Bilogic characteristics of fibroblast cells cultured from the knee ligaments

OBJECTIVE: To culture fibroblast cells from the knee ligaments and to study the biological characteristics of these cells. METHODS: Cells of the anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) from New Zealand white rabbit were cultured in vitro. Cellular growth and expression of the collagen were analyzed. Moreover, an in vitro wound closure model was established and the healing of the ACL and the MCL cells was compared. RESULTS: Maximal growth for all these cells were obtained with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, but RPMI 1640 and Ham's F12 media were not suitable to maintain these cells. Morphology of both ACL and MCL cells from New Zealand white rabbit was alike in vitro, but the MCL cells grew faster than the ACL cells. Both cell types produced similar amount of collagen in culture, but the ratio of collage type I to type III produced by ACL cells was higher than that produced by MCL cells. Wound closure assay showed that at 36 hours after injury, cell-free zones created in the ACL cultures were occupied partially by the ACL cells; in contrast, the wounded zone in the MCL cultures was almost completely covered by the cells. CONCLUSIONS: Although the ACL cells and the MCL cells from New Zealand white rabbit show similar appearance in morphology in culture, the cellular growth and the biochemical synthesis of collagen as well as the healing in vitro were significantly different. These differences in intrinsic properties of the two types of cells in vitro might contribute to the differential healing potentials of these ligaments in vivo.     

Intrinsic healing response of the human anterior cruciate ligament: An histological study of reattached ACL remnants

A reattachment of the tibial remnant of the torn anterior cruciate ligament (ACL) to the posterior cruciate ligament is sometimes observed during surgery and apparently implies that the human ACL does have a healing response. The aim of this study was to investigate whether this reattachment tissue has similar histological characteristics of a healing response as the medial collateral ligament (MCL), which can heal spontaneously. Standard histology and immunostaining of α‐smooth muscle actin and collagen type 3 was performed. The results shows that the reattached tissue has typical characteristics of a healing response: the reattached ACL remnant could not be released by forceful traction; microscopy showed that the collagen fibers of the reattached tissue were disorganized with no preferred direction; increased neovascularization; the presence of lipid vacuoles; the mean number of cells within the biopsy tissue was 631 ± 269 cells per mm²; and 68 ± 20% was expressing α‐SMA; semi‐quantitative analysis of collagen type 3 expression showed that collagen type 3 had an high expression with an average score of 3. In conclusion, this study shows that the human proximal 1/3 ACL has an intrinsic healing response with typical histological characteristics similar to the MCL. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:296–301, 2014.     

Characterization of the intrinsic properties of the anterior cruciate and medial collateral ligament cells: an in vitro cell culture study.

The poor healing abilities of the anterior cruciate ligament (ACL) in contrast to those of the medial collateral ligament (MCL) are well known. Different intrinsic properties of the constituent cells of these ligaments have been proposed to be one of the factors in the differential repair mechanisms. To examine this hypothesis, we have established primary cell lines of ACL and MCL from the tissue explants of approximately similar dimensions and have studied their behavior in vitro. The outgrowth of cells from ACL explants was slower than from MCL explants, as shown by the size of the surrounding clusters of cells. Both ACL and MCL cultures exhibited typical fibroblastic morphology. No significant differences were observed in either attachment or growth of cells from the attached explants derived from various segments of ACL and MCL. Growth curves of ACL and MCL cultures at both passage numbers 2 and 6 showed a slower rate of proliferation of ACL cells than MCL cells (p less than 0.005). DNA synthesis measured in terms of [3H]thymidine incorporation (CPM/10(3) cells) of both log phase (ACL = 607.5 +/- 5.4 vs. MCL = 1356.4 +/- 11.3) and confluent (ACL = 83.0 +/- 3.6 vs. MCL = 189.8 +/- 5.4) cultures, supports the conclusion that differential proliferation rates of these cells exist in culture. FITC-phalloidin staining (for actin) of later passage cultures (P3-P5) showed a spread-out appearance of ACL cells and an elongated appearance of MCL cells. Relatively more stress fibers were seen within ACL cells. SDS-PAGE and Western blot analysis of cellular proteins revealed higher actin (43 kDa) content in ACL cells than in MCL cells. In vitro wound closure assay was performed by creating a uniform wound of 0.6 mm width in the confluent layer of ACL and MCL cultures. By 48 h postwounding, cell-free zones created in ACL cultures were occupied partially by single cells in a nonconfluent fashion. In contrast, the wounded zone in the MCL cultures was almost completely covered by the cells. Results presented in this report demonstrate a lower proliferation and migration potential of ACL cells in comparison with MCL cells. These differences in intrinsic properties of ACL and MCL cells that were observed in vitro might contribute to the differential healing potentials of these ligaments in vivo.     

