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-beta1 (TGF-beta1) (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 cellular 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.     

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- &#103 1 (TGF- &#103 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.     

Changes in Gene Expression of Matrix Constituents with Respect to Passage of Ligament and Tendon Fibroblasts

Trauma to the knee joint often results in injury to one or more supporting soft tissue structures, such as the medial collateral (MCL) and anterior cruciate (ACL) ligaments. Also, a portion of the patellar tendon (PT) is frequently used as a replacement graft for the ACL, resulting in a PT defect. The healing responses of these tissues are dramatically different and range from spontaneous healing to little or no healing. Studies have suggested that native cell behavior could be responsible for differences in healing potential. However, it is difficult to make comparisons as the reported results are based on different cellular passages which could have a dramatic effect on their potential to form healing tissues. Therefore, the objective of this study was to quantify the gene expression of collagen and other matrix constituents of fibroblasts from the MCL, ACL, and PT to document how they change with cell passage. We hypothesized that MCL fibroblasts would possess higher potential for matrix production through passages than ACL and PT cells because the MCL mounts a robust healing response unlike the ACL and PT. These differences in matrix expression would be dependent on passage because at earlier passages all cells would mostly be proliferating while at later passages they would tend to become senescent. Cells were isolated from the MCL, ACL, and PT of three rats and passaged a total of five times (Passage 1 to Passage 5). Using real time RT-PCR, expression of all genes of interest (Collagen Type I (ligament/tendon’s main matrix constituent), Collagen Type III, Fibronectin, Metalloprotease-13 [MMP-13], and Tissue Inhibitor of Metallopreotease-1 [TIMP-1]) were quantitatively assessed. It was found that cell number for all three fibroblast types remained high from Passage 1 to Passage 5. There was a statistically significant increase in Collagen Type I of rat MCL fibroblasts throughout passage (p < 0.05). This was evident in the higher relative abundance (to GAPDH) at Passages 3 and 4 (14.5 ± 2.2 fold and 15.3 ± 6.9 fold, respectively) than at Passage 1 (3.3 ± 2.6 fold) (p < 0.05). On the other hand, Collagen Type I expression for ACL and PT fibroblasts were lower than that of MCL fibroblasts and remained at 2.5 ± 2.0 fold and 1.7 ± 0.8 fold, respectively. Interestingly, the gene expressions of Collagen Type III, Fibronectin, MMP-13, and TIMP-1 for MCL, ACL, and PT fibroblasts were all relatively constant throughout passage and were not significantly different from one another. The findings of this study indicate that passage does affect the Collagen Type I gene expression of rat MCL fibroblasts and further show that for in vitro ligament tissue engineering efforts, MCL fibroblasts have a more robust potential for ligament remodeling and repair due to the increase in collagen gene expression.     

Novel method for the quantitative assessment of cell migration: a study on the motility of rabbit anterior cruciate (ACL) and medial collateral ligament (MCL) cells

A novel method of quantitating cell migration has been proposed for the potential utilization of tissue engineered scaffolds. Applying Alt's conservation law to describe the motion of first passage ACL and MCL cells, we have developed a quantitative method to assess innate differences in the motility of cells from these two ligamentous tissues. In this study, first passage ACL and MCL cells were cultured from four mature New Zealand white rabbits. One side of the cell monolayer was scraped completely away to create a wound model. The cell moved into the cell-free area, and cell density profiles were analyzed at 6 h and 12 h. Values of the random motility coefficient (mu) were then estimated by curve fitting the 6 h and 12 h data to a mathematical model, derived from the conservation law of cell flux. During 6 h of incubation in medium supplemented with 1% FBS, MCL cells (mu(MCL) = 4.63 +/- 0.65 X 10(-6) mm(2)/sec) were significantly (p < 0.05) more mobile than ACL cells (mu(ACL) = 2.51 +/- 0.31 X 10(-6) mm(2)/sec). At 12 h, the MCL cells also appeared to move faster (mu(ACL) = 4.39 +/- 0.63 X 10(-6) mm(2)/sec, mu(MCL) = 6.59 +/- 1.47 X 10(-6) mm(2)/sec), but the difference was not statistically significant (p = 0.18). Exposure of the cells to growth factors PDGF-BB or bFGF for 6 h had no significant effect on the migration of the ACL and MCL cells. However, exposure of the ACL cells (p < 0.05) and the MCL cells (p = 0.19) to 1 ng/mL of PDGFBB for 12 h enhanced their migration. Incubation with a high concentration (100 ng/mL) of PDGF-BB or bFGF at concentrations tested (1 or 100 ng/mL) for 12 h, produced little or no migratory stimulation on these ligament cells. Our findings support the previous qualitative observations made by numerous investigators. The novel methodology developed in this study may provide a basis for tissue engineering, and the results may be applied to tissue reconstruction techniques of the knee ligaments.     

