Development of a Tissue-Engineered Artificial Ligament: Reconstruction of Injured Rabbit Medial Collateral Ligament With Elastin-Collagen and Ligament Cell Composite Artificial Ligament.

Ligament reconstruction using a tissue-engineered artificial ligament (TEAL) requires regeneration of the ligament-bone junction such that fixation devices such as screws and end buttons do not have to be used. The objective of this study was to develop a TEAL consisting of elastin-coated polydioxanone (PDS) sutures covered with elastin and collagen fibers preseeded with ligament cells. In a pilot study, a ring-type PDS suture with a 2.5 mm (width) bone insertion was constructed with/without elastin coating (Ela-coat and Non-coat) and implanted into two bone tunnels, diameter 2.4 mm, in the rabbit tibia (6 cases each) to access the effect of elastin on the bond strength. PDS specimens taken together with the tibia at 6 weeks after implantation indicated growth of bone-like hard tissues around bone tunnels accompanied with narrowing of the tunnels in the Ela-coat group and not in the Non-coat group. The drawout load of the Ela-coat group was significantly higher (28.0 ± 15.1 N, n = 4) than that of the Non-coat group (7.6 ± 4.6 N, n = 5). These data can improve the mechanical bulk property of TEAL through extracellular matrix formation. To achieve this TEAL model, 4.5 × 106 ligament cells were seeded on elastin and collagen fibers (2.5 cm × 2.5 cm × 80 µm) prior to coil formation around the elastin-coated PDS core sutures having ball-shape ends with a diameter of 2.5 mm. Cell-seeded and cell-free TEALs were implanted across the femur and the tibia through bone tunnels with a diameter of 2.4 mm (6 cases each). There was no incidence of TEAL being pulled in 6 weeks. Regardless of the remarkable degradation of PDS observed in the cell-seeded group, both the elastic modulus and breaking load of the cell-seeded group (n = 3) were comparable to those of the sham-operation group (n = 8) (elastic modulus: 15.4 ± 1.3 MPa and 18.5 ± 5.7 MPa; breaking load: 73.0 ± 23.4 N and 104.8 ± 21.8 N, respectively) and higher than those of the cell-free group (n = 5) (elastic modulus: 5.7 ± 3.6 MPa; breaking load: 48.1 ± 11.3 N) accompanied with narrowed bone tunnels and cartilage matrix formation. These data suggest that elastin increased the bond strength of TEAL and bone. Furthermore, our newly developed TEAL from elastin, collagen, and ligament cells maintained the strength of the TEAL even if PDS was degraded.

Mechanoreceptors distribution in the human medial collateral ligament of the elbow.

BACKGROUND: The human elbow maintains its stability mainly through its bony structure. Stability is enhanced by ligamentous structures. To allow the ligamento-muscular reflex, which protects against strain and stress, mechanoreceptors are embedded in the ligament. This report describes the existence and the distribution of the elbow medial collateral ligaments (MCLs) mechanoreceptors. HYPOTHESIS: The bony attachment site has the highest density of mechanoreceptors, and the anterior part has the highest density of mechanoreceptors. MATERIALS AND METHODS: Eight MCLs of elbow from fresh frozen cadavers were used. The MCLs were harvested deep to the periosteum from the medial epicondyle to the ulna. The fan-shaped ligaments were divided into six regions of interest (ROI) and stained with modified gold chloride stain. Specimens were evaluated under a light microscope. Golgi, Ruffini, and Pacinian corpuscles were found in every specimen. The number and the distribution of each mechanoreceptor in each ROI were recorded. The density of each mechanoreceptor was calculated in regards to its volume. RESULTS: Golgi, Ruffini, and Pacinian corpuscles were seen in the ligament with small nerve fibers. Ruffini corpuscles had the highest median density of all three corpuscles. The median corpuscle density was higher in the anterior than in the posterior part and higher in the bony attachment than in the mid-substance site except for Golgi corpuscle. CONCLUSION: The three typical types of mechanoreceptors were identified in human MCL with the anterior part and bony attachment as the dominant distribution site. LEVEL OF EVIDENCE: Basic Science Study.

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 2 mM sodium nitroprusside treatment, ACL cells were more prone to apoptosis at 1 mM. Based on flow cytometric analysis, DNA fragmentation at 1 mM 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.

