequences not only in altered fibrin degradation, but also in subsequent repair steps that lead to keloids and fibrosis.

In vitro fibroplasia: matrix contraction, cell growth, and collagen production of fibroblasts cultured in fibrin gels.

Extracellular matrix (ECM) reorganization, cell growth, and collagen synthesis/deposition are key features of fibroplasia during tissue repair. An in vitro fibrin gel culture model system simulating fibroplasia of wound repair was characterized. In the model system, fibrin gels were stabilized on plastic culture plates as hemispheres. In this way, fibroblasts were able to reorganize fibrin fibrils, resulting in a measurable decrease in gel thickness with no change in gel diameter, thereby producing a matrix with tension relevant to that of a repairing tissue. Within the study period, human dermal fibroblasts exhibited dynamic activities in cell growth and in reorganization and remodeling of the fibrin matrix. In the first 2 days of culture, fibroblasts quickly reorganized the fibrin matrix to 10% of its original thickness. Fibroblast proliferation occurred at a much slower rate compared to monolayer cultures. Proliferation continued at the same rate throughout the study in contrast to monolayer cultures, which ceased proliferation at confluence. Collagen synthesis was detected as early as the second day in culture. Type I collagen was the major collagen synthesized by fibroblasts with small amounts of type V and type III collagen. Collagen from either monolayer or fibrin gel cultures appeared identical when analyzed by two-dimensional peptide mapping of their CNBr fragments. Although collagen was detected biochemically from Day 2, organized collagen fibrils were apparently only in the later stage of cultures in transmission electron micrographs. Also, at this time, fibrin fibrils were largely removed and the matrix was filled with collagen fibrils and other filamentous ECM. The growth factor TGF-beta stimulated both fibrin gel contraction and collagen synthesis by fibroblasts. Therefore, using the model system, we have demonstrated that fibroblasts can actively reorganize the fibrin matrix and subsequently remodel it into a collagen-containing scar-like tissue. The unique features of this model system allow for creative designs in studying the complex mechanisms underlying tissue repair.

Engineering, expression and renaturation of targeted TGF-beta fusion proteins.

This study reports the expression, purification, and renaturation of biologically active Transforming Growth Factor-beta 1 (TGF-beta 1) fusion proteins from Escherichia coli (E. coli). A prokaryotic expression vector was engineered to produce tripartite fusion proteins consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen binding domain, and (iii) a cDNA sequence encoding the active fragment of human TGF-beta 1. The expressed fusion proteins TGF-B1-F1 and TGF-B1-F2, located in inclusion bodies, were solubilized with 8 M urea and renatured using a glutathione redox-coupled system and protracted dialysis under several experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion proteins on a Ni-NTA metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to inhibit mink lung (Mv1Lu) cell proliferation and/or to stimulate proliferation of NIH-3T3 mouse fibroblasts, where purified human platelet TGF-beta 1 served as a positive control. Purified TGF-B1-F1 and TGF-B1-F2 (collagen-binding) constructs exhibited anti-proliferative activities comparable to purified platelet TGF-beta 1, but at lower specific activities. Binding of the renatured TGF-B1-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column. The high affinity binding was also demonstrated by the binding of 3H-collagen to the TGF-B1-F2 protein immobilized on a Ni-NTA column. The TGF-B1-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration. Taken together, these results demonstrate that biologically active TGF-beta 1 fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms. Furthermore, these results support the concept that auxiliary domains may be used to modulate and/or target TGF-beta 1 for specific applications.

Human endothelial cells are defective in diabetic vascular disease.

Diabetic vascular disease is characterized pathologically by endothelial cell (EC) hyperplasia and basement membrane (BM) thickening. One key question regarding the pathogenesis of diabetic vascular disease is whether the EC or BM or both are primarily defective and responsible for these pathological changes. Previous studies, which took the approach of creating artificial diabetic conditions, have been inconclusive. It is known, however, that the extracellular matrix may be altered by glycosylation as a result of hyperglycemia, thereby altering EC function. To begin to address this question and more closely mimic the situation in vivo, we characterized human diabetic EC harvested from insulin-dependent diabetic mothers (IDDM) at the cellular and molecular levels. Human EC were isolated from both normal and IDDM umbilical cords and cellular functions evaluated using standard assays of attachment (% attached cells), proliferation (cpm/cell), resistance to detachment under shear stress (number of cells remaining attached), and glucose uptake (cpm/2 X 10(4) cells). Gene expression of major BM components (collagen type IV, laminin beta 1, and laminin beta 2) was quantified by Northern analysis. Diabetic EC demonstrated increased proliferation (two- to eightfold compared to normals), were 20-40% less resistant to shear stress and took up glucose 10-15% more slowly than normal EC. Furthermore, Northern analysis showed that the expression of major BM components was increased by an average of 10-18% in diabetic cells compared to normal cells. These results were consistent with in vivo observations and previously published data.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II binding to human embryonic lung fibroblasts causes transient changes in cytosolic calcium and promotes fibrin gel contraction.