Differential sensitivity to NO-induced apoptosis between anterior cruciate and medial collateral ligament cells

It is well known that the anterior cruciate ligament (ACL) of the knee joint has poorer healing responses than the medial collateral ligament (MCL). Nitric oxide (NO) induces free radicals and plays a key role in the induction of apoptosis in various wound-healing models. We hypothesized that the poor healing response of the ACL may be ascribed to high susceptibility to apoptosis, and we investigated the difference in susceptibility to apoptosis between ACL and MCL cells after treatment with sodium nitroprusside, a NO donor. Apoptosis was evaluated by phase contrast microscopy, electron microscopy, DNA gel electrophoresis, and flow cytometric analysis. Although morphological changes and DNA ladders were observed in both ACL and MCL cells after 2mM sodium nitroprusside treatment, ACL cells were more prone to apoptosis at 1mM. Based on flow cytometric analysis, DNA fragmentation at 1mM sodium nitroprusside was significantly greater in ACL cells than in MCL cells (58.6% ± 1.6% vs. 11.9% ± 2.2%). Caspase-3 inhibitor (Ac-Asp-Glu-Val-Asp-CHO) and caspase-9 inhibitor (Ac-Leu-Glu-His-Asp-CHO) completely inhibited this DNA fragmentation. In conclusion, the ACL and MCL cells exhibit essential differences, and the differential sensitivity to NO-induced apoptosis between the ACL and MCL cells may be a reflection of these differences.     

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 α2. Functional blocking analyses using anti‐integrin α2 antibodies revealed that cellular migration responding to growth factors depended on the integrin α2‐mediated adhesion on type I collagen. The expression of integrin α2 was also increased by growth factors in both cells. Our results demonstrate that GDF‐5/7 and bFGF stimulate cellular migration by modulating integrin α2 expression and integrin α2‐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 α2‐mediated cellular migration in response to growth factors. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:225–231, 2010     

Correlation of healing capacity with vascular response in the anterior cruciate and medial collateral ligaments of the rabbit.

In clinical terms, functional recovery after anterior cruciate ligament (ACL) injury is generally poorer than after medial collateral ligament (MCL) injury. In experimental studies of injury, the early phases of ligament healing require an augmented blood supply. We hypothesized that the differences in healing properties of the ACL and MCL would be reflected in the magnitude of their vascular responses to partial injury. This study is the first to quantify and define the time course of changes in blood flow and vascular volume following hemisection of the rabbit ACL and MCL.Adult female rabbits were assigned to control, sham operation, ACL hemisection or MCL hemisection groups. Standardized ACL or MCL injuries were surgically induced. About 2, 6 or 16 weeks later, blood flow and vascular volume of the ACL and MCL were measured.The MCL of the rabbit responded to hemisection with a large significant increase in blood flow and a substantial angiogenic response associated with inflammation and scar formation. During subsequent matrix remodelling, blood flow and vascular volume returned towards control values. In contrast, the ACL showed only a 2-fold increase in vascular volume, no increase in blood flow and atrophied after hemisection. The superior capacity of the MCL to increase its blood supply through angiogenesis and increased flow is essential for ligament healing to occur, and may be the major difference in healing potential between the ACL and MCL.     

