Anterior Cruciate Ligament Cells Research Articles

The differentiation of mesenchymal stem cells by mechanical stress or/and co-culture system

Differentiation of mesenchymal stem cells (MSCs) into anterior cruciate ligament (ACL) cells is regulated by many factors. Mechanical stress affects the healing and remodeling process of ACL after surgery in important ways. Besides, co-culture system had also showed the promise to induce MSCs toward different kinds of cells on current research. The purpose of this study was to investigate the gene expression of ACL cells’ major extracellular matrix (ECM) component molecules of MSCs under three induction groups. In addition, to follow our previous study, cell electrophoresis technique and mRNA level gene expression of MSC protein were also used to analyze the differentiation of MSCs. The results reveal that specific regulatory signals which released from ACL cells appear to be responsible for supporting the selective differentiation toward ligament cells in co-culture system and mechanical stress promotes the secretion of key ligament ECM components. Therefore, the combined regulation could assist the development of healing and remolding of ACL tissue engineering. Furthermore, this study also verifies that cell electrophoresis could be used in investigation of cell differentiation. Importantly, analysis of the data suggests the feasibility of utilizing MSCs in clinical applications for repairing or regenerating ACL tissue.

Proliferation of anterior cruciate ligament cells in vitro by photo‐immobilized epidermal growth factor

The purpose of this study was to explore the early treatment potential of anterior cruciate ligament (ACL) injuries using artificial juxtacrine stimulation by photo-immobilization of a growth factor. A photo-reactive epidermal growth factor complex (EGF-Az) was synthesized by conjugating EGF with N-(4-azidobenzoyloxy) succinimide followed by immobilization onto polystyrene culture plates using UV irradiation. ACL cells from human tissues (1 x 10(5)cells, 100 microl/well) were cultured as follows: control, no EGF; 50 microl native EGF; 50 microl EGF-Az immobilized; and 100 microl EGF-Az immobilized. The ACL cells were cultured long-term and evaluated for possible differences in their responses to EGF. An in vitro wound closure assay was developed to enable examination of cellular proliferation and migration. ACL cell proliferation was most evident in the photo-immobilized EGF culture group and was seen to increase in proportion to the amount of added EGF. In the in vitro wound closure assay, the lesioned area at 72 h after culture initiation was indistinguishable in the photo-immobilized cultures, but remained clearly visible in the controls. We conclude that photo-immobilized EGF induced rapid proliferation of ACL fibroblast cells by artificial juxtacrine stimulation and speculate that similar EGF immobilization onto bioabsorbable material (e.g., polyglycolic acid or polylactic acid) might contribute to a new therapy for the treatment of ACL injuries.

Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament

This study investigated four different connective tissue cell types to determine which cell type should be the source for seeding a tissue-engineered anterior cruciate ligament (ACL) replacement. Cells derived from the ACL, medial collateral ligament (MCL), achilles tendon (AT), and patellar tendon (PT) of New Zealand White rabbits were isolated and cultured. Each cell type was cultured in vitro after seeding on three-dimensional (3-D) braided polymer scaffolds and on tissue culture polystyrene that served as a control. Samples were evaluated and compared for their morphology, proliferation, and gene expression of fibronectin, type I and type III collagen. Scanning electron microscopy (SEM) photomicrographs verified cell attachment of all four types of connective tissue fibroblasts to the scaffolds. Preliminary results comparing proliferation indicate that cells obtained from the PT and AT have the fastest proliferation. Whereas gene expression of the phenotypic markers measured using real-time reverse transcription polymerase chain reaction (RT-PCR) indicates ACL cells have the highest gene expression for the matrix markers. This leads to the question of which cell type should be the cell source for tissue-engineering of ligament, the highly proliferating cells or the differentiated matrix producing cells. This study would suggest that ACL differentiated matrix producing cells are the most suitable cells for further study and development of a tissue-engineered ligament.

