Collagen Lattice Research Articles

Transplantation of Autologous Keratinocyte Suspension in Fibrin Matrix to Chronic Venous Leg Ulcers: Improved Long-Term Healing after Removal of the Fibrin Carrier

The transplantation of keratinocytes suspended in fibrin carrier represents a candidate regimen for chronic ulcer treatment in an outpatient setting. We evaluated the integration and survival of autologous individualized keratinocytes applied within fibrin matrix onto chronic venous leg ulcers in vivo. Parallel in vitro culture was used to validate keratinocyte survival and apoptosis in fibrin compared to collagen matrix carrier. Seven patients with chronic venous leg ulcers were transplanted with autologous keratinocytes suspended in fibrin sealant after isolation and expansion from full-skin biopsy. The fibrin carrier was removed in three patients after 7 days, whereas four patients served as control with fibrin remaining. In parallel in vitro cultures, primary keratinocyte movement in fibrin as well as viability in three-dimensional (3D) fibrin versus collagen lattices was examined. Complete ulcer healing was observed in four of seven ulcers after a mean duration of 14.5 weeks. If the fibrin layer was removed, complete wound healing occurred in three of three patients, compared to one of four in the control group. In vitro, keratinocytes formed a monolayer underneath but remained isolated and nonmobile within the fibrin matrix, suggesting reepithelialization along the lower fibrin interphase. Keratinocyte culture in 3D fibrin at clinically used concentration (90 mg/mL) caused high levels of apoptosis, similar to 3D collagen, which was prevented by diluting fibrin concentration to 3 mg/mL. Transplantation of autologous keratinocytes suspended in fibrin is efficient in the treatment of chronic venous leg ulcers. Due to an antimigratory and survival-compromising effect, the presently used fibrin carrier should be removed after a few days of transplantation.

Replicative Senescence Affects Fibroblast α‐Smooth Muscle Actin and Contractility

There are changes associated with aging including impaired wound healing. Senescent skin fibroblasts have characteristics similar to cells in aging skin. Our objective was to study telomere‐based senescence and its role in wound healing and whether telomerase (hTERT) expression could rescue the aging phenotype.Early and late passage fibroblasts were grown on coverslips with +/− Transforming Growth Factor‐β (TGF‐β) and stained for the myofibroblast marker, α‐smooth muscle actin (α‐sma). Collagen lattices were generated with the fibroblasts to compare contractility. Similar experiments were performed with hTERT fibroblasts.Results show that late passage cells demonstrated twice the amount of α‐sma as the early passage cells whereas hTERT fibroblasts expressed half the amount as the early passage cells. After two days in the collagen lattices, the early passage cells contracted less than the late passage cells.The results suggest that late passage cells have a greater contractile ability than early passage cells due to their demonstration of α‐sma and perhaps to lack of proliferation. Results indicate that hTERT may rescue the aging phenotype by demonstrating less α‐sma.Funding was provided by the NIH IDeA Network of Biomedical Research Excellence (INBRE)

Vascular tube formation on matrix metalloproteinase‐1 damaged collagen

Endothelial cells maintained on a three‐dimensional lattice of intact, polymerized type I collagen formed a monolayer of cells with a cobblestone morphology. When the collagen was exposed to organ culture fluid from human skin (previously characterized and shown to contain a high level of active matrix metalloproteinase‐1 [MMP‐1]), degradation of the collagen occurred. The major degradation products were the ¾ and ¼ sized fragments known to result from the action of MMP‐1 on type I collagen. When endothelial cells were maintained on the partially fragmented collagen, the cells organized into a network of vascular tubes. Pretreatment of the organ culture fluid with either tissue inhibitor of metalloproteinase‐1 (TIMP‐1) or neutralizing antibody to MMP‐1 prevented degradation of the collagen lattice and concomitantly inhibited endothelial cell organization into the vascular network. Purified (activated) MMP‐1 duplicated the effects of skin organ culture fluid but other enzymes including MMP‐2 (gelatinase A), MMP‐9 (gelatinase B), elastase or trypsin failed to produce measurable fragments from intact collagen and failed to promote vascular tube formation. Based on the findings described here, we suggest that damage to the connective tissue is a major promoter of new vessel formation. This study was supported in part by NIH grant GM77724 from the USPHS.

