Canine ACL fibroblast integrin expression and cell alignment in response to cyclic tensile strain in three-dimensional collagen gels.

Tissue-engineered ligament substitutes have the potential to become an alternative graft source for ligament reconstruction. If this approach is to become viable, one must first understand and define the mechanisms responsible for creation, maintenance, and remodeling of the native anterior cruciate ligament. It is well accepted that mechanical load alters fibroblast phenotypic expression in a variety of cell sources; however, the mechanosensitive pathways responsible for alteration in matrix production, remodeling, and alignment are unknown. We hypothesize that cell surface integrins play a role in this mechanotransduction process, and as such respond to application of cyclic tensile load. Linear 3D collagen gels containing canine ACL fibroblasts were created in Flexercell Tissue-Train Culture Plates. Gels were untethered (control), tethered without external strain (tethered), or tethered and exposed to 2.5% cyclic strain for 2 h per day for 4 days (strain). Quantitation of alpha1, alpha5, and beta1 integrin subunit was performed using flow cytometry. Cell and matrix alignment was studied using light, polarized light, and fluorescent microscopy. Expression of alpha5 and beta1 integrin subunits was increased significantly in fibroblasts in tethered and strained 3D collagen gels compared with the control, unloaded constructs (p < 0.05). These integrins are known to function as mechanotransducers in other tissues, implicating a similar role in mechanotransduction in ACL fibroblasts. Histologic analysis of the tethered and strained gels demonstrated a linear arrangement of cells and parallel collagen fibril architecture. In contrast, cell distribution and collagen alignment were disorganized in the control, unloaded gels. The alignment of cells and collagen in the 3D gels parallel to applied strain is similar to the in vivo state. These data add to our understanding of the behavior of ACL fibroblasts in vitro. The ability to manipulate signal transduction pathways may enhance our ability to engineer implantable ACL grafts or to modify ACL healing response.

Effect of cyclic strain and plating matrix on cell proliferation and integrin expression by ligament fibroblasts.

The role of cell surface integrins in cell migration, proliferation, and attachment to matrix molecules is well known. Integrin-matrix interactions have been implicated in mechanotransduction and load transmission from the outside to the inside of the cell. In this study, the effect of cyclic strain on the cell proliferation, attachment, and expression of integrin subunits beta1, beta3, and alpha5 was determined in anterior cruciate ligament (ACL) and medial collateral ligament (MCL) fibroblasts grown on polystyrene, Type I collagen, laminin, elastin, and fibronectin. ACL fibroblast proliferation was not affected by growth substrate whereas MCL cells reached confluence more rapidly on fibronectin compared with collagen or polystyrene. Exposure to 5% cyclic strain resulted in a significant decrease in ACL and MCL fibroblast proliferation on fibronectin and Type I collagen. MCL cells showed a greater strain-dependent inhibition of cells grown on a fibronectin substrate than those grown on collagen. This matrix-dependent effect of strain on cell proliferation was not seen with ACL cells. Attachment of ACL and MCL fibroblasts was stronger to fibronectin compared with Type I collagen, laminin, and polystyrene. In the absence of applied load, the expression of beta1, beta3, and alpha5 subunits was not substrate dependent and the expression of beta1 and alpha5 integrin subunits was higher in MCL cells than ACL cells on all substrates. In contrast, the expression of beta3 integrin subunit was higher in ACL cells than MCL cells. In response to 5% strain, beta1, and alpha5 expression increased in all fibroblasts with MCL cells having a higher magnitude of expression. beta3 expression showed a 90% increase in response to load when grown on laminin for both MCL and ACL fibroblasts and demonstrated no change in expression on Type I collagen or fibronectin. The duration of applied strain from 2 versus 22 h had no effect on cell proliferation or integrin expression.

Quantitation of estrogen receptors and relaxin binding in human anterior cruciate ligament fibroblasts.