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

The significantly higher incidence of anterior cruciate ligament (ACL) injuries in collegiate women compared with men may result from relative ligament laxity. Differences in estrogen and relaxin activity, similar to that seen in pregnancy, may account for this. We quantified estrogen receptors by flow cytometry and relaxin receptors by radioligand binding assay in human ACL cells and compared the presence of these receptors in males and females. ACL stumps were harvested from seven males and eight females with acute ACL injuries. The tissue was placed in M199 cell culture medium. Outgrowth cultures were obtained, and passage 2 cells were used for all studies. Estrogen receptor determination was performed using flow cytometry. Relaxin binding was performed in ACL cells derived from five female and male patients using I(125)-labeled relaxin. Estrogen receptors were identified by flow cytometry in 4 to 10% of ACL cells. Mean fluorescence of cells expressing estrogen receptors was approximately twice that of controls, with no significant differences between males and females. Relaxin studies showed low-level binding of I(125)-relaxin-labeled ACL cells. Relaxin binding was present in four out of five female ACL cells versus one out of five male ACL cells.

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

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

Characterization of sulfate, proline, and glucose transport systems in anterior cruciate and medial collateral ligament cells

The present study was undertaken to define the nature of key transport processes for sodium, glucose, proline, and sulfate in primary culture of canine anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells. Uptake studies using radiolabeled isotopes were performed and Na,K-ATPase activity was determined in cell lysates. At 25 degrees C both ACL and MCL cells showed a significant uptake of 86Rb. Ouabain inhibited Rb uptake by 55% in ACL cells and by 60% in MCL cells. The transport activity of Na,K-ATPase in intact cells was calculated to be 57 and 71 nmol.(mg protein)-1.(15 min)-1, respectively. The enzymatic activity of Na,K-ATPase in cell lysates was observed to be 104 for ACL cells and 121 nmol.(mg protein)-1.(15 min)-1 for MCL cells. Cytochalasin B, a known inhibitor of sodium-independent D-glucose transport, completely inhibited D-glucose uptake in ACL and MCL cells. Removal of Na+ or addition of 10-5 mol/L phlorizin, a potent inhibitor of the sodium-D-glucose cotransporter, did not alter D-glucose uptake, suggesting that glucose entered the cells using a sodium-independent pathway. Both ACL and MCL cells exhibited high sulfate uptake that was not altered by replacement of Na+ by N-methyl-D-glucamine, whereas DIDS, an inhibitor of sulfate/anion exchange abolished sulfate uptake in both cell types. Thus, neither cell type seems to possess a sodium-sulfate cotransport system. Rather, sulfate uptake appeared to be mediated by sulfate/anion exchange. Proline was rapidly taken up by ACL and MCL cells and its uptake was reduced by 85% when Na+ was replaced by N-methyl-D-glucamine, indicating that proline entered the cells via sodium-dependent cotransport systems. The data demonstrate that both ACL and MCL cells possess a highly active sodium pump, a secondary active sodium-proline cotransport system, and sodium-independent transport systems for D-glucose and sulfate.

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.

Anterior Cruciate Ligament Healing and Repair

The anterior cruciate ligament (ACL) fails to heal adequately after rupture, even after partial injuries or primary repair. This is vastly different from the extra-articular medial collateral ligament (MCL), which heals even without surgical intervention. The reasons for this involve cellular and structural factors. On a cellular level, ACL cells have decreased migration and different extracelluar matrix production profiles when compared with MCL cells; however, it is unlikely that these small differences are fully responsible for the stark dichotomy in healing response for the two ligaments. With regards to structural issues, recent experiments have shown a premature loss of the bridge between the ruptured ACL ends, and this structural defect is also likely a key factor in the failure of the ACL to heal. New approaches using a substitute bridge, or provisional scaffold, with implanted growth factors are being developed to stimulate the ACL to heal after primary repair. To lay the foundation for these new approaches, the four phases of the injury response in the ACL (inflammatory, epiligamentous regeneration, proliferative, and remodeling phases), and the differences in intrinsic ACL and MCL cellular response are reviewed in this article followed by a discussion of the manipulations currently being attempted to improve the healing response of the ACL, including hyaluronic acid, implanted growth factors, and tissue engineered scaffolds.

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 1 mM. Based on flow cytometric analysis, DNA fragmentation at ImM 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.