Reduced expansion rate of abdominal aortic aneurysms in patients with diabetes may be related to aberrant monocyte-matrix interactions

Diabetes increases the risk of atherothrombosis, but reduces the risk of abdominal aortic aneurysm (AAA). The reason for this difference is unknown. We examined the role of diabetes and glycation on AAA expansion and extracellular matrix-monocyte interactions. We followed 198 patients (20 with diabetes) who had 30-45 mm AAAs with yearly aortic ultrasound for 3 years. Diabetes was independently associated with reduced AAA growth (beta = -0.17, P = 0.01; OR for expansion above median 0.18, 95% confidence interval 0.06-0.57). In vitro incubation of resting human monocytes with glycated bovine serum albumin or monomeric type I collagen increased matrix metalloproteinase (MMP) secretion. In contrast, exposure of activated monocytes to glycated type I collagen lattices induced a marked reduction in MMP and interleukin-6 secretion. This de-activating effect was also demonstrated in cross-linked non-glycated collagen lattices, healthy decellularized aortic media, and decellularized aortic media from diabetes patients with atherosclerosis. In contrast, decellularized aortic media from patients with atherosclerosis, but no diabetes, induced increased MMP secretion. These findings confirm that the progression of AAA is slower in patients with diabetes and suggest a mechanism by which the aortic media may be protected from degradation in these individuals.

Heparan sulfate–dependent ERK activation contributes to the overexpression of fibrotic proteins and enhanced contraction by scleroderma fibroblasts

To investigate the contribution of heparan sulfate proteoglycan and Ras/MEK/ERK to the overexpression of profibrotic proteins and the enhanced contractile ability of dermal fibroblasts from patients with systemic sclerosis (SSc; scleroderma). The effects of the MEK/ERK inhibitor U0126, the heparan sulfate side chain formation inhibitor beta-xyloside, and soluble heparin on the overexpression of profibrotic genes were compared in fibroblasts from lesional skin of patients with diffuse SSc and fibroblasts from healthy control subjects. Identified protein expressions were compared with the contractile abilities of fibroblasts while they resided within a collagen lattice. Forces generated were measured using a culture force monitor. Inhibiting MEK/ERK with U0126 significantly reduced expression of a cohort of proadhesive and procontractile proteins that normally are overexpressed by scleroderma fibroblasts, including integrin alpha4 and integrin beta1. Antagonizing heparan sulfate side chain formation with beta-xyloside or the addition of soluble heparin prevented ERK activation, in addition to reducing the expression of these proadhesive/contractile proteins. Treatment with either U0126, beta-xyloside, or heparin resulted in a reduction in the overall peak contractile force generated by dermal fibroblasts. Blocking platelet-derived growth factor receptor with Gleevec (imatinib mesylate) reduced overall contractile ability and the elevated syndecan 4 expression and ERK activation in SSc fibroblasts. The results of this study suggest that heparan sulfate-dependent ERK activation contributes to the enhanced contractile ability demonstrated by dermal fibroblasts from lesional skin of patients with scleroderma. These results are consistent with the notion that the MEK/ERK procontractile pathway is dysregulated in scleroderma dermal fibroblasts. Additionally, the results suggest that antagonizing the MEK/ERK pathway is likely to modulate heparan sulfate proteoglycan activity, which in turn may have a profound effect on the fibrotic response in SSc.

Gadodiamide contrast agent ‘activates’ fibroblasts: a possible cause of nephrogenic systemic fibrosis

