The protective effect of hesperetin in osteoarthritis: an in vitro and in vivo study.

Osteoarthritis (OA), a progressive joint disorder, is principally characterized by the degeneration and destruction of articular cartilage. Previous research studies demonstrated that inflammation and ECM degradation play a major role in OA development. Hesperetin, the aglycone of neohesperidin found in the peel of Citrus aurantium L. (Rutaceae), demonstrated in several studies potential anti-inflammatory activity in a variety of diseases. However, the mechanisms by which hesperetin plays a protective role in osteoarthritis (OA) are not completely understood. In this study, we found the anti-inflammatory effects of hesperetin in the progression of OA in both in vitro and in vivo experiments. In vitro, IL-1ß-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nitric oxide (NO), tumor necrosis factor alpha (TNF-α), prostaglandin E2 (PGE2), and interleukin-6 (IL-6) were inhibited by hesperetin. Moreover, hesperetin down-regulated the IL-1ß-stimulated matrix metalloproteinase-13 (MMP-13) and thrombospondin motifs 5 (ADAMTS-5) while up-regulating collagen type II and aggrecan. Mechanistically, we revealed that hesperetin suppressed nuclear factor kappa B (NF-κB) signaling by activating the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in IL-1ß-induced chondrocytes. Hesperetin-induced repression of OA development is shown using a DMM model. Taken together, our findings suggest that hesperetin may be a novel potential therapeutic agent for repressing the development of OA.

Distinctive collagen maturation process in fibroblasts derived from rabbit anterior cruciate ligament, medial collateral ligament, and patellar tendon in vitro.

PURPOSE: Differences in the tissue-specific collagen maturation process between tendon and ligament are still unknown. Collagen cross-link formation is crucial for the collagen maturation process. The aim of this study is to examine collagen maturation processes of anterior cruciate ligament (ACL), medial collateral ligament (MCL), and patellar tendon (PT) in vitro, in order to determine the optimal cell source for tissue engineering of ligament. METHODS: Cells derived from the ACL, MCL, and PT of New Zealand white rabbits were isolated. Each cell type was cultured for up to 4 weeks after reaching confluence. Cell-matrix layers were evaluated and compared for their morphology, collagen cross-links, and gene expression levels of lysine hydroxylase 1 and 2, lysyl oxidase (LOX), tenomodulin, collagen1A1 (Col1A1), and collagen3A1 (Col3A1). RESULTS: Transmission electron microscopy photomicrographs verified that collagen fibrils were secreted from all three types of fibroblasts. A higher ratio of dihydroxylysinonorleucine/hydroxylysinonorleucine was evident in the ligament compared to the tendon, which was consistent with lysine hydroxylase 2/lysine hydroxylase 1 gene expression. The gene expression of LOX, which regulates the total amount of enzymatic cross-linking, and the gene expression levels of Col1A1 and Col3A1 were higher in the ACL matrix than in the MCL and PT matrices. CONCLUSION: ACL, MCL, and PT cells have distinct collagen maturation processes at the cellular level. In addition, the collagen maturation of ACL cells is not necessarily inferior to that of MCL and PT cells in that all three cell types have a good ability to synthesize collagen and induce collagen maturation. This bioactivity of ACL cells in terms of ligament-specific mature collagen induction can be applied to tissue-engineered ACL reconstruction or remnant preserving procedure with ACL reconstruction.

TNF-α induced down-regulation of lysyl oxidase family in anterior cruciate ligament and medial collateral ligament fibroblasts.