Effects of osteogenic protein-1 (OP-1, BMP-7) on gene expression in cultured medial collateral ligament cells.

Osteogenic protein-1 (OP-1, also called BMP-7), a member of the BMP family and the TGF-beta superfamily, induces formation of new bone and cartilage, but also regulates a wide array of processes. In the present study, the expression of several characteristic biochemical markers of ligaments, such as Six1, Scleraxis, aggrecan, and type I collagen in primary cultures of adult rat medial collateral ligament (MCL) cells was determined. The effects of OP-1 on cell proliferation and on gene expression were subsequently examined. OP-1 stimulated cell proliferation, alkaline phosphatase (AP) activity, and the steady-state mRNA levels of the transcription factor Runx2/Cbfa1 in a dose- and time-dependent manner. The mRNA levels of type I collagen only increased slightly, but the activity of the cloned collagen promoter increased by 2-fold in transiently transfected MCL cells. OP-1 also stimulated aggrecan mRNA expression. The mRNA levels of Six1 and Scleraxis were not detectably altered by OP-1. In control cultures, the steady-state mRNA levels of ActR-I, BMPR-IA, BMPR-IB, and BMPR-II increased as a function of time in culture. The mRNA levels of BMP-1 and -4 increased significantly after 12 days, but those of BMP-2 and -6 did not change. The GDF-1, -3, -5, -6, and -8 mRNA levels in the control cultures also increased as a function of time. OP-1 treatment stimulated mRNA expression of BMPR-IA and BMPR-II, but had little effect on ActR-I and BMPR-IB mRNA expression. OP-1 lowered the BMP-1, -2, and -6 mRNA levels without changing the BMP-4 mRNA level. OP-1 treatment also reduced the mRNA levels of GDFs detected. In summary, the present study demonstrated that OP-1 stimulated cell proliferation and mRNA expression of several biochemical markers in this ligament cell culture model and established the spatial and temporal appearance of several members of the TGF-beta superfamily.

Scanning electron microscopic characterization of healing and normal rat ligament microstructure under slack and loaded conditions.

The objective of this study was to observe and compare behavior of the collagen fiber microstructure in normal and healing ligaments, both in situ and ex vivo, in order to add insight into the structure-function relationship in normal and healing ligaments. Fifty-two ligaments from 26 male rats were investigated. Eleven animals underwent surgical transection of both medial collateral ligaments (MCLs) (22 ligaments), which were allowed to heal for a period of 2 weeks. An additional 15 animals (30 ligaments) were used as normals. Ligaments were placed into six groups: Slack (n = 6 control, n = 6 healing), Reference (n = 4 control, n = 4 healing), Loaded (n = 4 control, n = 4 healing), 15 degrees Flexion (n = 4 control, n = 4 healing), 120 degrees Flexion (n = 4 control, n = 4 healing), and Tissue Strain vs. Flexion Angle (n = 8 normals). All ligaments, except those in the Tissue Strain vs. Flexion Angle group, were prepared for scanning electron microscopy. Tissues were harvested, mounted in a load frame, and chemically fixed in one of five states: (1). slack, (2). reference (onset of loading), (3). loaded, (4). 15 degrees knee flexion, or (5). 120 degrees knee flexion. After fixation the tissues were prepared for electron microscopy (SEM). The micrographs from the slack, reference, and loaded groups show fiber straightening with loading in normal ligaments as well as in both scar and "retracted" regions of healing ligaments. Collagen fibers' diameter and crimp patterns were dramatically changed in the scar region of healing ligaments: Width decreased from 19.4 +/- 1.7 microm to 6.5 +/- 2.1 microm (p <.000001), period from 51.4 +/- 15.1 microm to 11.0 +/- 2.4 microm (p <.000001), and amplitude from 9.8 +/- 0.8 microm to 3.9 +/- 0.8 microm (p <.000001). Normal ligaments fixed in situ show wavy regions at 120 degrees but less so at 15 degrees flexion. Healing ligaments fixed in situ show regions of fiber waviness in the scar region at 120 degrees and also at 15 degrees flexion, indicating ligament laxity persists toward both extremes of the range of motion. The data suggest that straightening of crimped fibers is a functionally relevant phenomenon, not only in normal but also in healing ligaments.