Nephrogenic systemic fibrosis (NSF) is a fibrotic disease generating intense interest due to its recent discovery, and unknown cause. It appears confined to patients with renal disease and presents as grossly thickened, indurated, tight skin that is woody to palpation. Histologically, the dermis contains thickened collagen bundles, numerous plump fibroblast-like cells, and elevated hyaluronan expression. Recent data suggest a link between the use of gadolinium chelate as an MRI contrast agent and the onset of the disease. Fibroblasts from the lesions of six NSF patients, all of whom were exposed to gadodiamide, were compared with control fibroblasts for hyaluronan and collagen synthesis. Serum from NSF patients was assessed for fibroblast hyaluronan-stimulating activity, collagen synthesis, and gadodiamide for its effect on fibroblast proliferation and matrix synthesis. NSF fibroblasts synthesized excess levels of hyaluronan and collagen compared with control fibroblasts, with up to 2.8-fold and 3.3-fold increases, respectively. NSF patient serum stimulated control fibroblast hyaluronan synthesis by up to 7-fold, and collagen synthesis by up to 2.4-fold. 1 mM gadodiamide added to culture medium stimulated fibroblast growth in a dose-dependent manner, decreasing their doubling time from 28 h to 22 h, and increasing the maximum cell density. Even a short exposure to gadodiamide stimulated cell growth, suggesting that the cells were activated by the gadodiamide. The growth of fibroblasts within contracted collagen lattices was also significantly stimulated by gadodiamide, while fibroblasts exposed to gadodiamide synthesized increased levels of hyaluronan. Control fibroblasts exposed to gadodiamide, and NSF fibroblasts exhibited an extensive pericellular coat of hyaluronan, and expressed alpha-smooth muscle actin. Gadolinium chloride did not affect fibroblast growth. This report demonstrates that NSF fibroblasts synthesize excess levels of hyaluronan and collagen, and that gadodiamide stimulates control fibroblast growth, matrix synthesis, and differentiation into myofibroblasts, suggesting a possible role for gadodiamide in the pathophysiology of NSF.

Effect of angiotensin II and angiotensin II type 1 receptor antagonist on the proliferation, contraction and collagen synthesis in rat hepatic stellate cells

Angiotensin II (Ang II) is a very important vasoactive peptide that acts upon hepatic stellate cells (HSCs), which are major effector cells in hepatic cirrhosis and portal hypertension. The present study was aimed to investigate the effects of Ang II and angiotensin II type 1 receptor antagonist (AT(1)RA) on the proliferation, contraction and collagen synthesis in HSCs. HSC-T6 rat hepatic stellate cell line was studied. The proliferation of the HSC cells was evaluated by MTT colorimetric assay while HSC DNA synthesis was measured by (3)H-thymidine incorporation. The effects of angiotensin II and AT(1)RA on HSCs contraction were studied by analysis of the contraction of the collagen lattice. Cell culture media were analyzed by RT-PCR to detect secretion of collagen I (Col I), collagen III (Col III) and transforming growth factor beta1 (TGF-beta1) by enzyme linked immunosorbent assay. HSC was harvested to measure collagen I, collagen III and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression. Ang II ((1 x 10(-10) - 1 x 10(-4)) mol/L) stimulated DNA synthesis and proliferation in HSCs compared with untreated control cells. AT(1)RA inhibited angiotensin II induced proliferation of HSCs. A linear increase in the contractive area of collagen lattice correlated with the concentration of angiotensin II (1 x 10(-9) - 1 x 10(-5) mol/L) and with time over 48 hours. AT(1)RA blocks angiotensin II induced contraction of collagen lattice. Col I, Col III and TGF-beta1 levels of the Ang II group were higher than those of control group and this increase was downregulated by AT(1)RA. The mRNA expressions of Col I, Col III and TIMP-1 were higher in HSCs from the Ang II group than the control group and downregulated by AT(1)RA. Angiotensin II increased DNA synthesis and proliferation of HSCs in a dose-dependent manner, stimulated the contraction of HSCs dose- and time-dependently. Angiotensin also promoted excretion of Col I, Col III and TGF-beta1 levels and stimulated Col I, Col III and TIMP-1 expression in HSCs. Angiotensin acts via the angiotensin II receptor because all of these effects are blocked by angiotensin II type 1 receptor antagonist.

Confocal Laser Scanning Microscopic Analysis of Collagen Scaffolding Patterns in Cranial Sutures

