Early expression of marker genes in the rabbit medial collateral and anterior cruciate ligaments: the use of different viral vectors and the effects of injury.

Gene therapy is a technique that may offer advantages over current methods of cytokine delivery to ligaments. To determine if implanted genes could be expressed in normal and injured knee ligaments, the medial collateral ligament and anterior cruciate ligament were studied in 18 rabbits. A retroviral ex vivo technique using allograft medial collateral ligament and anterior cruciate ligament fibroblasts and an adenoviral in vivo technique were compared as methods for delivering the LacZ marker gene to knee ligaments. Bilateral knee surgeries were performed, and the rabbits were equally divided into three groups. Group 1 received the retrovirus and the medial collateral ligament was ruptured, Group 2 received the adenovirus and the medial collateral ligament was ruptured, and Group 3 received the adenovirus and the medial collateral ligament was not injured. The anterior cruciate ligament was not injured in any group. The medial collateral and anterior cruciate ligaments of the right knees received 10(6) allografted, transduced ligament fibroblasts or 10(9) adenovirus particles, whereas the ligaments of the left knee received a similar volume of saline solution only. Equal numbers of rabbits were killed at 10 days, 3 weeks, and 6 weeks following the procedure. Ligament samples were stained with X-gal to detect the expression of the LacZ gene product, beta-galactosidase. LacZ gene expression was evident in ruptured and uninjured medial collateral ligaments as well as in the anterior cruciate ligament. The expression lasted between 10 days and 3 weeks in the medial collateral and anterior cruciate ligaments with use of the retrovirus and between 3 and 6 weeks in the medial collateral ligament and at least 6 weeks in the anterior cruciate ligament with the adenovirus. The length of gene expression in the ruptured and uninjured medial collateral ligaments did not differ. These preliminary studies indicate that gene transfer to normal and injured knee ligaments is possible.     

Medial collateral ligament healing one year after a concurrent medial collateral ligament and anterior cruciate ligament injury: An interdisciplinary study in rabbits

The optimal treatment for concurrent injuries to the medial collateral and anterior cruciate ligaments has not been determined, despite numerous clinical and laboratory studies. The objective of this study was to examine the effect of surgical repair of the medial collateral ligament on its biomechanical and biochemical properties 52 weeks after such injuries. In the left knee of 12 skeletally mature New Zealand White rabbits, the medial collateral ligament was torn and the anterior cruciate ligament was transected and then reconstructed. This is an experimental model previously developed in our laboratory. In six rabbits, the torn ends of the medial collateral ligament were repaired, and in the remaining six rabbits, the ligament was not repaired. Fifty-two weeks after injury, we examined varus-valgus and anterior-posterior knee stability; structural properties of the femur-medial collateral ligament-tibia complex; and mechanical properties, collagen content, and mature collagen crosslinking of the medial collateral ligament. We could not detect significant differences between repair and nonrepair groups for any biomechanical or biochemical property. Our data support clinical findings that when the medial collateral and anterior cruciate ligaments are injured concurrently and the anterior cruciate ligament is reconstructed, conservative treatment of the ruptured medial collateral ligament can result in successful healing.     

Healing of the medial collateral ligament after a combined medial collateral and anterior cruciate ligament injury and reconstruction of the anterior cruciate ligament: Comparison of repair and nonrepair of medial collateral ligament tears in rabbits

The optimal treatment for a combined injury of the medial collateral and anterior cruciate ligaments is controversial, and the question remains as to whether repair of the medial collateral ligament and reconstruction of the anterior cruciate ligament improves healing of the medial collateral ligament. We compared reconstruction of the anterior cruciate ligament with and without repair of the medial collateral ligament in a rabbit model of a combined injury of these two ligaments. The anterior-posterior translation and varus-valgus rotation of the knee, the structural properties of the femur-medial collateral ligament-tibia complex, and the mechanical properties of the midsubstance of the medial collateral ligament were evaluated immediately after surgery and at 6 and 12 weeks postoperatively. Repair of the medial collateral ligament led to significantly less varus-valgus rotation of the knee than did no repair, but the anterior-posterior translation of the knees in the repair and nonrepair groups were not significantly different at any study time. At 12 weeks, the cross-sectional area and ultimate load in the repair group were 60 and 53% greater, respectively, than in the nonrepair group. Among 12 specimens that were repaired (six specimens at 6 weeks and six specimens at 12 weeks), failure occurred within the midsubstance in four (two at each time period); in all of the specimens that were not repaired, failure occurred at the tibial insertion site. There was no significant difference between the modulus of the midsubstance in the repaired and the nonrepaired medial collateral ligaments. Thus, the improved structural properties of the femur-medial collateral ligament-tibia complexes that were repaired resulted from an increase in cross-sectional area of the repaired medial collateral ligament and healing of the tibial insertion site. Postoperative healing time had little effect on the tensile properties. In this rabbit model, repair of the medial collateral ligament with reconstruction of the anterior cruciate ligament may lead to better healing of the medial collateral ligament in the early phase than does reconstruction of the anterior cruciate ligament alone.     