Vascular structure and function in the medial collateral ligament of anterior cruciate ligament transected rabbit knees

To determine if decreased vascular responsiveness in the medial collateral ligament (MCL) of anterior cruciate ligament transected (ACL‐t) rabbit knees is due to pericyte deficiency associated with angiogenesis. Vascular responses to potassium chloride (KCl), phenylephrine, acetylcholine, and sodium nitroprusside (SNP) were evaluated in ACL‐t rabbit knees (n = 6) and control knees (n = 5) using laser speckle perfusion imaging. Ligament degeneration was determined by ultrasound imaging. Vascular and pericyte volume were measured using quantitative immunohistochemical volumetric analysis using CD31 and α‐smooth muscle actin antibodies with co‐localization analysis. Perfusion was increased in the ACL‐t rabbits 2.5‐fold. Responsiveness to phenylephrine, SNP, and acetylcholine was significantly decreased in the ACL knee while no change in KCl responses was seen. MCL ultrasound imaging revealed decreased collagen organization, increased ligament thickness, and increased water content in the ACL‐t MCL. Vascular Volume was increased fourfold in ACL deficient knees, while pericyte volume to endothelial volume was not changed. No difference in CD31 and α‐SMA co‐localization was found. Blood vessels in the MCL of ACL‐t knees do not lack smooth muscle. The MCL vasculature can undergo constrictive response to KCl, but have impaired receptor mediated responses and impaired nitric oxide signaling. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1104–1110, 2014.     

Type I procollagen gene expression in normal and early healing of the medial collateral and anterior cruciate ligaments in rabbits: an in situ hybridization study

The purpose of this study was to compare the levels of procollagen type I messenger RNA (mRNA) in normal and healing medial collateral ligament (MCL) and anterior cruciate ligament (ACL) in a rabbit model. Our method of injury involved a surgical model with identical partial lacerations in the midsubstance of the MCL and ACL. Paraffin sections of normal ligaments, and ligaments 3, 7, 14, and 28 days postlaceration were studied by in situ hybridization to compare and follow the level of type I procollagen mRNA in the two ligaments. A complementary DNA (cDNA) probe corresponding to alpha 1(I) procollagen mRNA was labeled with [32P]d-CTP. After hybridization, autoradiography, and staining of the sections, the level of procollagen mRNA was assessed by microscopic examination. A higher level of procollagen mRNA was consistently detected in normal MCL than in normal ACL, suggesting higher collagen synthetic activity in the MCL. At the injury sites of the MCL and ACL, the levels of type I procollagen mRNA increased at all post-laceration periods, reaching its highest level at 14 days postsurgery. The MCL healing site had a considerably higher level of procollagen mRNA than the ACL healing site (i.e., injury site) at all postoperative intervals. The results demonstrate that procollagen mRNA levels in MCL tissue are higher than those in ACL tissue under normal conditions, as well as in response to injury. The differences in the procollagen mRNA levels of MCL and ACL may reflect the synthesis of collagen in these tissues, and may contribute to the differences in their healing capacities.     

Immediate ACL reconstruction prevents microvascular pathophysiology in the Uninjured MCL that is not fully reversed by delayed ACL reconstruction