Although recent studies indicate that regional dura mater influences the fate of the overlying cranial suture, little is known about the assembly of extracellular matrix (ECM) molecules within the patent and fusing murine cranial suture complexes. Confocal laser scanning microscopy was used to study ECM assembly within patent and fusing cranial suture complexes. Coronal sections (20 microm thick) of patent sagittal (SAG) and fusing posterior frontal (PF) sutures from postnatal 8-, 14-, and 18-day-old Sprague-Dawley rats were scanned in 0.5-microm increments, and images were collected consecutively to create a z-series for three-dimensional reconstruction. Spatial and temporal collagen arrangements were compared between SAG and PF sutures by measuring interfiber distance, fiber thickness, and total collagen surface area at each time point. We demonstrate that on day 8 (before the onset of suture fusion), collagen bundles are randomly arranged in both the SAG and PF sutures. By day 14 (midfusion period), there was a statistically significant reduction in total collagen surface area (80.5% versus 67.4%; P < 0.05) as the collagen bundles were organized into orthogonal lattices along the anterior and endocranial margins of the PF suture. Furthermore, new bone matrix deposition was observed along the edges of these organized collagen bundles. In contrast, collagen within the SAG suture remained randomly arranged and unossified. By day 18 (late fusion period), the PF suture was completely fused except for the posterior-ectocranial portion. This patent section of the PF suture contained a highly organized mineralizing orthogonal collagen lattice. The total collagen surface area in the day-18 PF suture continued to decline compared with the day-8 PF suture (80.5% versus 55.6%; P < 0.05). In the day-18 SAG suture, the collagen bundles remained randomly arranged, and the total surface area did not change. The same analysis was performed in a human pathologic fusing and patent suture. Similar results were observed. The total collagen surface area significantly decreased in the pathologic fusing human suture compared with the patent suture (92.8% versus 60.6%; P < 0.05). Moreover, the pathologically fusing suture contained a highly organized mineralizing orthogonal collagen lattice. This is the first analysis of collagen patterns in patent and fusing cranial sutures.

Extracellular matrix metalloproteinase inducer/CD147 promotes myofibroblast differentiation by inducing α‐smooth muscle actin expression and collagen gel contraction: implications in tissue remodeling

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein enriched on tumor cells and normal epithelia. It is mainly known for its ability to induce matrix metalloproteinase production in fibroblasts following epithelial-stromal interaction. We sought to examine whether EMMPRIN has a broader role promoting fibroblast-to-myofibroblast differentiation. Because alpha-smooth muscle actin (alphaSMA) is considered a marker of this differentiation process, we analyzed the effect of EMMPRIN on its expression in corneal and skin fibroblasts by Western blots, immunocytochemistry, and a functional assay of collagen lattice contraction. Increasing EMMPRIN expression by cDNA transfection or by treatment with exogenously added recombinant EMMPRIN resulted in an up-regulation of alphaSMA expression. EMMPRIN also increased the contractile properties of the treated fibroblasts as demonstrated by the immunohistochemical appearance of stress fibers and by the accelerated contraction of fibroblast-embedded collagen lattices. Blocking EMMPRIN expression by small interfering RNA inhibited alphaSMA and collagen gel contraction induced not only by EMMPRIN but also by transforming growth factor-beta, a major mediator of myofibroblast differentiation that also regulated EMMPRIN expression. These findings, combined with the fact that EMMPRIN and alphaSMA colocalized to the same cells in the stroma of pathological corneas, expand on the mechanism by which EMMPRIN remodels extracellular matrix during wound healing and cancer.

Collagen scaffolds for tissue engineering

There are two major approaches to tissue engineering for regeneration of tissues and organs. One involves cell-free materials and/or factors and one involves delivering cells to contribute to the regeneraion process. Of the many scaffold materials being investigated, collagen type I, with selective removal of its telopeptides, has been shown to have many advantageous features for both of these approaches. Highly porous collagen lattice sponges have been used to support in vitro growth of many types of tissues. Use of bioreactors to control in vitro perfusion of medium and to apply hydrostatic fluid pressure has been shown to enhance histogenesis in collagen scaffolds. Collagen sponges have also been developed to contain differentiating-inducing materials like demineralized bone to stimulate differentiation of cartilage tissue both in vitro and in vivo.

Contraction of tubulointerstitial fibrosis tissue in diabetic nephropathy, as demonstrated in an in vitro fibrosis model

Tubulointerstitial fibrosis in diabetic nephropathy (DN) was investigated using an in vitro tissue model of remodeling, to determine the pathogenic mechanism of fibrosis that leads to renal atrophy, i.e., renal failure. The remodeling model consisted of a renal fibroblast-populated collagen lattice (FPCL). The overexpression of transforming growth factor (TGF)-beta1 in the diabetic kidney gave rise to FPCL contraction. FPCL relaxation was induced by the subsequent addition of cytochalasin D. The FPCL failed to contract when exposed to TGF-beta1 plus Y27632, a Rho kinase inhibitor. TGF-beta1 induced the phosphorylation of myosin light chains, and Y27632 blocked this activity. TGF-beta1-induced FPCL contraction was suppressed by the addition of 2,3-butanedione monoxime, a myosin ATPase inhibitor. As shown in the video, the contraction rate of the projections of the cells in the FPCL was significantly greater in the TGF-beta1 group than in the control group. Collectively, these results indicate that TGF-beta1-induced FPCL contraction is attributable to actin-myosin interactions in the fibroblasts through the activation of Rho kinase, the phosphorylation of myosin light chains, and the subsequent activation of myosin ATPase. We propose that via these mechanisms, tubulointerstitial fibrosis generates tissue contraction that leads to renal atrophy and renal failure in DN.