The behavior of ligament cells cultured on elastin and collagen scaffolds

The ruptured anterior cruciate ligament (ACL) does not heal spontaneously. Therefore, the development of new healing techniques employing tissue engineering is vital. One of the aspects related to tissue-engineered artificial ligaments is the type of cell to be used for the artificial ligament. In this study, ligament cells from the ACL and periodontal ligament (PDL) were evaluated. In addition, we prepared highly oriented extracellular matrix (ECM) fiber scaffolds that mimicked the structure of the ligament and examined the cellular responses to these scaffolds. Elastin-A and collagen were used as the ECM proteins. Although the cells from the PDL (PDL fibroblasts [PDLFs]) showed approximately 2.1-fold higher expression of alkaline phosphatase (ALP; marker of osteogenic differentiation) than the ACL cells, the expression of ligament-related genes (for type I collagen, type III collagen, and tenomodulin) did not differ between PDLFs and ACL cells. Furthermore, the cellular responses (expression pattern of ligament-related genes and ALP activity) to the ECM were similar between ACL cells and PDLFs. In particular, elastin-A upregulated ALP and downregulated tenomodulin (TeM; a ligament marker) in ligament cells. In contrast, collagen maintained TeM expression in ligament cells. These results suggest that elastin-A promotes the osteogenic differentiation of ligament cells and that collagen maintains the phenotype of ligament cells.     

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. 1–20 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.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.     

Effects of applied DC electric field on ligament fibroblast migration and wound healing

Applied electric fields (static and pulsing) are widely used in orthopedic practices to treat nonunions and spine fusions and have been shown to improve ligament healing in vivo. Few studies, however, have addressed the effect of electric fields (EFs) on ligament fibroblast migration and biosynthesis. In the current study, we applied static and pulsing direct current (DC) EFs to calf anterior cruciate ligament (ACL) fibroblasts. ACL fibroblasts demonstrated enhanced migration speed and perpendicular alignment to the applied EFs. The motility of ligament fibroblasts was further modulated on type I collagen. In addition, type I collagen expression increased in ACL fibroblasts after exposure to pulsing EFs. In vitro wound-healing studies showed inhibitory effects of static EFs, which were alleviated with a pulsing EF. Our results demonstrate that applied EFs augment ACL fibroblast migration and biosynthesis and provide potential mechanisms by which EFs may be used for enhancing ligament healing and repair.     

鼠膝关节前交叉韧带和内侧付韧带的损伤拉伸研究

本研究是为了观察鼠膝关节前交叉韧带(Anterior cruciate ligament,ACL)和内侧副韧带(Medial collateral ligament,MCL)在拉伸损伤后的病理变化.股骨-ACL-胫骨和股骨-MCL-胫骨复合物取自20只雄性Wistar大鼠,复合物拉伸应变分别为10% 或20%,拉伸时间分别为10 min或30 min.将拉伸后的样本于10%的福尔马林中固定、石蜡包埋、切片,分别进行奥新兰-PAS及HE染色.结果发现未拉伸的对照组, ACL基质较MCL者含有更多的黏多糖; 当拉伸应变为10%时,ACL的大部分细胞被拉长呈纺锤形,并出现细胞核固缩,时间越长,核固缩现象越严重,MCL除细胞核改变外还伴有胶原的损伤;当拉伸应变为20%时,ACL出现核溶解及胶原撕裂,而MCL在拉伸不到20%时自胫骨连接处撕脱.拉伸后的 ACL和MCL样品从外观上依然完好而在显微镜下可明显看到细胞或胶原的损伤.韧带拉伸损伤首先发生于韧带细胞,进一步的拉伸会扩展到胶原纤维,细胞的严重受损对韧带的修复极其不利.     

