Erik Attia

QUANTITATION OF ESTROGEN RECEPTORS AND RELAXIN BINDING IN HUMAN ANTERIOR CRUCIATE LIGAMENT FIBROBLASTS

SummaryThe 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 I125-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 I125-relaxin-labeled ACL cells. Relaxin binding was present in four out of five female ACL cells versus one out of five male ACL cells.

The Effect of Estrogen on Ovine Anterior Cruciate Ligament Fibroblasts Cell Proliferation and Collagen Synthesis

Abstract 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 17β-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 3H-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 17β-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 ofthe anterior cruciate ligament in vivo. The etiology of noncontact anterior cruciate ligament injuries is complex and multifactorial in nature, meriting further investigation.

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 (α-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, α-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 α-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 × 10− 6), 3-fold in transected AM samples (P = 3.3 × 10 −6), and 2-fold in the intact PL bundle samples as compared with controls. α-SMA was increased 7.5-fold and 5-fold in complete transection and transected AM samples, respectively (P = .004, P = 2.2 × 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 α-SMA expression at the early time point (1 week) and an increase in α-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.

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.

Cyclooxygenase-2 Inhibitor Decreases Extracellular Matrix Synthesis in Stretched Renal Fibroblasts

Background/Aims: Both TGF-β 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-β 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-β was quantified by ELISA, and protease activity determined by a colorimetric assay. Results: Bothcollagen and TGF-β synthesis were increased following a 48-hour stretch of NRK-49F. Meloxicam significantly decreased the collagen and TGF-β 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-β-dependent process.

CELL-MATERIAL SYSTEMS FOR ANTERIOR CRUCIATE LIGAMENT REGENERATION

Dear Editor: Injuries and defects of the anterior cruciate ligament (ACL) frequently result in disabilities that are permanent and disabling. Currently, autografts and allografts are the most commonly used methods to repair these injuries. The disadvantages of autogenous grafts and the increased risk associated with allografts have fueled the search for alternative repair options, including the use of synthetic ligaments (1,2,6,7). Cell-material composite systems are of particular interest as an alternative, having additional possible advantages for tissue revascularization and remodeling. Currently, various homogenous nondegradable polymers have been used as synthetic ligaments. These include the Gore-Tex prosthetic ligament made of polytetrafluorethylene, the ligament-augmentation device (LAD) composed of polypropylene, and the Stryker-Dacron ligament prosthesis, which is made of Dacron tapes wrapped in a Dacron sleeve (8). These polymers alone though are unable to duplicate the mechanical and surface properties of the complex ACL (7). Brody et al. (3) examined the effects of seeding canine fibroblast cells onto the surface of a knitted Dacron ligament prostheses prior to implantation. Results showed that prostheses seeded with fibroblast cells demonstrated a more uniform and abundant encapsulation with connective tissue than did unseeded prostheses. As an alternative to the currently used nondegradable polymers, investigators have begun to examine biodegradable materials that would provide immediate stabilization to the repaired ligament, but would also act as a scaffold for the ingrowth and/or replacement by host cells (9). The only completely degradable material currently reported under investigation for ACL repair is polyglycolic acid (PGA) (4). Our laboratory has begun to examine the possible use of the copolymer PLAGA poly(D,L-lactide-co-glycolide) in conjunction with ligament cells as an alternative synthetic material in the use of soft tissue grafts. Candidate cell types to be used for soft tissue engineering purposes must be able to attach, grow, and maintain their phenotype on their newly seeded surfaces. The purpose of this study was to characterize the ability of various soft tissue cells to adhere to nondegradable and degradable PLAGA surfaces and to examine the morphology of these various cells during the process of attachment. The degradable polymer used in these experiments was 50:50 PLAGA (Medisorb, DuPont, Wilmington, DE). PLAGA matrices (diameter 14 mm, 0.5 mm thickness) were prepared by a solvent casting technique described previously (5). Six canine fibroblast cellular types were studied: anterior cruciate ligament (ACL) cells, posterior cruciate ligament (PCL) cells, medial collateral ligament (MCL) cells, lateral collateral ligament (LCL) cells, patellar tendon (PT) cells, and Achilles tendon (AT) cells. The ligaments and tendon were harvested using aseptic techniques and placed in medium M199 (GIBCO, Life Technologies, Inc., Grand Island, NY) containing 1% antibiotic antimycotic (GIBCO) for 30 min. They were then finally minced and placed in medium M199 containing 10% fetal calf serum (FCS) and 1% antibiotic-antimycotic. Explant outgrowth cultures were created with ceils visible by 1 wk, with cells reaching confluency by 2 wk. Cells were then seeded onto PLAGA matrices and TCPS controls at a density of 1 × 10 a. Cell morphology was determined on both PLAGA discs and TCPS controls using an AMR Model 1000 environmental scanning electron microscope (ESEM). Cell adhesion kinetics were studied using all six types of fibroblast cells listed above. Each of the six types of fibroblasts were incubated with 10 Ci/ml 3H thymidine for 24 h. They were then mixed thoroughly to remove unincorporated 3H and seeded at a density of 1 ×

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.