Involvement of ERK signaling in halofuginone-driven inhibition of fibroblast ability to contract collagen lattices

Halofuginone, an alkaloid isolated from the plant Dichroa febrifuga, has been shown to be a potent inhibitor of tissue fibrosis. We herein demonstrate that, at concentrations below 10(-7) M, halofuginone does not affect the cell cycle but efficiently induces extracellular signal-regulated kinases(1,2) (ERK(1,2)), p38 and Jun NH2-terminal kinases(1,2) (JNK(1,2)) phosphorylation. In addition, at these non cytotoxic concentrations, halofuginone diminishes the capacity of fibroblasts to contract mechanically unloaded collagen lattices, an effect that is specifically blocked by the ERK inhibitors PD98059 and U0126, not by inhibitors of the JNK or p38 pathways. These data thus indicate that the inhibitory effect of halofuginone on fibroblast contractile activity, a key function for wound healing implicated in the development of tissue fibrosis, is an ERK-mediated mechanism.

Increased TGF‐β–producing CD4+ T lymphocytes in postburn patients and their potential interaction with dermal fibroblasts in hypertrophic scarring

The development of hypertrophic scar involves a complex interplay between cells and cytokines. Although the mechanism underlying its pathogenesis is not well understood, a polarized T-helper type 2 immune response has been reported, indicating a role for CD4+ T lymphocytes in hypertrophic scarring. Here, we report an increased frequency of CD4+/transforming growth factor-beta (TGF-beta)-producing T cells in the peripheral blood and hypertrophic scar tissue of burn patients. These cells may play an indirect regulatory role in hypertrophic scar by affecting the functions of dermal fibroblasts. Our results show an increase in cell proliferation and collagen synthesis by dermal fibroblasts treated with medium derived from burn patient CD4+ T lymphocytes but not from the CD4+ T cells of normal subjects. Using confocal microscopy and immunoblotting, we found the level of alpha-smooth muscle actin to be elevated in these treated dermal fibroblasts, which also showed an enhanced ability to contract collagen lattices. TGF-beta levels in medium conditioned by the culture of CD4+ T lymphocytes from burn patients were significantly higher than in the conditioned medium from CD4+ T lymphocytes of normal subjects. In addition, the application of a TGF-beta-neutralizing antibody significantly reduced the effect of burn patient CD4+ T lymphocyte medium on dermal fibroblast proliferation and collagen lattice contraction. Our study suggests that CD4+/TGF-beta-producing T lymphocytes may play an important role in postburn hypertrophic scarring.

Human DESC1 serine protease confers tumorigenic properties to MDCK cells and it is upregulated in tumours of different origin

Proteolysis of the extracellular matrix components plays a crucial role in the regulation of the cellular and physiological processes, and different pathologies have been associated with the loss or gain of function of proteolytic enzymes. DESC1 (differentially expressed in squamous cell carcinoma gene 1), a member of the TTSP (type II transmembrane serine protease) family of serine proteases, is an epithelial-specific enzyme that has been found downregulated in squamous cell carcinoma of the head and neck region. We describe new properties of DESC1 suggesting that this protease may be involved in the progression of some type of tumours. Thus, this enzyme hydrolyses some extracellular matrix components, such as fibronectin, gelatin or fibrinogen. Moreover, Madin–Darby canine kidney (MDCK) cells expressing exogenous human DESC1 acquire properties associated with tumour growth such as enhanced motility and an increase of tubular forms in a 3D collagen lattice following HGF treatment. Finally, we generated polyclonal anti-DESC1 antibodies and immunohistochemical analysis in tissues different from head and neck region indicated that this protease was overexpressed in tumours of diverse origins. Taken together, our results suggest that DESC1 could be considered as a potential therapeutic target in some type of tumours.