Anterior cruciate ligament regeneration using braided biodegradable scaffolds: in vitro optimization studies

The anterior cruciate ligament (ACL) is the most commonly injured intra-articular ligament of the knee, and limitations in existing reconstruction grafts have prompted an interest in tissue engineered solutions. Previously, we reported on a tissue-engineered ACL scaffold fabricated using a novel, three-dimensional braiding technology. A critical factor in determining cellular response to such a graft is material selection. The objective of this in vitro study was to optimize the braided scaffold, focusing on material composition and the identification of an appropriate polymer. The selection criteria are based on cellular response, construct degradation, and the associated mechanical properties. Three compositions of poly- α-hydroxyester fibers, namely polyglycolic acid (PGA), poly- L-lactic acid (PLLA), and polylactic- co-glycolic acid 82:18 (PLAGA) were examined. The effects of polymer composition on scaffold mechanical properties and degradation were evaluated in physiologically relevant solutions. Prior to culturing with primary rabbit ACL cells, scaffolds were pre-coated with fibronectin (Fn, PGA-Fn, PLAGA-Fn, PLLA-Fn), an important protein which is upregulated during ligament healing. Cell attachment and growth were examined as a function of time and polymer composition. While PGA scaffolds measured the highest tensile strength followed by PLLA and PLAGA, its rapid degradation in vitro resulted in matrix disruption and cell death over time. PLLA-based scaffolds maintained their structural integrity and exhibited superior mechanical properties over time. The response of ACL cells was found to be dependent on polymer composition, with the highest cell number measured on PLLA-Fn scaffolds. Surface modification of polymer scaffolds with Fn improved cell attachment efficiency and effected the long-term matrix production by ACL cells on PLLA and PLAGA scaffolds. Therefore based on the overall cellular response and its temporal mechanical and degradation properties in vitro, the PLLA braided scaffold pre-coated with Fn was found to be the most suitable substrate for ACL tissue engineering.

Application of PLGA-collagen hybrid mesh for three-dimensional culture of canine anterior cruciate ligament cells

Canine anterior cruciate ligament (ACL) cells were isolated from the anterior cruciate ligament and subcultured in DMEM containing 10% FBS. After being subcultured twice, the ACL cells were seeded in a PLGA-collagen hybrid mesh and cultured in DMEM containing 10% FBS in vitro for 16 days. After 1 day, the cells adhered and spread well on collagen microsponges of the hybrid mesh. The cells proliferated and excreted extracellular matrices with culture time. After 16 days of culture, the cells and extracellular matrices occupied the spaces in the scaffold. At that point, the cell/mesh constructs were rolled up and implanted subcutaneously in the dorsum of nude mouse. The implants were harvested after 2, 4, 8, and 12 weeks of implantation and examined by histological staining. The implants were macroscopically white. Histological examination of cross sections of the implant by hematoxylin and eosin staining showed a large population of cells and newly deposited collagen in the rolled implants seeded with ACL cells. The cells were surrounded with bundles of collagen fibers. The layers in the rolled implant became integrated after 8 weeks. The polymer fibers could still be seen 8 weeks after implantation and gradually degraded and eventually disappeared after 12 weeks.

Enhanced Repair of the Anterior Cruciate Ligament by in Situ Gene Transfer: Evaluation in an in Vitro Model

The inability of the ruptured anterior cruciate ligament (ACL) of the knee joint to heal spontaneously presents numerous clinical problems. Here we describe a novel, gene-based approach to augment ACL healing. It is based upon the migration of cells from the ruptured ends of the ligament into a collagen hydrogel laden with recombinant adenovirus. Cells entering the gel become transduced by the vector, which provides a basis for the local synthesis of gene products that aid repair. Monolayers of bovine ACL cells were readily transduced by first-generation, recombinant adenovirus, and transgene expression remained high after the cells were incorporated into collagen hydrogels. Using an in vitro model of ligament repair, cells migrated from the cut ends of the ACL into the hydrogel and were readily transduced by recombinant adenovirus contained within it. The results of experiments in which GFP was used as the transgene suggest highly efficient transduction of ACL cells in this manner. Moreover, during a 21-day period GFP+ cells were observed more than 6 mm from the severed ligament. This distance is ample for the projected clinical application of this technology. In response to TGF-β1 as the transgene, greater numbers of ACL cells accumulated in the hydrogels, where they deposited larger amounts of type III collagen. These data confirm that it is possible to transduce ACL cells efficiently in situ as they migrate from the ruptured ACL, that transduction does not interfere with the cells' ability to migrate distances necessary for successful repair, and that ACL cells will respond in a suitable manner to the products of the transgenes they express. This permits optimism over a possible clinical use for this technology.