The significantly higher incidence of anterior cruciate ligament (ACL) injuries in collegiate women compared with men may result from relative ligament laxity. Differences in estrogen and relaxin activity, similar to that seen in pregnancy, may account for this. We quantified estrogen receptors by flow cytometry and relaxin receptors by radioligand binding assay in human ACL cells and compared the presence of these receptors in males and females. ACL stumps were harvested from seven males and eight females with acute ACL injuries. The tissue was placed in M199 cell culture medium. Outgrowth cultures were obtained, and passage 2 cells were used for all studies. Estrogen receptor determination was performed using flow cytometry. Relaxin binding was performed in ACL cells derived from five female and male patients using I(125)-labeled relaxin. Estrogen receptors were identified by flow cytometry in 4 to 10% of ACL cells. Mean fluorescence of cells expressing estrogen receptors was approximately twice that of controls, with no significant differences between males and females. Relaxin studies showed low-level binding of I(125)-relaxin-labeled ACL cells. Relaxin binding was present in four out of five female ACL cells versus one out of five male ACL cells.

Cyclooxygenase-2 inhibitor decreases extracellular matrix synthesis in stretched renal fibroblasts.

BACKGROUND/AIMS: Both TGF-beta and cyclooxygenase-2 have been implicated in the pathogenesis of interstitial fibrosis in unilateral ureteral obstruction (UUO). Cyclic tensile stretch has been used in vitro to mimic the changes in intrarenal pressure in UUO. We sought to determine the effect of meloxicam (a selective cyclooxygenase-2 inhibitor) on extracellular matrix and TGF-beta synthesis in stretched renal fibroblasts (NRK-49F). METHODS: NRK-49F cells were subject to cyclic stretch (6 cycles/min, 15% elongation) using a Flexcell apparatus. Cells were stretched in the absence or presence of meloxicam for 48 h, and then cells and supernatants were isolated. Collagen was quantified by the Sircol assay; fibronectin and laminin were visualized using immunofluorescence. TGF-beta was quantified by ELISA, and protease activity determined by a colorimetric assay. RESULTS: Both collagen and TGF-beta synthesis were increased following a 48-hour stretch of NRK-49F. Meloxicam significantly decreased the collagen and TGF-beta response to stretch. Stretch-induced fibronectin and laminin synthesis was also decreased by meloxicam. NRK-49F protease activity was decreased by stretch; this was unaffected by meloxicam. CONCLUSIONS: Stretch of NRK-49F results in extracellular matrix synthesis, a process which may be activated in UUO and contribute to interstitial fibrosis. Inhibition of cyclooxygenase-2 may reduce fibrosis through a TGF-beta-dependent process.

The effect of estrogen on ovine anterior cruciate ligament fibroblasts: cell proliferation and collagen synthesis.

UNLABELLED: Estrogen has been implicated as a causal factor for anterior cruciate ligament injuries in women. Studies have demonstrated a decrease in anterior cruciate ligament fibroblast proliferation and collagen synthesis at supraphysiologic levels of estrogen in a rabbit model. HYPOTHESIS: The authors hypothesized that physiologic levels of estrogen would have no significant effect on anterior cruciate ligament fibroblast proliferation and collagen synthesis in an ovine model. METHODS: Anterior cruciate ligament fibroblasts were isolated from sheep knees using routine cell culture methods. The cells were exposed to 17beta-estradiol at physiologic concentrations of 2.2, 5, 15, 25, 250, and 2500 pg/ml. Cell proliferation was determined by cell counts on days 4 and 6. Collagen synthesis was determined by (3)H-proline incorporation on day 4. Immunohistochemistry was performed to detect estrogen receptors. RESULTS: Immunohistochemistry demonstrated the presence of estrogen receptors in ovine anterior cruciate ligament fibroblasts. There was no significant difference in anterior cruciate ligament fibroblast proliferation or collagen synthesis regardless of 17beta-estradiol concentration. CONCLUSIONS: Based on results of this study, and given the low turnover of collagen in ligaments, it is unlikely that a 2- to 3-day per month increase in circulating estrogen would result in rapid, clinically significant alterations in material properties of the anterior cruciate ligament in vivo. The etiology of noncontact anterior cruciate ligament injuries is complex and multifactorial in nature, meriting further investigation.