BACKGROUND: The lysyl oxidase (LOX) family has the capacity to catalyze the cross-linking of collagen and elastin, implicating its important fundamental role in injury healing. Tumor necrosis factor alpha (TNF-α) is considered to be an important chemical mediator in the acute inflammatory phase of the ligament injury. The role of the lysyl oxidase family induced by TNF-α in the knee ligaments' wound healing process is poorly understood. Our purpose was to determine the different expressions of the LOXs in poorly self-healing anterior cruciate ligament (ACL) and well functionally self-healing medial collateral ligament (MCL) induced by TNF-α. METHODS: Semi-quantitative PCR, quantitative real-time PCR and western blot were performed for original research. RESULTS: The results showed that all LOX family members were expressed at higher levels in MCL than those in ACL fibroblasts; the significant differences existed in the down-regulations of the LOXs induced by TNF-α; and the TNF-α-mediated down-regulations of the LOXs were more prominent in ACL than those in MCL fibroblasts. 1-20 ng/ml TNF-α down-regulated mRNA levels in ACL and MCL fibroblasts by up to 76% and 58% in LOX; 90% and 45% in LOXL-1; 97.5% and 90% in LOXL-2; 89% and 68% in LOXL-3; 52% and 25% in LOXL-4, respectively. Protein assay also showed LOXs had lower expressions in ACL than those in MCL. CLINICAL RELEVANCE: Based on these results, the differential expressions of the LOXs might help to explain the intrinsic differences between the poorly self-healing ACL and well functionally self-healing MCL.

Enhanced medial collateral ligament healing using mesenchymal stem cells: dosage effects on cellular response and cytokine profile.

Mesenchymal stem cells (MSCs) have potential therapeutic applications for musculoskeletal injuries due to their ability to differentiate into several tissue cell types and modulate immune and inflammatory responses. These immune-modulatory properties were examined in vivo during early stage rat medial collateral ligament healing. Two different cell doses (low dose 1 × 10(6) or high dose 4 × 10(6) MSCs) were administered at the time of injury and compared with normal ligament healing at days 5 and 14 post-injury. At both times, the high dose MSC group demonstrated a significant decrease in M2 macrophages compared to controls. At day 14, fewer M1 macrophages were detected in the low dose group compared to the high dose group. These results, along with significant changes in procollagen I, proliferating cells, and endothelialization suggest that MSCs can alter the cellular response during healing in a dose-dependent manner. The higher dose ligaments also had increased expression of several pro-inflammatory cytokines at day 5 (IL-1ß, IFNγ, IL-2) and increased expression of IL-12 at day 14. Mechanical testing at day 14 revealed increased failure strength and stiffness in low dose ligaments compared to controls. Based on these improved mechanical properties, MSCs enhanced functional healing when applied at a lower dose. Different doses of MSCs uniquely affected the cellular response and cytokine expression in healing ligaments. Interestingly, the lower dose of cells proved to be most effective in improving functional properties.

Cellular senescence occurring in the rabbit medial collateral ligament during healing.

Medial collateral ligament (MCL) healing proceeds in a temporally ordered fashion after injury. Despite the critical roles of fibroblasts during ligament repair, the phenotypic features of these healing fibroblasts have not been well characterized. Here, we show that healing MCL fibroblasts obtained from rabbits at 3-week postinjury exhibited higher rates of senescent phenotypes and produced higher levels of TGF-ß1, collagens, α-SMA, and matrix metalloproteinases (MMPs), than the corresponding fibroblasts from sham-operated MCLs. Mechanical stretch further enhanced the cellular senescence and the expression of TGF-ß1, collagens, α-SMA, and MMPs in both sham and healing MCL fibroblasts. In addition to MCL fibroblasts at 3-week postinjury, the increased cellular senescence was also detected in healing MCL fibroblasts obtained at 4- and 6-week postinjury. Most importantly, the association between the cellular senescence and ligament healing was confirmed in tissue sections by the senescence-associated ß-galactosidase (SA-ß-gal) staining. Using a recombinant TGF-ß1 and a neutralizing antibody, we found that those phenotypic changes, such as cellular senescence and the expression of collagens and MMPs, in MCL fibroblasts under mechanical loading conditions were regulated through TGF-ß1. Taken together, our results propose that cellular senescence and turnover of extracellular matrixes regulated by TGF-ß1 in MCL fibroblasts are critical for ligament healing.

Differential expressions of lysyl oxidase family in ACL and MCL fibroblasts after mechanical injury.