Identification of membrane and secreted proteins in anterior cruciate ligament derived cells using “signal-sequence-trap”, a retrovirus-mediated expression screening method

Ligament injuries are one of most common injuries to the knee joint; however, little is known about how ligaments heal and what kinds of precursor cells participate in ligament repair. Ligament cells were thought to be members of one of the lineages from mesenchymal stem cells (MSCs). However, there have been no reports on the differentiation mechanism of MSC to ligament cells because they do not have known phenotypic markers. To identify the anterior cruciate ligament (ACL)-specific phenotypic marker, we screened a cDNA library derived from rabbit ACL cells, using the signal sequence trap by the retrovirus-mediated expression screening (SST-REX) method. We detected known clones representing 135 independent proteins. Among them, 120 were secreted proteins and 15 were membrane proteins. Some of the clones encoded proteins that have never been reported to be expressed in ACLs. Therefore, the data could provide a better understanding of ACL cells and could help in finding their phenotypic markers.

The effect of selected growth factors on human anterior cruciate ligament cell interactions with a three-dimensional collagen-GAG scaffold

Our work focuses on development of a collagen-glycosamimoglycan (CG) scaffold to facilitate ligament healing in the gap between the ruptured ends of the human anterior cruciate ligament (ACL). In the present investigation, we evaluated the effects of selected growth factors on human ACL cell responses important in tissue regeneration, namely cell migration, proliferation, collagen production, and expression of α-smooth muscle actin (SMA). Methods: Explants from six human ACLs were cultured on top of a CG scaffold. Culture conditions were with either 2% FBS (control), or 2% FBS supplemented with TGF-β1, PDGF-AB, EGF, or FGF-2. Histologic cell distribution, total DNA content, proliferation rate, rate of collagen synthesis, scaffold diameter and percentage of SMA positive cells were determined at two, three and four weeks. Results: The addition of TGF-β1 to the culture medium resulted in increased cell number, increased collagen production and increased expression of SMA within the scaffold. Supplementation with PDGF-AB resulted in increased cell proliferation rates within the scaffold and increased collagen production. The addition of FGF-2 resulted in increased cell proliferation rates and slowed rates of scaffold shrinkage when compared with the control group. Discussion: This work suggests that certain growth factors can alter the biologic functions of human ACL cells in a CG scaffold implanted as a bridge at the site of an ACL rupture. Based on these findings, the addition of selected growth factors to an implantable CG scaffold may facilitate ligament healing in the gap between the ruptured ends of the human ACL.

Characterization of anterior cruciate ligament cells and bone marrow stromal cells on various biodegradable polymeric films

In this study, the adhesion, proliferation and morphology of rabbit anterior cruciate ligament (ACL) cells and bone marrow stromal cells (bMSCs) on synthetic biodegradable polymeric films were investigated. Tissue culture polystyrene (TCP) was used as control. Seven biodegradable polymers were used; they are as follows: poly(ε-caprolactone) (PCL), poly( dl-lactide) ( d-PLA), poly( l-lactide) ( l-PLA), PLA/PCL (50:50), PLA/PCL (75:25), high molecular weight (HMW) poly( dl-lactide–co-glycolide (PLGA50:50) and HMW PLGA75:25. Polymeric film substrates were manufactured using solvent spin-casting technique. After 8 h of cell culture, a high percentage of ACL cells was found attached to PLGA50:50 (38.6±8.4%) and TCP (39.3±6.1%) as compared to the other six polymeric films ( p≤0.001). As for bMSCs, 76.4±10%, 76.3±16% and 76.1±19% of seeded bMSCs were adhered to TCP, PLGA50:50 and PLGA75:25, respectively. These were significantly more than those of the other five polymeric films ( p<0.001). At Day 5, bMSCs were found to proliferate faster on TCP (by 7±0.8-fold of initial cell seeding number), d-PLA (by 5.6±1.6-fold), PLGA50:50 (by 9.3±1.3-fold) and PLGA75:25 (by 5.8±1.3-fold) than on PCL, PLLA and PCL/PLA (50:50, 25:75) ( p<0.001). ACL cells had a greater fold expansion on TCP (by 3.5±0.2-fold), PLGA50:50 (by 3.1±0.4-fold) and PLGA75:25 (by 3.9±0.4-fold) than on the other five polymer substrates ( p<0.001). From these results, HMW PLGA (50:50, 75:25) was shown more likely to allow bMSCs and ACL cells to attach and proliferate, and bMSCs attached and proliferated faster than ACL cells.