Contraction of granulation tissue is promoted by the peptide vasoconstrictor angiotensin II (AII), which presumably acts on the fibroblasts present in granulation tissue. Direct effects of AII on fibroblast contraction were examined using an in vitro fibrin gel contraction model. Fibrin gels were formed by mixing cultured human embryonic lung fibroblasts with fibrinogen, in the presence of thrombin and measurement of gel volume was used to determine the extent of fibrin gel contraction by fibroblasts. AII stimulated an additional 28.5 +/- 3% decrease in gel size after 24 hr, AII is also shown to specifically bind to human embryonic lung fibroblasts and stimulate rapid transient increases in cytosolic calcium. AII stimulates contraction of human embryonic lung fibroblasts as reflected by acceleration of fibrin gel contraction. Fibrin clot contraction contributes to extra cellular matrix reorganization during initial stages of wound repair and AII-stimulated fibroblast contraction may accelerate this process.

Modulation of collagen synthesis by transforming growth factor-beta in keloid and hypertrophic scar fibroblasts.

Keloid and hypertrophic scars are fibrous growths characterized by overabundant collagen deposition. We examined the effect of transforming growth factor-beta (TGF-beta), a known stimulant for the production of connective tissue matrices, on the rate of collagen synthesis in keloid fibroblasts (KFs), hypertrophic scar fibroblasts (HSFs), and normal skin fibroblasts (NSFs). Fibroblasts were cultured in three-dimensional fibrin-gel matrices in the presence or absence of TGF-beta (5 ng/ml) or anti-TGF-beta neutralizing antibody (50 micrograms/ml). Secreted collagen levels, labeled with 3H-proline, were measured after 48 hours. KFs produced up to 12 times more collagen than NSFs, and up to 4 times more than HSFs. Although KFs increased their rate of collagen production by up to 2.7 times in response to TGF-beta, HSFs and NSFs did not (p = 0.065). Anti-TGF-beta antibody reduced the rate of collagen synthesis of KFs by 40% (p = 0.003), although it did not suppress collagen production in HSFs (p = 0.06) and NSFs (p = 0.75). We conclude that although KFs and HSFs are similar in that they both overproduce collagen, they are different in that only KFs display a marked sensitivity to TGF-beta, which is abundant during the proliferative phase of wound healing.

Dermal fibroblasts activate keratinocyte outgrowth on collagen gels.

The effects of dermal fibroblasts on keratinocyte outgrowth on collagen substrata was studied using an in vitro keratinocyte-collagen gel composite model. Skin fibroblasts were seeded inside collagen gels, which remained attached to the cell culture plastic substratum. Fibroblasts incorporated in collagen gels were either kept viable throughout the study, or were lysed hypotonically with water at different time intervals (2 hours and 5 days). Results show that very little keratinocyte outgrowth occurred on either plain collagen gels or gels that had previously contained viable fibroblasts for 2 hours. A 3- to 4-fold increase in keratinocyte outgrowth occurred on collagen gels that had previously contained viable fibroblasts for 5 days. A striking increase (20-fold) in keratinocyte outgrowth was observed on collagen gels that contain viable fibroblasts. The effect of fibroblast diffusible factors on keratinocyte outgrowth was further studied with a co-culture system using Millicell inserts. It was found that the co-culture of fibroblasts with the composite enhanced keratinocyte outgrowth on collagen gels that had previously contained viable fibroblasts for 5 days. Among all, however, the keratinocyte outgrowth was far better on gels containing viable fibroblasts. Addition of keratinocyte growth factor or its neutralizing antibody did not affect keratinocyte outgrowth. These results suggest that dermal fibroblasts can activate keratinocyte outgrowth on collagen matrices through some diffusible factors other than keratinocyte growth factor, and epithelial-mesenchymal interactions exert some special effects on keratinocyte outgrowth on collagen gels.

Cellular responses to silicone and polyurethane prosthetic surfaces.