Characterization of total and active matrix metalloproteinases-1, -3, and -13 synthesized and secreted by anterior cruciate ligament fibroblasts in three-dimensional collagen gels.

Anterior cruciate ligament (ACL) injury and subsequent reconstructive surgery is increasing with an estimated 200,000 reconstructions performed yearly in the United States. Current treatment requires reconstruction with autograft or allograft tissue with inherent disadvantages. The development of tissue-engineered ligament replacements or scaffolds may provide an alternative treatment method minimizing these issues. The study of ligament fibroblast catabolic and anabolic responses to mechanical and biologic stimuli in three-dimensional (3D) cell culture systems is critical to the development of such therapies. A 3D cell culture system was used to measure the total content and active forms of matrix metalloproteinases (MMPs)-1, -3, and -13 to assess the potential role of the mechanical environment in regulation of matrix turnover by ligament fibroblasts. The production, retention, and secretion of MMPs by ACL fibroblasts in 3D culture were measured over a 14-day period. The total MMP content and MMP activity were determined. The level of all MMPs studied increased over 7-10 days and then reached a steady state or decreased slightly in both the collagen gels and the media. This system will now permit the study of externally applied cyclic and static strains, strain deprivation, and the potential combined role of the cytoskeleton and MMPs in matrix turnover in ligaments.

Fluoroquinolones impair tendon healing in a rat rotator cuff repair model: a preliminary study.

BACKGROUND: Recent studies suggest that fluoroquinolone antibiotics predispose tendons to tendinopathy and/or rupture. However, no investigations on the reparative capacity of tendons exposed to fluoroquinolones have been conducted. HYPOTHESIS: Fluoroquinolone-treated animals will have inferior biochemical, histological, and biomechanical properties at the healing tendon-bone enthesis compared with controls. STUDY DESIGN: Controlled laboratory study. METHODS: Ninety-two rats underwent rotator cuff repair and were randomly assigned to 1 of 4 groups: (1) preoperative (Preop), whereby animals received fleroxacin for 1 week preoperatively; (2) pre- and postoperative (Pre/Postop), whereby animals received fleroxacin for 1 week preoperatively and for 2 weeks postoperatively; (3) postoperative (Postop), whereby animals received fleroxacin for 2 weeks postoperatively; and (4) control, whereby animals received vehicle for 1 week preoperatively and for 2 weeks postoperatively. Rats were euthanized at 2 weeks postoperatively for biochemical, histological, and biomechanical analysis. All data were expressed as mean ± standard error of the mean (SEM). Statistical comparisons were performed using either 1-way or 2-way ANOVA, with P < .05 considered significant. RESULTS: Reverse transcriptase quantitative polymerase chain reaction (RTqPCR) analysis revealed a 30-fold increase in expression of matrix metalloproteinase (MMP)-3, a 7-fold increase in MMP-13, and a 4-fold increase in tissue inhibitor of metalloproteinases (TIMP)-1 in the Pre/Postop group compared with the other groups. The appearance of the healing enthesis in all treated animals was qualitatively different than that in controls. The tendons were friable and atrophic. All 3 treated groups showed significantly less fibrocartilage and poorly organized collagen at the healing enthesis compared with control animals. There was a significant difference in the mode of failure, with treated animals demonstrating an intrasubstance failure of the supraspinatus tendon during testing. In contrast, only 1 of 10 control samples failed within the tendon substance. The healing enthesis of the Pre/Postop group displayed significantly reduced ultimate load to failure compared with the Preop, Postop, and control groups. There was no significant difference in load to failure in the Preop group compared with the Postop group. Pre/Postop animals demonstrated significantly reduced cross-sectional area compared with the Postop and control groups. There was also a significant reduction in area between the Preop and control groups. CONCLUSION: In this preliminary study, fluoroquinolone treatment negatively influenced tendon healing. CLINICAL RELEVANCE: These findings indicate that there was an active but inadequate repair response that has potential clinical implications for patients who are exposed to fluoroquinolones before tendon repair surgery.