Anterior cruciate ligament (ACL) injury induces maladaptive vascular responses that degrade medial collateral ligament (MCL) function. The purpose of this study was to determine if early or delayed ACL reconstruction can prevent or reverse the abnormal changes in vascular function that occur in the uninjured MCL after ACL injury. Twenty‐four rabbits were divided into four groups (n = 6); control, ACL‐deficient (ACL‐X), immediate ACL reconstructed (ACL‐IR) and delayed ACL reconstructed (ACL‐DR). After 8 weeks, MCLs were assessed for blood flow, responses to acetylcholine (ACh) and phenylephrine (Phe) and autoregulatory responses, using laser speckle perfusion imaging. In ACL‐X knees, blood flow in the MCL increased by 2.5‐fold compared to control. MCL hyperemia was diminished in ACL‐DR knees and was unaltered in ACL‐IR knees. MCL vasculature was unresponsive to ACh and Phe in ACL‐X. These responses were partially restored by ACL reconstruction. Autoregulatory responses were not significantly different between groups. ACL‐DR decreased hyperemia in the MCL and partially attenuated abnormal MCL vascular responses. ACL‐IR was more effective at preventing MCL hyperemia and preserving vascular responsiveness to ACh and Phe. This suggests that the vascular alterations in the uninjured rabbit MCL are largely caused by abnormal mechanical loading resulting from ACL deficiency and can be prevented through early reconstruction. Early ACL reconstruction could limit the progression of microvascular dysfunction of the MCL, and preserve physiological joint homeostasis. This might prevent joint degeneration and delay the progression of osteoarthritis. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1390–1396, 2011     

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.     

Vascular physiology and long-term healing of partial ligament tears.

Functional outcomes of anterior cruciate ligament (ACL) injury are generally poorer than those of medial collateral ligament (MCL) tears. Following ligament damage, all phases of ligament healing require an adequate blood supply. We hypothesized that the differences in healing properties of the ACL and MCL would reflect their vascular responses to joint injury. This paper examines the long-term changes in blood flow and vascular volume of rabbit knee ligaments after direct injury, and under conditions of chronic joint instability induced by section of the posterior cruciate ligament (PCL). Standardized injuries were surgically induced in adult rabbit knee ligaments: partial MCL transection, partial ACL transection, or complete PCL transection (joint instability). Sixteen weeks later the blood flow and vascular volume of the ACL and MCL were measured and compared to control and sham-operated animals. Direct ligament injury induced significant increases in standardized blood flow and vascular volume of both ACL and MCL after 16 weeks; however, the vascular volume of the ACL was not higher than the control levels in the MCL. We conclude that direct injury to both the anterior cruciate and MCLs induces long-term physiological responses. Joint laxity is a common sequel to PCL injury. Chronic joint laxity failed to induce adaptive vascular responses in the ACL, while the MCL shows significant amplification of blood supply. Although both MCL and ACL showed increased weight after PCL transection, the lack of a long-term vascular response in the ACL may be a major factor in its the diminished healing potential.     

成纤维细胞生长因子和表皮生长因子及复合因子对兔ACL、MCL体外增殖作用

目的观察酸性成纤维细胞生长因子(acid fibroblast growth factor，aFGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)和表皮生长因子(epidermal growth factor，EGF)以及复合因子对兔前交叉韧带(anterior cruciate ligament，ACL)和内侧副韧带(medial collateral ligament，MCL)的促增殖作用。方法分离传代培养10周龄新西兰大白兔骨关节韧带的ACL和MCL的成纤维细胞，接种96孔板，并加入浓度为0(对照组)、1、5、10、50、100ng／ml的aFGF或bFGF，浓度为0(对照组)、1．56、3．13、6．25、12．50、25、50ng／ml的EGF，单独或aFGF EGF两种因子联用与细胞(n=4)共同培养48h，以XTT方法测定其促细胞增殖作用。结果3种生长因子单独应用对ACL和MCL都有促进作用，aFGF对两种细胞均存在量效关系；bFGF 1ng／ml，EGF 5ng／ml对ACL作用最好，而对MCL则是bFGF和EGF均存在量效关系。浓度为5ng／ml的aFGF与50ng／ml的EGF联合1ng／ml aFGF与3．13ng／mlEGF作用于ACL或MCL均有协同作用。结论3种生长因子对ACL和MCL均有促进作用，单独应用aFGF或联用EGF优于单一因子促进兔ACL和MCL细胞的增殖，并且提示低浓度的aFGF联用EGF优于单一生长因子。     

ACL transection influences mRNA levels for collagen type I and TNF-alpha in MCL scar.