α11β1 Integrin-Dependent Regulation of Periodontal Ligament Function in the Erupting Mouse Incisor

The fibroblast integrin alpha11beta1 is a key receptor for fibrillar collagens. To study the potential function of alpha11 in vivo, we generated a null allele of the alpha11 gene. Integrin alpha11(-/-) mice are viable and fertile but display dwarfism with increased mortality, most probably due to severely defective incisors. Mutant incisors are characterized by disorganized periodontal ligaments, whereas molar ligaments appear normal. The primary defect in the incisor ligament leads to halted tooth eruption. alpha11beta1-defective embryonic fibroblasts displayed severe defects in vitro, characterized by (i) greatly reduced cell adhesion and spreading on collagen I, (ii) reduced ability to retract collagen lattices, and (iii) reduced cell proliferation. Analysis of matrix metalloproteinase in vitro and in vivo revealed disturbed MMP13 and MMP14 synthesis in alpha11(-/-) cells. We show that alpha11beta1 is the major receptor for collagen I on mouse embryonic fibroblasts and suggest that alpha11beta1 integrin is specifically required on periodontal ligament fibroblasts for cell migration and collagen reorganization to help generate the forces needed for axial tooth movement. Our data show a unique role for alpha11beta1 integrin during tooth eruption.

Formation of Cartilage In Vivo with Immobilized Autologous Rabbit Auricular Cultured Chondrocytes in Collagen Matrices

The availability of generated cartilage de novo is one of the needs of reconstructive surgery. In this study, the authors constructed a matrix formed by autologous immobilized chondrocytes using collagen gel as a scaffold. Furthermore, the ability of these matrices to engraft and generate new cartilage was examined. Biopsy specimens of elastic cartilage were surgically obtained from the ears of eight New Zealand White rabbits. After collagenase II digestion of cartilage, chondrocytes were isolated and propagated in culture medium. Chondrocytes were immobilized into bovine collagen lattices and implanted, replacing pieces of removed native cartilage. Five weeks after implantation, the rabbits were killed and the ears were examined macroscopically and analyzed by means of histochemical methods. The results show the formation of new cartilage from implanted lattices with chondrocytes. Gross analysis of the ears shows similarities in appearance, consistency, texture, and histology between native and new cartilage. Fluorescence of the nucleus from bisbenzimide-labeled chondrocytes was detected in newly formed tissue, pointing out its in vitro culture origin. No signs of an inflammatory reaction attributable to implants were found in either the control or the chondrocyte lattices. The authors suggest that this approach is of value for future clinical use.

The Rho-Kinase Inhibitor H-1152P Suppresses the Wound-Healing Activities of Human Tenon’s Capsule Fibroblasts In Vitro

To analyze the role of Rho-kinase signaling in the wound-healing activities of human Tenon's capsule fibroblasts by using H-1152P, a potent inhibitor of this kinase, in vitro. The optimal concentration of H-1152P was determined by MTT test. Cell proliferation was measured by BrdU incorporation and Ki-67 immunostaining, whereas cell viability was investigated by ethidium homodimer-1 dye exclusion. The actin cytoskeleton organization was demonstrated by alpha-smooth muscle actin (SMA) immunostaining and Alexa 488-phalloidin staining. Cell migration was studied on restrained collagen gels and in a scratch-wound assay followed by Ki-67 and fibronectin immunostaining. The effect of H-1152P on contraction was analyzed in floating collagen gels populated with fibroblasts, which were subsequently processed for fibronectin immunostaining. The levels of adducin and the protein kinase A (PKA)-dependent phosphorylation of this protein were detected by immunoblot analysis, to rule out interference with PKA. Incorporation of BrdU and upregulation of Ki-67 were reduced by 80% to 90% in cells incubated with 10 microM of this inhibitor for 4 days (P < 0.01). H-1152P caused the disassembly of stress fibers in a dose-dependent manner without exerting toxic effects and without a considerable interference with the PKA-pathway. H-1152P also significantly suppressed cell migration 3- to 3.5-fold and the contraction of collagen lattices fivefold with a dose-dependent impairment in the assembly of the fibronectin network. These findings imply a role for Rho-kinase in the wound-healing activities of human Tenon's capsule fibroblasts and show the potential of H-1152P as a safe and specific means to suppress these events.