Activation of MKK3/6, SAPK, and ATF-2/c-jun in ACL fibroblasts grown in 3 dimension collagen gels in response to application of cyclic strain.

Signal transduction pathways involved in response to cyclic tensile strain and strain deprivation in anterior cruciate ligament (ACL) fibroblasts grown in 3D collagen gels were investigated. Application of cyclic tensile strain resulted in significant activation (phosphorylation) of MKK3/6, SAPK and their downstream target transcription factors, ATF-2 and c-jun, while strain deprivation resulted in a decrease in these kinases and transcription factors. These data suggest that ACL fibroblasts cultured in 3D collagen gels respond to the mechanical environment and provide a useful system for determination of the molecular mechanisms involved in the regulation of proliferation and matrix turnover by mechanical load.

Patterns of gene expression in a rabbit partial anterior cruciate ligament transection model: the potential role of mechanical forces.

BACKGROUND: The inconsistency in healing after anterior cruciate ligament (ACL) repair has been attributed to ACL fibroblast cellular metabolism, lack of a sufficient vascular supply, and the inability to form a scar or scaffold after ligament rupture because of the uniqueness of the intra-articular environment. Hypotheses (1) Stress deprivation in the surgically transected ACL will increase matrix metalloproteinase (MMP) and alpha smooth muscle actin (alpha-SMA) expression. (2) Stress deprivation will decrease collagen expression. (3) The transected anteromedial bundle of the ACL will demonstrate a pattern of gene expression similar to the completely transected ACL, while gene expression profiles in the intact posterolateral bundle will be similar to the sham-operated controls. STUDY DESIGN: Controlled laboratory study. METHODS: Thirty-six New Zealand White rabbits underwent a partial ACL surgical transection separating the anteromedial (AM) and posterolateral (PL) bundles and transecting the AM bundle. Contralateral ACLs were either sham operated or completely transected. Ligament tissue was harvested at 1, 2, or 6 weeks after surgery, and real-time PCR was performed using primers for collagen I, collagen III, alpha-SMA, MMP-1, and MMP-13. RESULTS: At 1 week, a 28- and 29-fold increase in MMP-13 expression was seen in the complete transection and the transected AM bundle specimens when compared with sham-operated controls (P = .049, P = .018), respectively. There was no significant difference in MMP-13 between the sham controls and the intact PL bundle specimens. A 22- and 23-fold increase in alpha-SMA was seen (P = .03, P = .009) in the complete transection and transected AM bundle specimens, respectively, while no difference was seen between the intact PL bundle and controls. No significant differences were seen in collagen I (Col I) or collagen III (Col III) gene expression at 1 week. At 6 weeks, Col I expression increased 5-fold in complete transection samples (P = 3.9 x 10(-6)), 3-fold in transected AM samples (P = 3.3 x 10(-6)), and 2-fold in the intact PL bundle samples as compared with controls. alpha-SMA was increased 7.5-fold and 5-fold in complete transection and transected AM samples, respectively (P = .004, P = 2.2 x 10(-6)), while no significant change was seen in the intact PL bundle samples compared with controls. Complete transection specimens showed a 3-fold increase in MMP-1 expression. Col III increased 5.4-, 2.6-, and 2.4-fold in the complete transection, transected AM, and intact PL groups, respectively (P = .003, P = .004, P = .04). CONCLUSION: Partial or complete surgical transection of the rabbit ACL with resultant loss of mechanical stimuli results in an increase in MMP-13 and alpha-SMA expression at the early time point (1 week) and an increase in alpha-SMA, Col I, and Col III expression at the later time point (6 weeks). These data provide support for the hypothesis that there is a time-dependent alteration of anabolic and catabolic matrix gene expression after injury/loss of ligament integrity. Clinical Relevance Identification of pathways that respond to mechanical stress in the intact ACL and after surgical transection may permit development of novel therapies to alter healing of the partial ACL injury or to assist in the development of biomechanical active ''smart'' scaffolds for tissue-engineered ligament replacements.