Lysyl oxidase (LOX) family has the capacity to catalyse the cross-linking of collagen and elastin, implicating its important fundamental roles in tissue development and injury healing. However, the variations in expression of the LOX family in the normal and injured anterior cruciate ligament (ACL) are not fully known. To better understand the role of LOX family in the self-healing inability mechanism of injured ACL, this study is to measure the LOX family's differential expressions in ACL and medial collateral ligament (MCL) fibroblasts after mechanical injury induced by using an equi-biaxial stretching chamber. The cells received various degrees of mechanical stretch 0% (resting state), 6% (physiological state) and 12% (injurious state), respectively. The gene profile and protein expressions were analysed by semi-quantitative PCR, quantitative real-time PCR and Western blotting. At physiological state, gene expression showed LOX in ACL was 2.6-5.2 folds higher than that in MCL in all culture time periods, LOXL-4 1.2-3.6 folds, but LOXL-3 in MCL showed 1.1-4.8 folds higher than that in ACL. In injurious state, MCL gene expressions were 2.8-29.6 folds higher than ACL in LOX, LOXL-2, LOXL-3 and LOXL-4 at 2, 6 and 12h periods. These differential expression profiles of the LOX family in the two ligament tissues were further used to explain the intrinsic differences between ACL and MCL, and why injured ACL could not be amenable to repair itself, whereas MCL could.

Differential response to CoCl2-stimulated hypoxia on HIF-1α, VEGF, and MMP-2 expression in ligament cells.

The adult human anterior cruciate ligament (ACL) has a poor functional healing response, whereas the medial collateral ligament (MCL) does not. The difference in intrinsic properties of these ligament cells can be due to their different response to their located microenvironment. Hypoxia is a key environmental regulator after ligament injury. In this study, we investigated the differential response of ACL and MCL fibroblasts to hypoxia on hypoxia-inducible factor-1α, vascular endothelial growth factor, and matrix metalloproteinase-2 (MMP-2) expression. Our results show that ACL cells responded to hypoxia by up-regulating the HIF-1α expression significantly as compared to MCL cells. We also observed that in MCL fibroblasts response to hypoxia resulted in increase in expression of VEGF as compared to ACL fibroblasts. After hypoxia treatment, mRNA and protein levels of MMP-2 increased in both ACL and MCL. Furthermore we found in ACL pro-MMP-2 was converted more into active form. However, hypoxia decreased the percentage of wound closure for both ligament cells and had a greater effect on ACL fibroblasts. These results demonstrate that ACL and MCL fibroblasts respond differently under the hypoxic conditions suggesting that these differences in intrinsic properties may contribute to their different healing responses and abilities.

Role of angiogenesis after muscle derived stem cell transplantation in injured medial collateral ligament.

We performed this study to investigate the therapeutic role of vascular endothelial growth factor (VEGF) in medial collateral ligament (MCL) healing. Murine muscle derived stem cells (MDSCs) obtained via the preplate technique were retrovirally transduced to express: (1) VEGF and nLacZ (MDSC-VEGF), (2) soluble fms-like tyrosine kinase-1 (sFLT1, a VEGF-specific antagonist) and nLacZ (MDSC-sFLT1), and (3) nLacZ (MDSC-nLacZ). After transecting the MCL of immunodeficient rats, 5 × 10(5) cells of each of the transduction groups list above were transplanted into the MCL injury site. A control group was injected with phosphate-buffered saline (PBS) only. Immunohistochemical staining demonstrated that there were more Isolectin B4 and ß-galactosidase double positive cells in the rats transplanted with MDSC-VEGF transduced cells than the other groups at week 1. Capillary density was significantly higher in the MDSC-VEGF group than the other groups at week 2; however, there were no significant differences in the biomechanical assessment between the MDSC-VEGF and MDSC-nLacZ groups. On the other hand, the MDSC-sFLT1 group revealed a lower capillary density than the other two groups and the functional ligament healing of the MDSC-sFLT1 group was significantly decreased compared to the other groups when assessed biomechanically. The findings of the present study suggest that angiogenesis plays a critical role in the healing process of injured MCL.

Up-regulation expressions of lysyl oxidase family in Anterior Cruciate Ligament and Medial Collateral Ligament fibroblasts induced by Transforming Growth Factor-Beta 1.