The effect of ciprofloxacin on tendon, paratenon, and capsular fibroblast metabolism

Am J Sports Med 2000: 28: 364–369 The effect of ciprofloxacin on tendon, paratenon, and capsular fibroblast metabolism R. J. Williams III, E. Attia, T. L. Wickiewicz, J. A. Hannafin Laboratory for Soft Tissue Research, Sports Medicine & Shoulder Service, Hospital for Special Surgery, New York, New York, USA The pathologic mechanisms underlying fluoroquinolone-induced tendinopathy are poorly understood. The observed incidence of tendinitis and tendon rupture in patients treated with ciprofloxacin hydrochloride suggests that the fluoroquinolone antibiotics alter tendon fibroblast metabolism. The purpose of this study was to examine the effect of ciprofloxacin on fibroblast metabolism in vitro. Canine Achilles tendon, paratenon, and shoulder capsule specimens were maintained in culture with ciprofloxacin (5, 10, or 50 mg/ml). Fibroblast proliferation, collagen synthesis, proteoglycan synthesis, and matrix-degrading activity were analyzed. Incubation of Achilles tendon, Achilles paratenon, and shoulder capsule fibroblasts with ciprofloxacin resulted in a statistically significant 66% to 68% decrease in cell proliferation compared with control cells at day 3 in culture. Ciprofloxacin caused a statistically significant 36% to 48% decrease in collagen synthesis compared with controls in all fibroblast cultures. Ciprofloxacin caused a statistically significant 14% to 60% decrease in proteoglycan synthesis in all fibroblast cell lines. Compared with unstimulated control fibroblasts, culture media from Achilles tendon, paratenon, and shoulder capsule cells that were exposed to ciprofloxacin demonstrated statistically significant increases in matrix-degrading proteolytic activity after 72 hours in culture. This study demonstrates that ciprofloxacin stimulates matrix-degrading protease activity from fibroblasts and that it exerts an inhibitory effect on fibroblast metabolism. The increase in protease activity and the inhibition of both cell proliferation and the synthesis of matrix ground substance may contribute to the clinically described tendinopathies associated with ciprofloxacin therapy. 61 Muscle Nerve 2000: 23: 1576–1581 Effects of resistance training on neuromuscular junction morphology M. R. Deschenes, D. A. Judelson, W. J. Kraemer, V. J. Meskaitis, J. S. Volek, B. C. Nindl, F. S. Harman, D. R. Deaver Department of Kinesiology, College of William and Mary, Williamsburg, Virginia 23187-8795, USA The aim of this study was to determine the impact of resistance exercise on neuromuscular junction (NMJ) architecture. Eighteen Sprague-Dawley rats either participated in a 7-week resistance training program or served as untrained controls. Following the experimental period, the NMJs of soleus muscles were visualized with immunofluorescent techniques, and muscle fibers were stained histochemically. Results indicate that resistance training significantly (P,0.05) increased endplate perimeter length (15%) and area (16%), and significantly enhanced the dispersion of acetylcholine receptors within the endplate region. Preand post-synaptic modifications to resistance exercise were well-coupled. No significant alterations in muscle fiber size or fiber type were detected. The data presented here indicate that the stimulus of resistance training was sufficiently potent to remodel NMJ structure, and that this effect cannot be attributed to muscle fiber hypertrophy or fiber type conversion. Copyright 2000 John Wiley & Sons, Inc. J Appl Physiol 2000: 89: 1365–1379 IGF-I restores satellite cell proliferative potential in immobilized old skeletal muscle M. V. Chakravarthy, B. S. Davis, F. W. Booth Department of Integrative Biology, University of Texas Medical School, Houston, Texas 77030, USA One of the key factors responsible for the age-associated reduction in muscle mass may be that satellite cell proliferation potential (number of doublings contained within each cell) could become rate limiting to old muscle regrowth. No studies have tested whether repeated cycles of atrophy-regrowth in aged animals deplete the remaining capacity of satellite cells to replicate or what measures can be taken to prevent this from happening. We hypothesized that there would be a pronounced loss of satellite cell proliferative potential in gastrocnemius muscles of aged rats (25to 30-mo-old FBN rats) subjected to three cycles of atrophy by hindlimb immobilization (plaster casts) with intervening recovery periods. Our results indicated that there was a significant loss in gastrocnemius muscle mass and in the proliferative potential of the resident satellite cells after just one bout of immobilization. Neither the muscle mass nor the satellite cell proliferation potential recovered from their atrophied values after either the first 3-wk or later 9-wk recovery period. Remarkably, application of insulin-like growth factor I onto the atrophied gastrocnemius muscle for an additional 2 wk after this 9-wk recovery period rescued ∂46% of the lost muscle mass and dramatically increased proliferation potential of the satellite cells from this muscle.