Biologic 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.

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.

Analysis of strain distribution in the medial collateral ligament using a photoelastic coating method.

The strain distribution over the entire medial collateral ligament (MCL) was measured using a photoelastic coating method. This new approach utilized a polyurethane monomer as a photoelastic coating film. The initial experiments investigating MCL strain measurement showed that this film had a high sensitivity for strain and good adhesion to the ligament. It was confirmed that strain distribution could be obtained qualitatively over the entire ligament using this method. The mechanism of MCL injury was studied by applying this polyurethane coating film to the entire MCL in a femur-MCL-tibia complex. When simple tension was applied to the complex, strain concentrations were centred at the tibial insertion site, and all the specimens ruptured at the MCL tibial insertion site. With application of a valgus bending moment, increased strain was seen in the MCL from the medial femoral condyle to the medial epicondyle. Histological analysis demonstrated midsubstance ligament ruptures in this same region. For both tests, rupture sites and increased strain concentration sites correlated. In addition, an impingement phenomenon of the MCL on the medial femoral condyle can be seen during application of valgus force, and this phenomenon may explain the higher incidence of MCL injuries on the femoral side seen in the clinical setting. This polyurethane coating method allows for direct and visual measurements, and can qualitatively measure the strain behaviour over the entire MCL surface. This new technique represents a significant improvement over previous point-by-point strain measurement methods.

Anatomic and histologic studies of lateral collateral ligament complex of the elbow joint.

We studied the gross and histologic anatomic characteristics of the lateral collateral ligament complex of the elbow joint from 15 cadavers to demonstrate its cross-sectional anatomy. The lateral ulnar collateral ligament adheres closely to the supinator, the extensor muscles, its intermuscular fascia, and the anconeus muscle and lies posterior to the radial collateral ligament. The lateral ulnar collateral ligament itself was identified with microscopy as a slender, poor structure consisting of the thick area of the posterolateral capsuloligamentous layer and a poorer structure than the anterior bundle of the medial collateral ligament as the primary stabilizer of the elbow joint. We believe that the lateral ulnar collateral ligament contributes to rather than is a major constraint to the posterolateral rotatory instability as part of the lateral collateral ligament complex with the surrounding tissues.

Structural, material, and anatomic characteristics of the collateral ligaments of the canine cubital joint.

OBJECTIVE: To document gross and microscopic anatomic features of the collateral ligaments of the canine cubital joint and to determine their structural and material properties. ANIMALS: 37 canine cadavers. PROCEDURE: After measurement of ligament dimensions, the bone-collateral ligament-bone specimens were loaded in tension until failure, using a materials testing machine. Data from the load-displacement curves were used to determine the structural and material properties of the ligaments. Gross anatomic features were studied during dissection of the specimens from the medial collateral ligament (MCL) and lateral collateral ligament (LCL), which then were saved for microscopic examination. RESULTS: Failure load and stiffness values for the LCL were significantly (P < 0.05) greater than those for the MCL. The LCL had obvious cranial and caudal components that attached to the radius and ulna, respectively. The MCL also had cranial and caudal components; however, the cranial component was indistinct, appearing only as a slight thickening of the joint capsule. The caudal component was more prominent; as it extended distad, it had minor attachments to the interosseous and annular ligaments and attached principally on the caudolateral surface of the proximal portion of the radius. The caudal component did not have substantial attachment to the ulna in any of the specimens studied. Both ligaments were composed of closely packed, parallel fascicles of dense collagen, with scant amounts of fibrocartilage and no detectable elastin. CONCLUSIONS: Gross anatomic features of the collateral ligaments of the canine cubital joint indicate that they provide principal structural support to the joint; microscopic anatomic features are typical of other ligaments. The LCL is stronger and stiffer than the MCL; however, their material properties are similar. CLINICAL RELEVANCE: Knowledge of the sites of attachment of collateral ligament components is essential for surgeons undertaking repair or reconstruction of these structures.

Effect of growth factors on the proliferation of ligament fibroblasts from skeletally mature rabbits.