PURPOSE: The lysyl oxidase (LOX) family plays a crucial role in the formation and stabilisation of extracellular matrix (ECM) by catalysing the cross-linking of collagen and elastin, implicating its important fundamental roles in injury healing. A high level of transforming growth factor-ß(1) (TGF-ß(1)) accompanies the inflammatory phase of an injury of the knee joint. Our purpose was to detect the expressions of the LOX family in anterior cruciate ligament (ACL) and medial collateral ligament (MCL) response to TGF-ß(1). METHODS: This study used reversed transcript PCR, real time quantitative PCR and Western blot for analyses. RESULTS: The results showed significant increases in mRNA levels of LOX family members. At 5 ng/ml concentration of TGF-ß(1,) the gene profiles of LOXs showed most active, and LOX and LOXL-3 showed increasing peaks at 12 hours after TGF-ß(1) treatment (LOX: 7.2, 8.8-fold and LOXL-3: 3.8, 5.4-fold compared with normal controls in ACL and MCL, respectively); LOXL-1, LOXL-2 and LOXL-4 reached their highest amounts at six hours (LOXL-1: 1.9, 2.4-fold; LOXL-2: 14.8, 16.2-fold; LOXL-4: 2.5, 4.4-fold in ACL and MCL, respectively). Protein assays revealed that LOXs in ACL cells had relatively lower response to TGF-ß(1) compared with those in MCL cells. CONCLUSIONS: The differential expression and activities of LOXs might help to explain the intrinsic difference between ACL and MCL, and LOXs could imply a potential capability for ACL healing.

Combined use of bFGF and GDF-5 enhances the healing of medial collateral ligament injury.

Basic fibroblast growth factor (bFGF) and growth and differentiation factor (GDF)-5 stimulate the healing of medial collateral ligament (MCL) injury. However, the effect of isolated and combined use of bFGF/GDF-5 remains still unclear. We investigated cellular proliferation and migration responding to bFGF/GDF-5 using rabbit MCL fibroblasts. Rabbit MCL injury was treated by bFGF and/or GDF-5 with peptide hydrogels. Gene expression and deposition of collagens in healing tissues were evaluated. bFGF/GDF-5 treatment additively enhanced cell proliferation and migration. bFGF/GDF-5 hydrogels stimulated Col1a1 expression without increasing Col3a1 expression. Combined use of bFGF/GDF-5 stimulated type I collagen deposition and the reorganization of fiber alignment, and induced better morphology of fibroblasts in healing MCLs. Our study indicates that combined use of bFGF/GDF-5 might enhance MCL healing by increasing proliferation and migration of MCL fibroblasts, and by regulating collagen synthesis and connective fiber alignment.

GDF-5/7 and bFGF activate integrin alpha2-mediated cellular migration in rabbit ligament fibroblasts.

Cellular activities responding to growth factors are important in ligament healing. The anterior cruciate ligament (ACL) has poor healing potential compared to the medial collateral ligament (MCL). To assess the differences, we investigated the proliferation, migration, adhesion, and matrix synthesis responding to growth factors in rabbit ACL and MCL fibroblasts. ACL cell proliferation to basic fibroblast growth factor (bFGF), bone morphogenetic protein-2, growth and differentiation factor (GDF)-5, and GDF-7 treatment was similar to that of MCL cells. GDF-5 enhanced Col1a1 expression in ACL and MCL fibroblasts up to 4.7- and 17-fold levels of control, respectively. MCL fibroblasts showed stronger migration activities in response to bFGF and GDF-5 than ACL cells. GDF-5/7 and bFGF also changed the stress fiber formation and cellular adhesion by modulating the distribution of integrin alpha2. Functional blocking analyses using anti-integrin alpha2 antibodies revealed that cellular migration responding to growth factors depended on the integrin alpha2-mediated adhesion on type I collagen. The expression of integrin alpha2 was also increased by growth factors in both cells. Our results demonstrate that GDF-5/7 and bFGF stimulate cellular migration by modulating integrin alpha2 expression and integrin alpha2-dependent adhesion, especially in MCL fibroblasts. These findings suggest that the different healing potential between ACL and MCL may be caused by different cellular behavior in the integrin alpha2-mediated cellular migration in response to growth factors.