自噬在兔部分损伤前交叉韧带和内侧副韧带中的差异

目的：确定兔前交叉韧带和内侧副韧带部分损伤后自噬的存在,并比较损伤后不同时间两者之间自噬的表 达差异,探讨其愈合能力差异与自噬的关系。方法： 3 月龄健康雄性新西兰大白兔18 只,随机选取3 只作为对照 组,其余兔建立前交叉韧带和内侧副韧带部分损伤模型, 随机分为5 组,各组分别在造模后3 d、1 周、2 周、4 周、 6 周取材。H-E 染色观察损伤部位形态学变化,透射电镜观察损伤部位超微结构变化,免疫印迹检测自噬相关蛋 白Beclin 1、LC3Ⅱ/Ⅰ、p62 表达水平,RT-PCR 检测自噬相关基因Beclin 1、ATG-5 mRNA表达水平。结果：在 部分损伤后3 d、1 周、2 周、4 周、6 周5 个时间点,前交叉韧带和内侧副韧带均未表现出明显的愈合趋势,观察 到自噬小体的存在。与对照组相比,自噬相关蛋白Beclin 1、LC3Ⅱ/Ⅰ、p62 在前交叉韧带中的高表达更为显著, 在内侧副韧带中的表达更早恢复至正常水平,并继续降低;自噬相关基因ATG-5、Beclin 1 在前交叉韧带中始终 处于高表达状态,峰值出现在1 周,内侧副韧带高表达峰值出现在2 周,此后逐渐恢复至正常水平。结论： 兔前 交叉韧带、内侧副韧带部分损伤后自噬相关因子的表达随时间推移,总体呈现先升高后降低的变化趋势,损伤后 不同时间点前交叉韧带、内侧副韧带中自噬的表达存在明显差异,推测前交叉韧带损伤后自噬的过度激活可能导 致其内源性修复障碍。     

Tissue engineering of ligaments: a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source

Anterior cruciate ligament (ACL) reconstruction surgery still has important problems to overcome, such as "donor site morbidity" and the limited choice of grafts in revision surgery. Tissue engineering of ligaments may provide a solution for these problems. Little is known about the optimal cell source for tissue engineering of ligaments. The aim of this study is to determine the optimal cell source for tissue engineering of the anterior cruciate ligament. Bone marrow stromal cells (BMSCs), ACL, and skin fibroblasts were seeded onto a resorbable suture material [poly(L-lactide/glycolide) multifilaments] at five different seeding densities, and cultured for up to 12 days. All cell types tested attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I. On day 12 the scaffolds seeded with BMSCs showed the highest DNA content (p < 0.01) and the highest collagen production (p < 0.05 for the two highest seeding densities). Scaffolds seeded with ACL fibroblasts showed the lowest DNA content and collagen production. Accordingly, BMSCs appear to be the most suitable cells for further study and development of tissue-engineered ligament.     

In situ IGF-1 gene delivery to cells emerging from the injured anterior cruciate ligament

Ruptures of the anterior cruciate ligament (ACL) are common knee injuries that do not heal, even with surgical repair. Our research is directed towards developing novel, biological approaches that enable suture repair of this ligament. One promising strategy involves the insertion of a collagen hydrogel between the severed ends of the ACL. Cells migrate from the damaged ligament into the hydrogel and produce repair tissue. Here we have investigated the potential for augmenting this process by the transfer of insulin like growth factor (IGF) 1 cDNA to the repair cells using an adenovirus vector. The goal is to achieve direct, in situ gene delivery by loading the hydrogel with vector prior to its insertion into the defect. In a step-wise approach towards evaluating this process, we confirmed that monolayers of ACL fibroblasts were efficiently transduced by adenovirus vectors and continued to express transgenes when subsequently incorporated into the hydrogel; indeed, transgene expression persisted longer within collagen gels than in monolayer culture. Transfer of IGF-1 cDNA increased the cellularity of the gels and led to the synthesis and deposition of increased amounts of types I and III collagen, elastin, tenascin, and vimentin. The cells remained viable, even when subjected to high viral loads. Similar results were obtained when collagen hydrogels were preloaded with adenovirus prior to insertion into an experimental ACL lesion in vitro. These data confirm the promise of using vector-laden hydrogels for the in situ delivery of genes to cells within damaged ligaments and suggest novel possibilities for the biological repair of the ACL.     