To assess the mRNA expression of extracellular matrix genes which might correlate with or contribute to mechanically weaker medial collateral ligament (MCL) scars in the ACL-deficient rabbit knee joint compared to those in anterior cruciate ligament (ACL) intact knee joints, a bilateral MCL injury was induced in 10 skeletally mature female NZW rabbits. As part of the same surgical procedure, the ACL was transected in one of the knees while the contralateral knee had a sham procedure. The side having the combined MCL and ACL injury was randomly assigned. After six weeks, the rabbits were euthanized. Histological assessments were performed on samples of the MCL scars from each operated knee (n = 3 animals) and mRNA levels for collagen type I, III, V, decorin, biglycan, lumican, fibromodulin, TGF-beta, IL-1, TNF-alpha, MMP-1, MMP-13, and a housekeeping gene (GAPDH) were assessed using semiquantitative RT-PCR on RNA isolated from the MCL scar tissue of the remaining animals (n = 7 animals). Levels of mRNA for each gene were normalized using the corresponding GAPDH value. Results showed that the total RNA yield (per mg wet weight) in the MCL scar of the ACL-deficient knee was significantly greater than that in the MCL scar from the ACL-intact knee. Collagen type I mRNA levels were significantly lower and mRNA levels for TNF-alpha were significantly greater in the scars of ACL-deficient knees compared to scars from ACL-intact joints. There were no significant differences between ACL-deficient and ACL-intact knees with respect to MCL scar mRNA levels for the remaining genes assessed. Histologically, the "flaw" area, which has been shown to correlate with mechanical properties in previous studies, was significantly greater in MCL scars from ACL-deficient knees than in the ACL-intact MCL scars. The mean number of cells/mm2 in MCL scars from ACL-deficient knees was significantly greater than in MCL scars from ACL-intact knees. The present study suggests that MCL scar cell metabolism is differentially influenced by the combined injury environment.     

In-situ force in the medial and lateral structures of intact and ACL-deficient knees

The anterior cruciate ligament (ACL) is the major contributor to limit excessive anterior tibial translation (ATT) when the knee is subjected to an anterior tibial load. However, the importance of the medial and lateral structures of the knee can also play a significant role in resisting anterior tibial loads, especially in the event of an ACL injury. Therefore, the objective of this study was to determine quantitatively the increase in the in-situ forces in the medial collateral ligament (MCL) and posterolateral structures (PLS) of the knee associated with ACL deficiency. Eight fresh-frozen cadaveric human knees were subjected to a 134-N anterior tibial load at full extension and at 15°, 30°, 60°, and 90° of knee flexion. The resulting 5 degrees of freedom kinematics were measured for the intact and the ACL-deficient knees. A robotic/universal force-moment sensor testing system was used for this purpose, as well as to determine the in-situ force in the MCL and PLS in the intact and ACL-deficient knees. For the intact knee, the in-situ forces in both the MCL and PLS were less than 20 N for all five flexion angles tested. But in the ACL-deficient knee, the in-situ forces in the MCL and PLS, respectively, were approximately two and five times as large as those in the intact knee (P < 0.05). The results of this study demonstrate that, although both the MCL and PLS play only a minor role in resisting anterior tibial loads in the intact knee, they become significant after ACL injury. Received: December 3, 1999 / Accepted: July 19, 2000     

Effects of age and platelet‐rich plasma on ACL cell viability and collagen gene expression