Interleukin-6 (IL-6) modulates migration and matrix metalloproteinase function in dermal fibroblasts from IL-6KO mice

Interleukin-6-deficient (IL-6KO) mice display significantly delayed cutaneous wound healing characterized by decreased re-epithelialization, granulation tissue and wound closure. Dermal fibroblasts are one of the principal cell types found in granulation tissue and mediate numerous processes during healing. To investigate the effects that IL-6 might have on granulation tissue formation and fibroblast motility. As fibroblast motility is associated with matrix metalloproteinase (MMP) activity, the expression of MMP-2 and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2 were assessed. Punch biopsies (4 mm) were performed in the skin of IL-6KO and C57BL/6 mice. The expression of MMP-2, TIMP-1 and -2 in wound tissue was monitored over time. Cellular infiltration and granulation tissue formation was monitored by subcutaneous implantation of polyvinyl alcohol (PVA) sponges. A free-floating collagen lattice model was also used to investigate the direct effects of IL-6 treatment on isolated IL-6KO fibroblasts. The expression of MMP-2, and the inhibitors TIMP-1 and -2, were assessed via real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. IL-6KO wounds showed impaired granulation tissue formation 5 days postwounding and fewer fibroblasts had populated the PVA matrices 7 days after implantation in IL-6KO mice compared with wild-type C57BL/6. The mRNA and protein expression of MMP-2 and TIMP-2 mRNA was increased in IL-6KO mice compared with wild-type mice beyond 1 day postwounding, while the expression of TIMP-1 mRNA was transiently higher in IL-6KO only 3 days postwounding. Treatment of collagen lattices with various concentrations of rmIL-6 again showed a dose-response decrease in mRNA and protein expression of MMP-2 and TIMP-2 protein expression, compared with saline control, while TIMP-1 did not appear to be significantly modulated. These results indicate that IL-6 influences the function of fibroblasts in wounds, and one mechanism of this regulation may be through the modulation of MMP-2 and TIMP proteins.

Interleukin 6 modulates genes associated with fibroblast migration and function (101.10)

Abstract Interleukin 6 deficient mice (IL-6KO) display significantly delayed cutaneous wound healing, characterized by decreased granulation tissue formation. Dermal fibroblasts are one of the principal cell types found in granulation tissue and mediate numerous processes during healing including collagen deposition. Polyvinyl alcohol sponge matrices implanted seven days in IL-6KO mice showed far fewer infiltrating fibroblasts as compared to wild-type C57. It was also found that full thickness wounds from IL-6KO mice showed higher expression of MMP-2 and TIMP-2 as compared to wild-type from 1 to 5 days post wounding. Treatment of IL-6KO fibroblast populated free-floating collagen lattices with various concentrations of rmIL-6 showed a dose response decrease in MMP-2 TIMP-2 mRNA and protein expression. The expression of TGFβ’s 1, and 3 as well as TGFβRII, which can mediate collagen formation, were increased in IL-6KO mouse skin treated intradermally with rmIL-6 in a dose response manner. Expression of TGFβ1, 3, and RII were induced as well in IL-6KO fibroblast collagen lattice cultures in a similar manner. These results indicate that IL-6 may influence on collagen deposition during granulation tissue formation in skin wounds. The mechanism of this may involve both the inhibition of MMP function, and the induction of TGFβ genes. This research was supported by a grant from NIH/NIGMS (5R01GM067745-03).

Factors related to contraction and mechanical strength of collagen gels seeded with canine endotenon cells

Fibroblasts can construct a hydrated collagen lattice to a tissue-like structure that is greatly influenced by initial culture conditions. The purpose of this study was to investigate the effects of cell concentration and collagen concentration on the contraction kinetics and mechanical properties of resultant endotenon-derived fibroblast-seeded collagen lattice. The experiment was designed to evaluate the effect of cell concentration (0, 0.25, 0.5, and 1.0 x10(6) cells/mL) and collagen concentration (0.5, 1.0, 1.5, and 2.0 mg/mL). Collagen lattice contraction was recorded for 42 days, after which time the lattices were mechanically tested. The collagen lattices seeded with higher initial cell concentration had a shorter contraction lag phase (p < 0.01), and exhibited a higher ultimate stress (p < 0.01) and instantaneous and equilibrium modulus (p < 0.01) than those seeded with a lower initial cell concentration. The collagen lattices cultured with a lower initial collagen concentration also had a shorter contraction lag phase, and exhibited greater instantaneous and equilibrium modulus (p < 0.01) than those cultured with higher initial collagen concentration. The collagen lattices of initial 0.5 mg/mL collagen concentration had the highest value of ultimate stress (p < 0.03).