Medial Collateral Ligament Fibroblasts Research Articles

Influences of Tumor Necrosis Factor-α on Lysyl Oxidases and Matrix Metalloproteinases of Injured Anterior Cruciate Ligament and Medial Collateral Ligament Fibroblasts.

The anterior cruciate ligament (ACL) fails to heal after injury, even after a primary surgical attempt. In contrast, the medial collateral ligament (MCL) can heal relatively well and restore the full joint function. The difference in intrinsic properties of these ligament cells can be due to their different responses to their local factors. TNF-α is considered to be an important chemical mediator in the wound healing of the ligaments. However, TNF-α-induced expression of lysyl oxidases (LOXs) and matrix metalloproteinases (MMPs) after injury is poorly understood. In this study, we use equi-biaxial stretch chamber to realize 12% stretch, which could mimic the injury to the ACL and MCL fibroblasts in vitro, and aim to determine the intrinsic differences between injured ACL and MCL by characterizing the differential expressions of LOXs and MMPs in response to TNF-α. The methods included Semiquantitative PCR, quantitative real-time PCR, Western blot, and zymography. We found that the mRNAs of LOXs had temporal increases in injured ACL and MCL. Moreover, the increases were higher in injured MCL than those in injured ACL (up to 1.77 ± 0.13-fold in LOX, 1.73 ± 0.21-fold in LOXL-1, 2.23 ± 0.27-fold in LOXL-2, 1.95 ± 0.11-fold in LOXL-3, 1.97 ± 0.28-fold in LOXL-4). On the other hand, the expressions of MMPs in injured ACL were much more prominent than those in injured MCL fibroblasts (up to 2.63 ± 0.20-fold in MMP-1, 3.73 ± 0.18-fold MMP-2, 1.58 ± 0.11-fold MMP-3, 4.23 ± 0.31-fold MMP-12). Similar results were observed at the protein level. The differential expression of LOXs and MMPs between the injured ACL and MCL fibroblasts in this study may help explain the healing abilities of the two different ligaments.

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

BackgroundThe 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. MethodsSemi-quantitative PCR, quantitative real-time PCR and western blot were performed for original research. ResultsThe 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. 1–20ng/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. ConclusionsBased 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. Clinical relevance.

TGF-beta1 induces the different expressions of lysyl oxidases and matrix metalloproteinases in anterior cruciate ligament and medial collateral ligament fibroblasts after mechanical injury

The anterior cruciate ligament (ACL) is known to have a poor self-healing ability. In contrast, the medial collateral ligament (MCL) can heal relatively well and restore the joint function. Transforming growth factor-beta1 (TGF-β1) is considered to be an important chemical mediator in the wound healing of the ligaments. While the role of TGF-β1-induced expressions of the lysyl oxidases (LOXs) and matrix metalloproteinases (MMPs), which respectively facilitate the extracellular matrix (ECM) repair and degradation, is poorly understood. In this study, we used equibiaxial stretch chamber to mimic mechanical injury of ACL and MCL fibroblasts, and aimed to determine the intrinsic differences between ACL and MCL by characterizing the differential expressions of LOXs and MMPs in response to TGF-β1 after mechanical injury. By using semi-quantitative PCR, quantitative real-time PCR, western blot and zymography, we found TGF-β1 induced injured MCL to express more LOXs than injured ACL (up to 1.85-fold in LOX, 2.21-fold in LOXL-1, 1.71-fold in LOXL-2, 2.52-fold in LOXL-3 and 3.32-fold in LOXL-4). Meanwhile, TGF-β1 induced injured ACL to express more MMPs than injured MCL fibroblasts (up to 2.33-fold in MMP-1, 2.45-fold in MMP-2, 1.89-fold in MMP-3 and 1.50-fold in MMP-12). The further protein results were coincident with the gene expressions above. The different expressions of LOXs and MMPs inferred the intrinsic differences between ACL and MCL, and the intrinsic differences could help to explain their differential healing abilities.

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.

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-β1, 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-β1, 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 β-galactosidase (SA-β-gal) staining. Using a recombinant TGF-β1 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-β1. Taken together, our results propose that cellular senescence and turnover of extracellular matrixes regulated by TGF-β1 in MCL fibroblasts are critical for ligament healing.