Platelet‐rich plasma (PRP) has shown in vivo potential to stimulate anterior cruciate ligament (ACL) healing at early time points in large animal models. However, in animal models, the healing potential of the ACL is dependent on animal age. In this study, we hypothesized that there are age‐dependent differences in ACL cell metabolism, collagen gene expression, and the ability of the cells to respond to growth factors in PRP. To test this hypothesis, ACL cells were obtained from skeletally immature, adolescent and adult pigs, and cultured in a collagen type I hydrogel with or without PRP for 14 days. When cultured in collagen‐only hydrogel, ACL cells from adult pigs had a 19% lower apoptotic rate as compared to immature pigs (p = 0.001) and a 25% higher cellular metabolic activity as compared to adolescent pigs (p = 0.006). The addition of PRP to the collagen hydrogel resulted in a significantly increased cellular metabolic activity, reduced apoptotic rate, and stimulation of collagen production in the cells from the immature and adolescent animals (p < 0.05 for all comparisons) but had less effect on adult cells. These findings suggest that skeletal maturity may influence ACL cells' metabolic activity, apoptosis, collagen production, and response to PRP. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:79–85, 2012     

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.     

Effects of cigarette smoking on early medial collateral ligament healing in a mouse model.

Cigarette smoking delays the healing process and increases morbidity associated with many common musculoskeletal disorders such as medial collateral ligament (MCL) injury. In the current study, a murine model of MCL healing was used to test the hypothesis that smoking impairs extracellular matrix synthesis after injury. Mice were divided into two groups, a nonsmoking control group and a group exposed to smoke for 2 months prior to surgical MCL injury. Mice were euthanized at 3 and 7 days after surgery. Subsequently, propidium iodine staining was used to quantify cellular density of injured and sham ligaments. Immunohistochemical staining and in situ hybridization to mRNA were used to detect proliferation, apoptosis, and type I collagen gene expression at the site of injury. Cell density increased significantly from baseline to 7 days after injury in control mice. In mice exposed to cigarette smoke, there was a significantly lower cellular density compared to controls at this time point (p=0.01). There was no difference in proliferation between groups at the site of injury, and the low level of proliferation observed was not sufficient to account for the large increase in cell density by day 7. No evidence of apoptosis was observed in any of the groups at the site of injury. Type I collagen gene expression was higher in controls compared to smokers at day 7. Almost all of the cells in the substance of the injured MCL at day 7 were spindle-shaped and expressed type I collagen, suggesting that increased cell density from day 3 to day 7 represented an increase in ligament cells rather than an increased inflammatory response. We conclude that the decreased cellular density and type I collagen expression in the injured ligament of mice exposed to smoke begin to provide a cellular and molecular basis for delayed or deficient early healing in these animals.     

Intrinsic properties of anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells and their responses to growth factors (bFGF and TGF-β): An in vitro cell culture study

Different intrinsic properties of the constituent cells of the anterior cruciate ligament (ACL) and medial collateral ligament (MCL) have been proposed as factors involved in the differential repair mechanisms in these ligaments. To examine this hypothesis, we established primary cell lines from tissue explants. The outgrowth of cells from ACL explants was slower than that from MCL explants. The growth curves of ACL and MCL cultures at both passages 2 and 6 showed a slower rate of proliferation of ACL cells than of MCL cells (P<0.005). DNA synthesis supports the conclusion that these cells have differential proliferation rates in culture. An in vitro wound closure assay was performed by creating a uniform wound of 0.6-mm width in the confluent layer of ACL and MCL cultures. By 48h post-wounding, cell-free zones created in ACL cultures were, partially occupied by single cells in a non-confluent fashion. In contrast, the wounded zone in the MCL cultures was almost completely covered by the cells. Growth factors have been demonstrated to have both mitogenic and metabolic effects on cell cultures. We investigated the possible proliferative response of the ACL and MCL to growth factors, and we analyzed the in vitro effects of basic fibroblast growth factor (b-FGF) and transforming growth factor-β (TGF-β) on fibroblast cell cultures obtained from rabbit knee ligaments. Neither b-FGF nor TGF-β had any significant effect on the cell proliferation of ACL and MCL cultures after 48h. However, TGF-β did have an inhibitory effect on thymidine incorporation, especially at concentrations greater than 1 ng/ml, while b-FGF stimulated thymidine incorporation in ACL and MCL fibroblasts in a dose-dependent manner. b-FGF altered the cell phenotype to an elongated form, which suggests stimulation of cell migration. This in vivo, study implies a potential use for growth factors in the treatment of ACL and MCL injuries in vivo. Keynote Lecture, Japanese Orthopaedic Association, Chiba, Japan, June 30, 1995. Presented at the 21st Meeting of the Japanese Orthopaedic Society of Sports Medicine, Makuhari, Chiba     