Collagenase production by rabbit ligaments and tendon

Three periarticular connective tissues from normal rabbits were examined for collagenolytic activity. Enzyme activity was secreted by cultures of anterior cruciate ligament (ACL), medial collateral ligament (MCL) and patellar tendon (PT). A lag period of six days or more was often observed prior to the detection of active collagenase. We attributed this to the presence of an excess of inhibitor in the early days of culture. We quantitated the amount of enzyme and inhibitor produced in 13 days. The levels of collagenase in the ACL and MCL were comparable. The PT, however, consistently secreted more enzyme than the two periarticular (ACL and MCL) ligaments. The reaction products were analyzed for all three collagenases and compared to those generated by the rabbit skin enzyme. We observed the characteristic TCA and TCB collagen fragments for MCL and PT enzymes. Collagen cleavage by the ACL cultures resulted in a product with a molecular weight intermediate between the alpha 2 chain and the TCA piece. These data suggest that quantitative and qualitative differences exist in the ability of these similar connective tissues to degrade collagen.     

Novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering

To clarify the feasibility of using novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering, their mechanical properties and ability to promote cellular adhesion, proliferation, and extracellular matrix production were studied in vitro. Chitosan fibers and chitosan-based 0.05% and 0.1% hyaluronan hybrid fibers were developed by the wet spinning method. Hyaluronan coating significantly increased mechanical properties, compared to the chitosan fibers. Rabbit fibroblasts adhesion onto hybrid fibers was significantly greater than for the control and chitosan fibers. For analysis of cell proliferation and extracellular matrix production, a three-dimensional scaffold was created by simply piling up each fiber. At 1 day after cultivation, the DNA content in the hybrid scaffolds was higher than that in the chitosan scaffold. Scanning electron microscopy showed that the fibroblasts had produced collagen fibers after 14 days of culture. Immunostaining for type I collagen was clearly predominant in the hybrid scaffolds, and the mRNA level of type I collagen in the hybrid scaffolds were significantly greater than that in the chitosan scaffold. The present study revealed that hyaluronan hybridization with chitosan fibers enhanced fiber mechanical properties and in vitro biological effects on the cultured fibroblasts.     

Collagen fibrillogenesis and mRNA levels in the maturing rabbit medial collateral ligament and patellar tendon

This study compared collagen fibril diameter and mRNA changes in a subset of molecules involved in collagen fibrillogenesis during postnatal development and at maturity of rabbit medial collateral ligament (MCL) and patellar tendon (PT). Tissue was analyzed by RT-PCR for mRNA levels and collagen fibril diameters were measured using transmission electron microscopy. Collagen fibril diameters increased from 3 to 14 weeks with mean fibril diameters of PT significantly greater than MCL at 9, 12, and 14 weeks and maturity. RT-PCR analysis showed decorin and lumican mRNA levels were significantly higher in PT than MCL at all ages. Type I collagen, MMP-11, and procollagen C proteinase enhancer mRNA levels also were higher in the PT than the MCL between 3 and 14 weeks but not at maturity. Further understanding of collagen fibrillogenesis by studying protein synthesis and matrix turnover during maturation may provide insight into the mechanism(s) by which fibrils accrete in maturing connective tissues and how they are altered during healing following injury.     

The "epiligament" of the rabbit medial collateral ligament: a quantitative morphological study.

The thin layer of connective tissue covering ligaments--the epiligament--has not been well described. The aim of the present study was to define, describe, and quantify the structure of the epiligament of the rabbit medial collateral ligament (MCL) using polarized light, scanning-electron, transmission-electron microscopy, and computerized histomorphometry. Epiligament was composed of woven bundles of collagen fibers, 3 morphologically-distinct cell types (spinous-shaped cells, cuboidal-shaped cells, and fat cells), and a neurovascular network that periodically arborized into the MCL. The areal fraction of vessels was significantly greater in the epiligament than in the MCL. The epiligament was significantly thicker on the superficial surface of the MCL than the deep surface, and the thickness of epiligament changed significantly during skeletal growth. Based on these structural features we speculate that the epiligament serves several important functions including: (1) protecting the MCL against abrasion, (2) supporting the neurovasculature, (3) controlling water and metabolite flux into the epiligament and possibly the MCL, and (4) being a source of extracellular matrix, cells, and vasculature during ligament growth and during ligament healing.     