Gene expression of type I and type III collagen by mechanical stretch in anterior cruciate ligament cells.

Mechanical stretch affects the healing and remodeling process of the anterior cruciate ligament (ACL) after surgery in important ways. In this study, the effects of mechanical stress on gene expression of type I and III collagen by cultured human ACL cells and roles of transforming growth factor (TGF)-beta1 in the regulation of mechanical strain-induced gene expression were investigated. Uniaxial cyclic stretch was applied on ACL cells at 10 cycles/min with 10% length stretch for 24 h. mRNA expression of the type I and type III collagen was increased by the cyclic stretch. TGF-beta1 protein in the cell culture supernatant was also increased by the stretch. In the presence of anti-TGF-beta1 antibody, stretch-induced increase in type I and type III mRNA expression was markedly ablated. The results suggest that the stretch-induced mRNA expression of the type I and type III collagen is mediated via an autocrine mechanism of TGF-beta1 released from ligament cells.

Mechanical stretch in anterior cruciate ligament derived cells regulates type I collagen and decorin expression through extracellular signal-regulated kinase 1/2 pathway

Anterior cruciate ligament (ACL) is continuously exposed to tensile load, which is generally recognized to be an essential factor in maintaining the physiological homeostasis of the ACL fibers during daily activities. Although this kind of mechanical stimulation is clearly known to play an important role in quickening repair of ACL injury, responses of ACL cells to mechanical stimulation still remain unclear. We have investigated whether extracellular signal-regulated kinase 1/2 (ERK1/2) in human ruptured ACL-derived cells are phosphorylated and involved in type I collagen (Col I) and decorin gene expression by mechanical stimulation. Mechanical stretch stimulates time-dependent phosphorylation of ERK1/2 in human ruptured ACL-derived cells. While Col I gene expression levels were increased in 48-h continuous mechanical stretch, decorin gene expression levels were decreased for 24-h continuous stimulation. Decorin gene expression levels were increased, however, in mechanical stretch+PD98059, which is an inhibitor of ERK pathway, treated cells as compared with control cells at 24 h. Our results show that ERK pathway is stimulated by mechanical stretch, and may be involved in regulation of Col I and decorin gene expression in ACL-derived cells.

The migration of cells from the ruptured human anterior cruciate ligament into collagen-glycosaminoglycan regeneration templates in vitro