Anterior cruciate ligament-derived cells have high chondrogenic potential.

Anterior cruciate ligament (ACL)-derived cells have a character different from medial collateral ligament (MCL)-derived cells. However, the critical difference between ACL and MCL is still unclear in their healing potential and cellular response. The objective of this study was to investigate the mesenchymal differentiation property of each ligament-derived cell. Both ligament-derived cells differentiated into adipogenic, osteogenic, and chondrogenic lineages. In chondrogenesis, ACL-derived cells had the higher chondrogenic property than MCL-derived cells. The chondrogenic marker genes, Sox9 and alpha1(II) collagen (Col2a1), were induced faster in ACL-derived pellets than in MCL-derived pellets. Sox9 expression preceded the increase of Col2a1 in both pellet-cultured cells. However, the expression level of Sox9 and a ligament/tendon transcription factor Scleraxis did not parallel the increase of Col2a1 expression along with chondrogenic induction. The present study demonstrates that the balance between Sox9 and Scleraxis have an important role in the chondrogenic differentiation of ligament-derived cells.

Expression of MMPs and TIMPs family in human ACL and MCL fibroblasts.

The human ACL (anterior cruciate ligament) is susceptible to injury but has poor healing response, whereas an injured MCL (medial collateral ligament) can be repaired relatively well. Since MMPs (matrix metalloproteases) and TIMPs (tissue inhibitor of metalloproteases) are involved in this tissue remodeling process, investigation of different response of MMPs and TIMPs family in ACL and MCL fibroblasts might lead to understanding the differential matrix remodeling process as well as their different healing ability. The first step would be determination of whether these tissue remodeling effectors are present in ligaments. In this study, we designed primers for real-time RT-PCR and determined the expression of MMPs and TIMPs family in ACL and MCL fibroblasts with synovium as a positive control. Semiquantitative RT-PCR revealed that multiple MMPs and TIMPs expressed in human ACL and MCL fibroblasts except MMP-8, 10, 12, 13, 15, 16, 20, and 26. MMP-7 was present in MCL but not in ACL fibroblast. Quantitative real-time RT-PCR showed that mRNA levels of MMP-1, 2, 14, 17, 23A, and 23B and TIMP-4 are significantly higher in MCL than in ACL fibroblasts. However, MMP-3 is higher in ACL than in MCL fibroblasts. We conclude that numerous MMPs and TIMPs family members that are differentially expressed in ACL and MCL might be involved in the differential matrix remodeling process as well as the differential healing ability of ACL and MCL.

Effects of hyperbaric oxygen and platelet derived growth factor on medial collateral ligament fibroblasts.

PURPOSE: This study investigated hyperbaric oxygen (HBO2) and platelet-derived growth factor-BB (PDGF-BB) to determine their combined effects on fibroblasts from rabbit medial collateral ligament (MCL). METHOD: Cells were divided into four groups: (I) Control, (II) HBO2 treatment, (III) PDGF-BB treatment and (IV) HBO2 combined with PDGF-BB treatment. All hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber for 120 minutes per 48 hours. Measurement of cell growth was based on increase in cell number. Cell cycle modulations were analyzed by fluorescence-activated cell sorter (FACS). Quantity of Type I and Type III collagen was determined by western blotting and image analyzer. RESULTS: Treatment doses of HBO2 alone or PDGF-bb alone dependently increased cell growth. A combination of HBO2 treatment plus PDGF-bb treatment had an additive effect on cell growth in comparison with HBO2 treatment alone or PDGF-bb treatment alone. FACS analysis revealed that HBO2 alone, PDGF-bb alone and PDGF-bb plus HBO2 treatment increase the percentage of cells accumulated in S-phase. Western blotting analysis revealed that Type III collagen content was decreased significantly after HBO2 treatment alone or HBO2 plus PDGF-bb treatment but not in PDGF-bb treatment alone. In contrast, although Type I collagen content was increased after HBO2 treatment, the increase in Type I collagen (increase /original) was not statistically significant. CONCLUSION: HBO2 or HBO2 plus PDGF-bb treatment decreases the Type III collagen/Type I collagen content, which could result in mechanically stronger collagen fibrils. We propose HBO2 therapy as a potentially effective treatment for MCL healing.