酸性成纤维细胞生长因子及表皮生长因子对兔韧带细胞增殖的影响

目的：观察酸性成纤维细胞生长因子(aFGF)和表皮生长因子(EGF)对内侧副韧带(MCL)和前十字韧带(ACL)细胞增殖行为的影响。方法：培养10周龄新西兰白兔内侧副韧带和前十字韧带细胞，在培养液中分别加入aFGF和EGF，以XTT方法测定细胞的增殖行为。结果：aFGF在1ng／ml时即对两种细胞具有显著的促进增殖作用，其浓度达50ng／ml时，对MCL细胞的促进作用最大，达100ng／ml时对ACL细胞的促进作用最大。EGF在0．78ng／ml时即对MCL细胞有显著的促增殖作用，在1．56ng／ml时始对ACL细胞有显著的促增殖作用，其浓度达3．125ng／ml时对2种细胞的促进作用最大。aFGF和EGF在超过其最佳浓度后，随浓度升高促进作用均下降。结论：aFGF和EGF可以促进韧带成纤维细胞增殖。     

Healing of the medial collateral ligament following a triad injury: a biomechanical and histological study of the knee in rabbits.

The effect of a partial medial meniscectomy and anterior cruciate ligament (ACL) transection on medial collateral ligament (MCL) healing was studied in skeletally mature rabbits. Two groups of animals, group I (isolated MCL rupture) and group II (MCL rupture with ACL transection and partial medial meniscectomy), were examined. At 6 and 12 weeks postoperatively, histological examination of the healing MCL and biomechanical evaluation of the varus-valgus (V-V) knee rotation and tensile properties of the femur-MCL-tibia complex (FMTC) were performed. Group II animals experienced substantial joint degeneration by 6 weeks. Progressive osteophyte formation was observed adjacent to the MCL insertions along with proximal migration of the MCL tibial insertion between 6 and 12 weeks. Histologic examination of the healing MCL substance from both groups showed disorganized collagen, inflammation, and fibroblast proliferation that decreased over time. For group II knees, the V-V knee rotation was found to be significantly elevated (4.7 to 5.2 times the contralateral control), and did not decrease with time. In contrast, the V-V knee rotations of the group I specimens were 1.8 times greater than control immediately following injury, and approached control values by 12 weeks. Tensile testing of the FMTCs revealed that the ultimate load increased with time for both groups, but group I had significantly higher values than group II. The linear stiffness in group I was not different than that group II and did not increase with time. For the mechanical (material) properties of the healed MCL substance, the modulus of the healing tissue for group II was only 40% that of group I. The structural properties of the FMTC and the mechanical properties of the MCL substance from both groups at 6 and 12 weeks were significantly different from the contralateral controls. We further demonstrated that immediately after ACL reconstruction, the V-V rotation of group II knees could be restored to group I levels. Recent clinical studies of MCL healing following isolated complete ligament tears have suggested that nonoperative management without immobilization leads to excellent treatment outcome. However, in more severe injuries involving additional tissues, poor quality of the healed ligament tissue and articular degeneration are observed. Our results demonstrate the deleterious effects of an untreated triad injury on the healing of the MCL substance and its insertions. Examination of the MCL substance suggests that a much larger healing mass is formed following a triad injury, which partially compensates for inferior ligament mechanical properties.(ABSTRACT TRUNCATED AT 400 WORDS)