Growth factors have been shown to stimulate fibroblast proliferation during wound and ligament healing. In this study, we analyzed individual effects of eight growth factors in vitro on the proliferation of fibroblasts from the medial collateral (MCL) and anterior cruciate (ACL) ligaments of skeletally mature rabbits. We compared the proliferative response of growth factor-treated and nontreated fibroblasts of both ligaments. The growth-factor treated fibroblasts of the MCL and ACL were also compared. We found that the fibroblasts exposed to epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-BB proliferated significantly more than untreated fibroblasts. Acidic fibroblast growth factor at a dose of 1.0 ng/ml caused significant increases in fibroblast proliferation only in the MCL. Transforming growth factor-beta 1, insulin-like growth factor-1, platelet-derived growth factor-AA, and interleukin-1 alpha did not significantly stimulate fibroblast proliferation. MCL fibroblasts generally did not proliferate significantly more than ACL fibroblasts with the exception of MCL fibroblasts exposed to the highest doses of basic fibroblast growth factor, acidic fibroblast growth factor and platelet-derived growth factor-BB. The data were also compared with those obtained earlier using fibroblasts from skeletally immature rabbits (Schmidt et al., JOR 1995). The proliferative response of both the MCL and the ACL fibroblasts was found to decrease with skeletal maturation. Thus, our findings suggest that animal age and fibroblast origin are important factors in determining the proliferative response to growth factors.

Biochemical analysis of collagens at the ligament-bone interface reveals presence of cartilage-specific collagens.

The collagen fibrils of some ligaments attach to bone by passing through a zone that consists of nonmineralized and mineralized fibrocartilages. Very little, however, is known about the cells, the biochemical composition, the extracellular matrix organization, and function of these fibrocartilages. In this study, the collagens present in the fibrocartilages of the bovine medical collateral and anterior cruciate ligaments femoral attachments to bone were isolated, characterized, and their distribution at these sites was assessed by laser confocal microscopy. Types II, IX, and XI collagens were identified after pepsin digestion of the tissues in addition to the types I and V collagens. Immunoblotting using specific polyclonal antibodies confirmed the presence of types II and IX collagens at these sites. Immunofluorescence using confocal microscopy showed that type II collagen was prominent in the nonmineralized area and to a lesser extent in the mineralized zone of the insertion. Type IX collagen showed similar distribution as type II collagen. Type II collagen isolated from the ligament-bone interface contained half hydroxypyridinium cross-linking residues when compared to type II collagen isolated from articular cartilage of the same animals. These data indicate that the fibrocartilaginous zones at the ligament-bone interface are cartilaginous in nature. The cartilage collagens may play a role of anchoring the ligament to bone or the cartilage-like tissue may participate in the modulation of the mechanical stresses which are known to exist at the soft tissue-hard tissue interface.

Increased expression of the beta 1, alpha 5, and alpha v integrin adhesion receptor subunits occurs coincident with remodeling of stress-deprived rabbit anterior cruciate and medial collateral ligaments.

The biomechanical, biochemical, and morphological properties of the anterior cruciate and medial collateral ligaments are dramatically altered in response to deprivation of normal physical forces and joint motion. Integrin adhesion receptors are known to play important roles in the tissue remodeling that occurs in the course of normal wound repair. We propose that integrins play a similar role in the remodeling of the extracellular matrix in stress-deprived periarticular ligaments. This study tests the hypothesis that altered expression of integrins on ligament fibroblasts accompanies this remodeling. The left knees of 15 New Zealand White rabbits were surgically immobilized in acute flexion and the right knees served as controls (no operation). The anterior cruciate and medial collateral ligaments were harvested at 1, 3, 5, 9, or 12 weeks after immobilization. Sections from the ligaments were immunostained with monoclonal antibodies specific for the integrin subunits beta 1, alpha 5, alpha 6, and alpha v, as well as with a negative control antibody. Fibroblasts within both the stress-deprived anterior cruciate and medial collateral ligaments demonstrated markedly increased staining for the beta 1, alpha 5, and alpha v subunits, as compared with the controls. The increased staining was greatest at 9 weeks in the anterior cruciate ligament and at 12 weeks in the medial collateral ligament. Western blot study of ligament proteins extracted with sodium dodecyl sulfate demonstrated an increased amount of beta 1 subunit protein in both ligaments from knees that were stress deprived for 9 and 12 weeks, as compared with the control ligaments.(ABSTRACT TRUNCATED AT 250 WORDS)