Interleukin-1 beta influences on lysyl oxidases and matrix metalloproteinases profile of injured anterior cruciate ligament and medial collateral ligament fibroblasts

The anterior cruciate ligament (ACL) is known to have a poor healing ability, especially in comparison with the medial collateral ligament (MCL) which can heal relatively well. Interleukin-1beta (IL-1β) is considered to be an important chemical mediator in the acute inflammatory phase of ligament injury. The role of IL-1β-induced expressions of lysyl oxidases (LOXs) and matrix metalloproteinases (MMPs), which respectively facilitate extracellular matrix (ECM) repair and degradation, is poorly understood. In this study, we aim to determine the intrinsic differences between ACL and MCL by characterising the differential expressions of LOXs and MMPs in response to IL-1β in the injury process. Semi-quantitative polymerase chain reaction (PCR), quantitative real-time PCR, Western blot, and zymography were performed. We detected high expressions of IL-1β-induced LOXs in normal ACL and MCL. Then, we found IL-1β induced injured MCL to express more LOXs than injured ACL (up to 2.85-fold in LOX, 2.58-fold in LOXL-1, 1.89-fold in LOXL-2, 2.46-fold in LOXL-3 and 2.18-fold in LOXL-4). Meanwhile, we found IL-1β induced injured ACL to express more MMPs than injured MCL (up to 1.72-fold in MMP-1, 1.95-fold in MMP-2, 2.05-fold in MMP-3 and 2.3-fold in MMP-12). The further protein results coincided with gene expressions above. Lower expressions of LOXs and higher expressions of MMPs might help to explain the poor healing ability of ACL.

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.

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

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-β(1) (TGF-β(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-β(1). This study used reversed transcript PCR, real time quantitative PCR and Western blot for analyses. The results showed significant increases in mRNA levels of LOX family members. At 5ng/ml concentration of TGF-β(1,) the gene profiles of LOXs showed most active, and LOX and LOXL-3 showed increasing peaks at 12hours after TGF-β(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 sixhours (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-β(1) compared with those in MCL cells. 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 α2‐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.

Differential expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in anterior cruciate ligament and medial collateral ligament fibroblasts after a mechanical injury: Involvement of the p65 subunit of NF‐κB

The anterior cruciate ligament (ACL) is known to have a poor healing ability, especially in comparison with the medial collateral ligament, which can heal relatively well. In this study, we detected significant increases in the mRNA levels of multiple matrix metalloproteinases (MMPs) (MMP-1, -2, -7, -9, -11, -14, -17, -21, -23A, -24, -25, -27, and -28) and tissue inhibitors of metalloproteinases (TIMPs) (TIMP-1, -2, -3, and -4) in ACL fibroblasts after an in vitro injury with an equi-biaxial stretch chamber. However, only some MMPs (MMP-7, -9, -14, -21, and -24) showed increases in injured medial collateral ligament fibroblasts, and to a much lesser degree than that observed in the injured ACL fibroblasts. Zymography revealed a 6.3-fold increase of MMP-2 activity in injured ACL but not medial collateral ligament fibroblasts, which agrees with the global MMP activities assay. Bay-11 and curcumin can significantly decrease MMP-2 activities to 13% and 29% in injured ACL fibroblasts, respectively, which implies the involvement of p65 subunits of nuclear factor kappaB and AP-1 pathways. Furthermore, Bay-11 can decrease the global MMP activity released from injured ACL fibroblasts in a dose-dependent manner. In summary, the differential expression and activities of MMPs might help to explain the poor healing ability of ACL, and the p65 subunit of nuclear factor kappaB might be a potential target to facilitate the ACL repair.

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.

Omega-3 Fatty Acids Enhance Ligament Fibroblast Collagen Formation in Association with Changes in Interleukin-6 Production