Guided tissue regeneration of the ruptured anterior cruciate ligament (ACL) offers the potential benefits of retaining the complex footprints of the ACL and the proprioceptive nerve fibers of the tissue. For this approach to be successful, ACL cells must retain the ability to migrate into an adjacent regeneration template, or scaffold, after ligament rupture. Ruptured ACLs were obtained from the knees of four men, ages 25–35, at the time of ACL reconstruction. Explants of ACL tissue were taken from three locations along the longitudinal axis of the remnant: the rupture site, the middle of the remnant, and far from the rupture site. These three areas have been found to be distinct histologically, with the region far from the rupture site having a histologic appearance similar to the intact ligament. Explants from each area were cultured in conventional tissue culture dishes (2-D culture) and on porous collagen-glycosaminoglycan (CG) scaffolds. Two-dimensional outgrowth was measured 3 times a week, and the 3-D explant/scaffold constructs were examined at 1, 2, 3 and 4 weeks to assess outgrowth of cells into the scaffold. The cell number density and expression of α-smooth muscle actin (SMA) were determined at each time point. The decrease in the diameter of the scaffolds and non-seeded controls were determined as a function of time in culture. The outgrowth of cells onto the tissue culture dishes was observed to begin as early as 3 days and as late as 21 days, with outgrowth first detected at an average of 6.8±2.0 days after explantation. In general, there was a larger area of outgrowth at the 2-week time point from explants with higher cell number density and higher blood vessel density. The 2-week area of outgrowth also correlated with the percentage of SMA-positive cells in the explant. In the experimental constructs with CG scaffolds, fibroblasts were noted to migrate from the human ACL explants into the templates at the earliest time point recorded (1 week). The migration and proliferation of cells from the explants in the CG matrices resulted in an increase in the cell density in the scaffolds with time. There was a significant effect of the location from which the explant was taken on cell density in the scaffold, with a higher density of cells migrating from the explants from the rupture site of the ACL specimens. The percentage of cells staining positive for the SMA isoform varied from 0 to 50% of cells in the scaffold. Scaffolds co-cultured with explants showed a reduction in diameter that was significantly affected by time in culture and the location in the ACL from which the explant was taken. The percentage contraction attributed to the cells was 15% at 2 weeks, and increased to 27% for the injury-zone explant at 4 weeks. There was a significant correlation of the cell-mediated contraction of the matrices at 4 weeks with the cell density in the scaffolds, but not with the number of SMA-positive cells in the scaffolds. These data demonstrate that cells in the human ACL retain their ability to migrate into an adjacent CG scaffold in vitro, weeks after complete rupture. Moreover, the ACL-derived cells can express a contractile actin isoform and can contract a CG analog of extracellular matrix.

The expression of matrix metalloproteinases and inhibitors in acute rupture of the anterior cruciate ligament

Rapid degeneration of the anterior cruciate ligament (ACL) may occur after ligament rupture, making primary repair of the anterior cruciate ligament difficult. Nine completely ruptured anterior cruciate ligaments were collected by arthroscopic surgery performed within 6 months of injury. The authors studied the localization of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in ruptured anterior cruciate ligament using immunohistochemistry, and measured messenger ribonucleic acid expression using the reverse transcriptase polymerase chain reaction. Cells in residual ligament tissue seemed to contain matrix metalloproteinases 1 and 3 and tissue inhibitors of matrix metalloproteinases 1 and 2. Promatrix metalloproteinase-9 positive cells were observed in the perivascular area. Promatrix metalloproteinase-2 positive cells frequently were seen between irregular collagen bundles in stumps of ruptured ligaments. Tissue inhibitors of matrix metalloproteinase-2 positive cells commonly were observed in ruptured ligaments. Matrix metalloproteinase-1 and matrix metalloproteinase-3 messenger ribonucleic acid were highly expressed compared with matrix metalloproteinase-2 messenger ribonucleic acid. Tissue inhibitor of matrix metalloproteinase-2 messenger ribonucleic acid was highly expressed compared with tissue inhibitor of matrix metalloproteinase 1. The authors could not identify whether these intrinsic reactions mediated by anterior cruciate ligament cells were the causes of rapid degradation or the results of the degradation process. Various amounts of matrix metalloproteinase and inhibitor production of intrinsic ligament cells were observed in the ruptured anterior cruciate ligament. The biological reaction reported in this study may suggest that pronounced metabolism is undertaken in ruptured ACL cells, and provide useful insight concerning the possiblitiy of achieving the primary repair of ruptured ACL.

Ligament tissue engineering using synthetic biodegradable fiber scaffolds.

Tissue engineering offers the possibility of replacing damaged human ligaments with engineered ligament tissues. Hence, we attempted to culture in vitro ligament tissues by seeding human anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells onto synthetic biodegradable polymer fiber scaffolds. The ACL and MCL cells readily attached to the scaffold fibers. These cells and their secreted matrix soon surrounded the scaffold fibers and bridged the gaps in between. Beginning at 2 weeks, portions of the scaffolds were completely filled with tissue matrix. By 5 weeks, the scaffolds became single bundles of tissue. Thus the cell/fiber system appears to be a viable system for culturing ligament tissues. Additionally, cell proliferation under mechanical and biochemical stimuli was studied for up to 4 days. Whereas mechanical stimulus and transforming growth factor enhanced proliferation, inflammatory agents (lipopolysaccharide and complement C5a) had a negative effect. This work can thus contribute to a sound strategy for culturing replacement ligament tissues in vitro.