Prosthetic devices composed of silicone or polyurethane are commonly used in surgery. These devices elicit a soft tissue reaction which may frequently be complicated by capsule formation. Histologically the capsule comprises both cellular (fibroblasts and endothelial cells (EC)) and matrix components (predominantly collagen type I). We hypothesized that the function of the cellular elements is altered by exposure to prosthetic materials and that this alteration contributes to capsule formation. To test this hypothesis, we utilized specific in vitro assays of cell function (attachment, proliferation, matrix gel contraction), which closely mimic in vivo cellular events, in order to define the responses of EC and fibroblasts to prosthetic surfaces (foam polyurethane, flat silicone, and textured silicone). Morphologic changes were evaluated by scanning electron microscopy (SEM). Attachment of both cell types to all prosthetic surfaces was significantly decreased compared to control (HUVEC: control, 55 +/- 1; foam polyurethane, 19 +/- 4*; flat silicone, 25 +/- 3*; textured silicone, 36 +/- 1*; fibroblast: control, 93 +/- 6; foam polyurethane, 21 +/- 4*; flat silicone, 57 +/- 5*; textured silicone, 44 +/- 5* (*P < 0.05 = significant; units, percentage spread)). Fibroblast proliferation was significantly decreased on foam polyurethane (0.1 +/- 0.03*) and textured silicone (0.18 +/- 0.05*), but not on flat silicone (0.79 +/- 0.2; control = 0.96 +/- .2). In contrast, HUVEC proliferation was significantly decreased on both silicone surfaces but not on polyurethane (units, cpm/cell; control, 0.26 +/- 0.05; foam polyurethane, 0.15 +/- 0.05; flat silicone, 0.08 +/- 0.03*; textured silicone, 0.02 +/- 0.01*).(ABSTRACT TRUNCATED AT 250 WORDS)

New evidence and new hope concerning endothelial seeding of vascular grafts.

Endothelial cell (EC) seeding of prosthetic bypass grafts has been promoted as a method of improving graft patency. However, an efficient and reliable method of seeding vascular prostheses with ECs is lacking due to inefficient harvesting of ECs and poor attachment and proliferation of cells on the prosthetic surfaces. To investigate the effect of a commonly used prosthetic surface on EC attachment and proliferation, we measured the attachment and proliferation of ECs on polytetrafluoroethylene (PTFE) grafts uncoated or coated with gelatin, laminin, fibronectin, collagen type I and/or III, or RGD (arginine-glycine-aspartate)-containing peptide. EC attachment and proliferation were both significantly decreased on the untreated PTFE graft surface. Conversely, coating of PTFE with fibronectin, RGD, laminin, or gelatin significantly (p less than 0.05) improved the attachment of ECs, with the most striking increases occurring with laminin and gelatin. Similarly, all matrix components in this study improved EC proliferation compared with untreated PTFE, with RGD and gelatin producing the most significant improvement. PTFE adversely effects EC attachment and proliferation. These properties can be improved by treating PTFE graft surfaces with extracellular matrix components in relatively low concentrations. Future investigations are needed to determine whether there are combinations and concentrations of matrix components that will optimize these cellular functions on vascular prostheses.

Fibronectin and fibrinolysis are not required for fibrin gel contraction by human skin fibroblasts.

Human skin fibroblasts contracted fibrin gels in a time- and cell-dependent manner. Under optimal conditions, gel contraction amounted to more than 50% in 2 hr. Fibronectin did not promote contraction, and fibrinolysis was not required for contraction, although gels contracted without serum or aprotinin were lysed. Before contraction, fibrin was present in loosely packed, randomly organized fibrils. After contraction, the fibrils were more densely packed and aligned in the plane of cell spreading. Cycloheximide treatment of fibroblasts inhibited gel contraction in serum-free medium but not in serum-containing medium. Fibronectin could not substitute for serum in overcoming the cycloheximide effect. Binding sites for fibrin were distributed randomly over the cells' surfaces based on electron microscopic observations. Often small groups of fibrils were localized in indentations at the cell surface. Finally, peptides containing the arg-gly-asp-ser sequence inhibited gel contraction.

Cell adhesion and phagocytosis promoted by monoclonal antibodies not directed against fibronectin receptors.

In this report we describe cell adhesion and phagocytosis promoted by two monoclonal antibodies that were selected for immunofluorescence staining of non-permeabilized baby hamster kidney (BHK) cells. Anti-BHK1 staining was heaviest along cell margins, whereas anti-BHK2 staining was continuous along cell margins. Neither antibody stained elongated plaque structures such as were observed when cells were reacted with antibodies to fibronectin (FN) receptors. The monoclonal antibodies functioned as adhesion ligands in four different assays: attachment to culture dishes, spreading, binding of latex beads and phagocytosis. Anti-BHK1 and anti-BHK2 promoted attachment to culture dishes similarly, but anti-BHK2 was more effective at promoting cell spreading. Antibody-promoted cell spreading was inhibited by the peptides Ser-Asp-Gly-Arg and Gly-Arg-Gly-Asp-Ser-Pro but not by other, related, peptides tested. The monoclonal antibodies also promoted binding of latex beads, and the bead binding sites were motile, on the basis of their 'capping' response. Nevertheless, anti-BHK2 beads were phagocytosed by cells 5- to 20-fold more efficiently than anti-BHK1 beads. The binding sites for anti-BHK1 and anti-BHK2 were characterized by immunoprecipitation experiments. Anti-BHK1 binding sites contained 50K (K = 10(3) Mr) and 88K components under non-reducing conditions that migrated as a 51/53K doublet and a 93K component under reducing conditions. On the other hand, anti-BHK2 binding sites contained 88K and 110K components under non-reducing conditions that shifted to apparent 107K and 128K values when measured under reducing conditions.