Abstract. Altering dietary ratios of n-3 and n-6 polyunsaturated fatty acids (PUFA) represents an effective nonpharmaceutical means to improve systemic inflammatory conditions. An effect of PUFA on cartilage and bone formation has been demonstrated, and the purpose of this study was to determine the potential of PUFA modulation to improve ligament healing. The effects of n-3 and n-6 PUFA on the in vitro healing response of medial collateral ligament (MCL) fibroblasts were investigated by studying the cellular coverage of an in vitro wound and the production of collagen, PGE2, IL-1, IL-6, and TNF. Cells were exposed to a bovine serum albumin (BSA) control or either eicosapentaenoic acid (EPA, 20:5n-3) or arachidonic acid (AA, 20:4n-6) in the form of soaps loaded onto BSA for 4 days and wounded on Day 5. AA and EPA improved the healing of an in vitro wound over 72 hr. EPA increased collagen synthesis and the overall percentage of collagen produced, but AA reduced collagen production and total protein. PGE2 production was increased in the AA-treated group and decreased in the EPA-treated group, but was not affected by wounding. IL-1 was not produced at the time point evaluated, but TNF and IL-6 were both produced, and their levels varied relative to the PUFA or wounding treatment. There was a significant linear correlation (r2 = 0.57, P = 0.0045) between IL-6 level and collagen production. These results demonstrate that n-3 PUFA (represented by EPA in this study) positively affect the healing characteristics of MCL cells and therefore may represent a possible noninvasive treatment to improve ligament healing. Additionally, these results show that MCL fibroblasts produce PGE2, IL-6, and TNF and that IL-6 production is related to MCL collagen synthesis.

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.

Selection of Cell Source for Ligament Tissue Engineering

Use of appropriate types of cells could potentially improve the functionality and structure of tissue engineered constructs, but little is known about the optimal cell source for ligament tissue engineering. The object of this study was to determine the optimal cell source for anterior cruciate ligament (ACL) tissue engineering. Fibroblasts isolated from anterior cruciate ligament, medial collateral ligament (MCL), as well as bone marrow mesenchymal stem cells (MSC) were compared using the following parameters: proliferation rate, collagen excretion, expression of collagen type I, II, and III, as well as alpha-smooth muscle actin. Green fluorescent protein (GFP) transfected MSCs were used to trace their fate in the knee joints. MSC, ACL, and MCL fibroblasts were all highly stained with antibodies for collagen types I and III and alpha-smooth muscle actin while negatively stained with collagen type II. Proliferation rate and collagen excretion of MSCs were higher than ACL and MCL fibroblasts (p < 0.05), and MSCs could survive for at least 6 weeks in knee joints. In summary, MSC is potentially a better cell source than ACL and MCL fibroblasts for anterior cruciate ligament tissue engineering.

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.

Cultures of Ligament Fibroblasts in Fibrin Matrix Gel

The cellular properties of anterior cruciate ligament (ACL) and medial collateral ligament (MCL) fibroblasts have been analyzed in a three-dimensional fibrin matrix gel (FMG) system. The MCL fibroblasts proliferated significantly faster than ACL fibroblasts in 10% fetal bovine serum (FBS). FMG contraction resembles soft-tissue wound contraction. Transforming growth factor- g 1 (TGF- g 1) (5 ng/ml) caused a significantly faster rate of FMG contraction than control (0.5% FBS) in both ACL and MCL fibroblasts. Unlike the cells in 10% FBS, this faster rate of FMG contraction was achieved without increasing the initial cell number. In the FMG, the MCL fibroblasts demonstrated significantly higher collagen synthesis per cell than ACL fibroblasts between the days 2 and 6 of culture. These differences in properties of the ACL and MCL fibroblasts that were observed in vitro may explain the differences in the healing potential of these ligaments in vivo.

Fate of donor bone marrow cells in medial collateral ligament after simulated autologous transplantation.

A potential strategy to enhance ligament healing by transplantation of mesenchymal stem cells (MSCs), which are demonstrated to differentiate into fibroblast-like cells in vitro, is presented. The objective of this study was to follow transplanted nucleated cells from bone marrow, which contain MSCs, in the healing medial collateral ligament (MCL) over time, and to examine their phenotype and survivability. It was hypothesized that MSCs in nucleated cells from bone marrow would differentiate into fibroblast-like cells in the healing ligament following adaptation to the environment. The transplantation model employed in this study eliminates the immune response to a donor by the recipient using a transgenic rat (donor), which does not produce foreign protein from transgenes, and its wild-type rat (recipient) in order to simulate autologous transplantation. The MCL of the wild-type rat was ruptured, where 1 x 10(6) nucleated cells of bone marrow from the transgenic rat were injected. The transgenes in transplanted nucleated cells were detected throughout the healing MCL for 28 days by in situ hybridization. At 3 days, many donor cells were evident in the injury site and fascial pocket, and some were found in the midsubstance. Morphologically, transplanted cells with elongated nuclei were found at the ruptured edge of the midsubstance and surface of the unruptured site after 3 days. At 28 days, these cells continued to survive in the healing MCL. Their shapes were similar to those of surrounding recipient MCL fibroblasts. Thus, transplanted cells might differentiate into fibroblasts. Therefore, it was demonstrated that there is a potential for nucleated cells from bone marrow to serve as a vehicle for therapeutic molecules as well as to be a source in enhancing healing of ligaments.