Development of a useful technique to discriminate anterior cruciate ligament cells and mesenchymal stem cells--the application of cell electrophoresis

Mesenchymal stem cells (MSCs) can differentiate into multiple nonhematopoietic cell lineages, including osteoblasts, chondrocytes, and ligament cells. The purpose of this study is to identify the difference between MSCs and anterior cruciate ligament (ACL) cells for the application of distinguishing these two cells during the process of MSCs differentiating into ACL cells. Although culture of MSCs and ACL cells have been studied extensively, it was found that these two cells could not be distinguished from their appearance, expression of surface antigens (including CD105, CD34, CD45, CD29, CD44, and CD71), alpha-smooth muscle actin, and mRNAs for type I collagen, type III collagen, and tenascin-C, based on a series of traditional methods for cell identification. Cell electrophoresis, measuring the electrophoretic mobility (EPM) of cells, was proposed to investigate the discrepancy in surface charge properties of MSCs and ACL cells. Surprisingly, the EPM value of MSCs is significantly greater than that of ACL cells (p < 0.001). Although cell electrophoresis cannot determine the specific surface protein, it can reflect the net surface charge density of cell membrane, which can be influenced by the dissociation of functional groups of peripheral membrane proteins. Therefore, it is suggested that cell electrophoresis, while simple and cheap in manipulation, can serve as a useful research tool to assist in identification of MSCs differentiating into ACL cells.     

A biochemical study of the distribution of collagen and its crosslinks in knee ligaments and the patellar tendon

Purpose: The purpose of this study was to investigate biochemical differences in collagen crosslinks from different locations within the ligaments and a tendon of the human knee.

Materials and Methods: The anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), lateral collateral ligament (LCL), and patellar tendon (PT) were obtained from 24 cadavers (13 men and 11 women) whose average age at the time of death was 84.8 years. Ligaments and PT samples were obtained from the femoral and tibial insertions and the midsubstance. Hydroxyproline (Hyp) and collagen crosslinks, including pyridinoline (Pyr) and pentosidine (Pen), were compared among the different sites.

Results: The midsubstance Hyp concentration was greater than at the femoral and tibial insertions in the ACL (p?=?0.00124 and 0.000255, respectively) and PCL (p?=?0.00036 and 0.042, respectively). The Pyr:collagen ratio did not differ among sites in any of the ligaments or PT. The Pen:collagen ratio at the midsubstance was greater than at the femoral and tibial insertions in the ACL (p?=?0.00022 and 0.00025, respectively) and LCL (p?=?0.000081 and 0.000021, respectively) and was greater at the femoral insertion in the MCL (p?=?0.00010).

Conclusions: The mature collagen crosslink Pyr was not different in distribution in knee ligaments and the PT. Pen increased at the midsubstance ligaments and the PT. As increased Pen may represent ligament degeneration, this may indicate that degeneration may progress more rapidly at the midsubstance than at the insertion sites of a ligament.     

Enhanced differentiation of mesenchymal stem cells co-cultured with ligament fibroblasts on gelatin/silk fibroin hybrid scaffold

The differentiation of mesenchymal stem cells (MSCs) towards fibroblasts is a crucial issue in ligament tissue engineering. This study aims to investigate the feasibility of using co-culture system to induce the differentiation of MSCs for constructing the tissue-engineered ligament in vitro. A kind of silk cable-reinforced gelatin/silk fibroin hybrid scaffold was used to provide three-dimensional (3-D) culture environments for MSCs. The 3-D co-culture system was set up by culturing MSCs/scaffold and ligament fibroblasts in the transwell insert and lower chamber, respectively. The regulatory effects of fibroblasts on MSCs were determined. After 2 weeks of co-culture the MSCs showed faster proliferation and higher DNA content compared with MSCs non-co-cultured. The MSCs were distributed uniformly throughout the scaffold and showed good viability. The collagen production also increased significantly with culture time. The MSCs in co-culture system were proved to differentiate into ligament fibroblasts by expressing ligament extra-cellular matrix (ECM)-specific genes including collagen I, collagen III, and tenascin-C in mRNA and protein level. The immunohistochemistry staining also confirmed the synthesis of key ligament ECM components. This study reveals that specific regulatory signals released from fibroblasts in 3-D co-culture system can enhance the differentiation of MSCs for ligament tissue engineering.