Internal fluid flow increases cellular interconnects between Medial Collateral Ligament fibroblasts and cellular extensions within three-dimensional collagen matrixes.

The interconnectivity of fibroblasts within the ligamentous extracellular matrix has been largely overlooked. Studies on the cell-to-cell contacts with their neighbors via gap junctions in ligament fibroblasts, and works on the ability of fibroblasts to generate interconnected networks in vivo, suggest interfibroblastic interactions play an important role in fundamental biological processes, including homeostasis and wound healing. The current study examines how fluidic shear stresses imposed by internal flow can be used to mediate the formation of three-dimensional, interconnected fibroblast networks within collagen solutions. Several fibroblast-collagen solutions were exposed to shear stresses via Poiselle Flow. The consequent changes in cell networking, interconnections, and cell morphology within collagen matrixes exhibited by cells derived from Bovine Medial Collateral Ligaments were analyzed. Results illustrate that higher imposed stresses generate cells with more dendritic and/or branched morphologies, which form more visible three-dimensional networks within collagen matrixes than fibroblast-collagen solutions that were unexposed to shear stress.

Upregulated expression of inducible nitric oxide synthase plays a key role in early apoptosis after anterior cruciate ligament injury.

The reason that the anterior cruciate ligament (ACL) has a very poor healing potential after injury is not well understood. In this study, we investigated the role of nitric oxide (NO) in the apoptotic cell death of ACL cells using a rabbit model and in vitro cell culture. The apoptosis of ACL cells in vivo was analyzed by TUNEL assay and electron microscopy. NO synthase (NOS) expression was observed by immunohistochemical analysis. ACL cells were cultured and the susceptibility to NO-induced apoptosis was tested. Inducible NOS (iNOS) expression after treatment with cytokines was examined by immunohistochemical and RT-PCR analyses. Mitogen-activated protein kinase (MAPK) inhibitors were used for the analysis of downstream signals. A significant number of apoptotic cells were observed on days 1 to 3 after injury; the apoptotic rate returned to the control level by day 7. Upregulation of iNOS in the ACL remnant was observed at day 1. Intraarticular injection of NOS inhibitor suppressed the apoptotic rate. Isolated ACL cells showed much higher susceptibility to NO-induced apoptosis than did medial collateral ligament cells. IL-1beta stimulated ACL cells to upregulate iNOS mRNA and increase NO production. p38 MAPK inhibitor decreased NO-induced apoptosis. Rapid iNOS induction after injury contributes to the high apoptotic rate of ACL cells, and this may partly account for the poor healing capacity of this ligament. iNOS and NO production is suggested to be stimulated by IL-1beta, and NO activates the p38 MAPK pathway and triggers an apoptotic signal in ACL cells.

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.

Permeability of human medial collateral ligament in compression transverse to the collagen fiber direction.

This study quantified the apparent and intrinsic hydraulic permeability of human medial collateral ligament (MCL) under direct permeation transverse to the collagen fiber direction. A custom permeation device was built to apply flow across cylindrical samples of ligament while monitoring the resulting pressure gradient. MCLs from 5 unpaired human knees were used (donor age 55 +/- 16 yr, 4 males, 1 female). Permeability measurements were performed at 3 levels of compressive pre-strain (10%, 20% and 30%) and 5 pressures (0.17, 0.34, 1.03, 1.72 and 2.76 MPa). Apparent permeability was determined from Darcy's law, while intrinsic permeability was determined from the zero-pressure crossing of the pressure-permeability curves at each compressive pre-strain. Resulting data were fit to a finite deformation constitutive law [Journal of Biomechanics 23 (1990) 1145-1156]. The apparent permeability of human MCL ranged from 0.40 +/- 0.05 to 8.60 +/- 0.77 x 10(-16) m(4)/Ns depending on pre-strain and pressure gradient. There was a significant decrease in apparent permeability with increasing compressive pre-strain (p=0.024) and pressure gradient (p<0.001), and there was a significant interaction between the effects of compressive pre-strain and pressure (p<0.001). Intrinsic permeability was 14.14 +/- 0.74, 6.30 +/- 2.13 and 4.29 +/- 1.71 x 10(-16) m(4)/Ns for compressive pre-strains of 10%, 20% and 30%, respectively. The intrinsic permeability showed a faster decrease with increasing compressive pre-strain than that of bovine articular cartilage. These data provide a baseline for investigating the effects of disease and chemical modification on the permeability of ligament and the data should also be useful for modeling the poroelastic material behavior of ligaments.