Time-dependent Increases in Type-III Collagen Gene Expression in Medial Collateral Ligament Fibroblasts under Cyclic Strains

Numerous studies have demonstrated the capacity of mechanical strains to modulate cell behavior through several different signaling pathways. Understanding the response of ligament fibroblasts to mechanically induced strains may provide useful knowledge for treating ligament injury and improving rehabilitation regimens. Biomechanical studies that quantify strains in the anterior cruciate and medial collateral ligaments have shown that these ligaments are subjected to 4-5% strains during normal activities and can be strained to 7.7% during external application of loads to the knee joint. The objective of this study was to characterize the expression of types I and III collagen in fibroblast monolayers of anterior cruciate and medial collateral ligaments subjected to equibiaxial strains on flexible growth surfaces (0.05 and 0.075 strains) by quantifying levels of mRNA encoding these two proteins. Both cyclic strain magnitudes were studied under a frequency of 1 Hz. The results indicated marked differences in responses to strain regimens not only between types I and III collagen mRNA expression within each cell type but also in patterns of expression between anterior cruciate and medial collateral ligament cells. Whereas anterior cruciate ligament fibroblasts responded to cyclic strains by expression of higher levels of type-I collagen message with almost no significant increases in type-III collagen, medial collateral ligament fibroblasts exhibited statistically significant increases in type-III collagen mRNA at all time points after initiation of strain with almost no significant increases in type-I collagen. Furthermore, differences in responses by fibroblasts from the two ligaments were detected between the two strain magnitudes. In particular, 0.075 strains induced a time-dependent increase in type-III collagen mRNA levels in medial collateral ligament fibroblasts whereas 0.05 strains did not. The strain-induced changes in gene expression of these two collagens may have implications for the healing processes in ligament tissue. The differences may explain, in part, the healing differential between the anterior cruciate and medial collateral ligaments in vivo.

Time-dependent increases in type-III collagen gene expression in medical collateral ligament fibroblasts under cyclic strains.

Numerous studies have demonstrated the capacity of mechanical strains to modulate cell behavior through several different signaling pathways. Understanding the response of ligament fibroblasts to mechanically induced strains may provide useful knowledge for treating ligament injury and improving rehabilitation regimens. Biomechanical studies that quantify strains in the anterior cruciate and medial collateral ligaments have shown that these ligaments are subjected to 4-5% strains during normal activities and can be strained to 7.7% during external application of loads to the knee joint. The objective of this study was to characterize the expression of types I and III collagen in fibroblast monolayers of anterior cruciate and medial collateral ligaments subjected to equibiaxial strains on flexible growth surfaces (0.05 and 0.075 strains) by quantifying levels of mRNA encoding these two proteins. Both cyclic strain magnitudes were studied under a frequency of 1 Hz. The results indicated marked differences in responses to strain regimens not only between types I and III collagen mRNA expression within each cell type but also in patterns of expression between anterior cruciate and medial collateral ligament cells. Whereas anterior cruciate ligament fibroblasts responded to cyclic strains by expression of higher levels of type-I collagen message with almost no significant increases in type-III collagen, medial collateral ligament fibroblasts exhibited statistically significant increases in type-III collagen mRNA at all time points after initiation of strain with almost no significant increases in type-I collagen. Furthermore, differences in responses by fibroblasts from the two ligaments were detected between the two strain magnitudes. In particular, 0.075 strains induced a time-dependent increase in type-III collagen mRNA levels in medial collateral ligament fibroblasts whereas 0.05 strains did not. The strain-induced changes in gene expression of these two collagens may have implications for the healing processes in ligament tissue. The differences may explain, in part, the healing differential between the anterior cruciate and medial collateral ligaments in vivo.