Differential MMP-2 activity of ligament cells under mechanical stretch injury: an in vitro study on human ACL and MCL fibroblasts.

Recent studies have revealed that following injuries, ligament tissues such as anterior cruciate ligaments (ACL), release large amounts of matrix metalloproteinases (MMPs). These enzymes have a devastating effect on the healing process of the injured ligaments. Although these enzymes are produced following ligament injuries, because of different healing capacities seen between the medial collateral ligament (MCL) and ACL, we were curious to find if the MMP activity was expressed and modulated differently in these tissues. For this purpose ACL and MCL fibroblasts were seeded on equi-biaxial stretch chambers and were stretched in different levels. The stretched cells were assayed using Zymography, Western Blot and global MMP activity assays. The results showed that within 72 h after injurious stretch, production of 72 kD pro-MMP-2 increased in both ACL and MCL. However, the ACL fibroblasts generated significantly more pro-MMP-2 than the MCL fibroblasts. Furthermore we found in ACL pro-MMP-2 was converted more into active form. With 4-aminophenyl mercuric acetate (APMA) treatment, large amounts of pro-MMP-2 were converted into active form in both. This indicates that there is no significant difference between ACL and MCL fibroblasts in post-translational modification of MMP-2. The fluorescent MMP activity assays revealed that the MMP family activities were higher in the injured ACL fibroblasts than the MCL. Since the MMPs are critically involved in extracellular matrix (ECM) turnover, these findings may explain one of the reasons why the injured ACL hardly repairs. The higher levels of active MMP-2 seen in the ACL injuries may disrupt the delicate balance of ECM remodeling process. These results suggest that the generation and modulation of MMP-2 may be directly involved in the different responses seen in ACL and MCL injuries.

[The effect of fibroblast growth factors and epidermal growth factors on the proliferation of rabbit anterior cruciate and medial collateral ligament cell in vitro].

OBJECTIVE: To investigate the effect of acid, basic fibroblast, growth factor (aFGF, bFGF) and epidermal growth factor (EGF), and their combination on the proliferation of rabbit anterior cruciate ligament (ACL) and medial collateral ligament (MCL) in vitro. METHODS: The cells of ACL and MCL were isolated and subcultured from the knee joints of ten-week-old New Zealand white rabbits. The cells were seeded into 96-well corning cluster plates. Three growth factors of different concentration alone or in combination were added into the culture medium respectively, which were 0, 1, 5, 10, 50 and 100 ng/ml for aFGF, bFGF and 0, 1.56, 3.13, 6.25, 12.5, 25 and 50 ng/ml for EGF. The proliferation of the fibroblasts was measured for 48 h with XTT method. RESULTS: All of the three growth factors alone promoted the cell proliferation of ACL and MCL fibroblasts. The concentration of aFGF had a significant effect on the proliferation of both ACL and MCL fibroblasts. The concentration of 1 ng/ml bFGF and 5 ng/ ml EGF was most effective in promoting the proliferation of ACL, and both bFGF and EGF had a significant effect on MCL. 5 ng/ml aFGF with 50 ng/ml EGF had effect on ACL. 1 ng/ml aFGF with 3.13 ng/ml EGF had effect on MCL. CONCLUSION: The three growth factors may promote the cell proliferation of ACL and MCL. These findings suggest that topical application of aFGF, either alone or in combination with EGF may have the potential to promote the proliferation of rabbit ACL and MCL,and aFGF of low concentration in combination with EGF is more effective than single growth factor.