Adhesion strength differential of human ligament fibroblasts to collagen types I and III.

Fibroblasts embedded in the amorphous healing tissue matrix of ligaments migrate into damaged sites during the inflammatory process that precedes the formation of new connective tissue after ligament injury. Cell motility involved in this migration is strongly influenced by cellular adhesion to proteins of the extracellular matrix. The adhesion mechanism of interest in this study is the attachment of fibroblasts from the anterior cruciate and medial collateral ligaments to types I and III collagen, two fibrillar collagens secreted by fibroblasts during tissue repair. Types I and III collagen constitute a major portion of these ligaments and are assembled by fibroblasts into long cable-like fibrils in the extracellular space. In this study, a micropipette aspiration technique was used to measure the force required to separate fibroblasts of the anterior cruciate and medial collateral ligaments from substrates composed of type I or III collagen, each at a concentration of 2 or 5 microg/ml. Approximately 1,200 fibroblasts from the anterior cruciate ligament and 1,600 from the medial collateral ligament were used, and the adhesion force and area of these cells were determined. Fibroblasts from the anterior cruciate ligament exhibited greater adhesion force than did those from the medial collateral ligament for all concentrations of types I and III collagen. In addition, the adhesiveness of fibroblasts from both ligaments was dependent on seeding time for all experimental conditions. To determine the adhesiveness per unit area, defined here as the adhesion strength, the adhesion force was normalized by the adhesion area. At early seeding times (15-45 minutes), fibroblasts from the anterior cruciate ligament exhibited greater adhesion strength on surfaces coated with type-I collagen than did those from the medial collateral ligament. However, for both collagen substrates, adhesion strength for fibroblasts from the anterior cruciate ligament decreased with seeding time whereas that for fibroblasts from the medial collateral ligament remained relatively constant for all seeding periods (15-75 minutes).     

The effects of transection of the anterior cruciate ligament on healing of the medial collateral ligament. A biomechanical study of the knee in dogs

The effect of concurrent injury to the anterior cruciate ligament on the healing of injuries of the medial collateral ligament was studied in dogs. In Group I, isolated transection of the medial collateral ligament was performed; in Group II, transection of the medial collateral ligament with partial transection of the anterior cruciate ligament; and in Group III, complete transection of both the medial collateral ligament and the anterior cruciate ligament. The three groups of animals were examined six and twelve weeks postoperatively with respect to varus-valgus rotation of the knee and tensile properties of the femur-medial collateral ligament-tibia complex. The varus-valgus rotation of the knee was found to be the largest in Group-III specimens at all time-periods and was 3.5 times greater than the control values at twelve weeks. Group-I and Group-II specimens also showed large varus-valgus rotations at time zero, but the rotations returned to the control values by twelve weeks. For the structural properties of the femur-medial collateral ligament-tibia complex, the values for ultimate load for Groups I and II reached the control values by twelve weeks, while that for Group III remained at only 80 per cent of the control value. Both energy absorbed at failure and linear stiffness for all three groups were less than those for the controls at six weeks, and only linear stiffness returned to the control values by twelve weeks. For the mechanical (material) properties of the healed ligament substance, the values for modulus and tensile strength were markedly lower than the control values for all groups at six weeks. By twelve weeks, the tensile strength of Group-I specimens had increased to 52 per cent of the control value, while those of Groups II and III were only 45 and 14 per cent, respectively. Our results demonstrate that healing of the transected medial collateral ligament is adversely affected by concomitant transection of the anterior cruciate ligament. Both varus-valgus rotation and mechanical properties of the healed ligament failed to recover in knees that had combined transection of the anterior cruciate and medial collateral ligaments. The structural properties of the femur-medial collateral ligament-tibia complex in tension recovered more rapidly as a consequence of the large mass of reparative tissue that formed in the medial collateral ligament of the anterior cruciate-deficient knees.     

Migration and healing of ligament cells under inflammatory conditions

It is well documented that the adult human medial collateral ligament has a functional healing response, whereas the anterior cruciate ligament does not. The differential healing responses of the medial collateral and anterior cruciate ligaments could be due to factors caused by different biological conditions and locations in vivo. In addition, different intrinsic properties of the constituent cells of these ligaments may contribute to their different healing abilities. Ligament healing follows an orderly process of hemorrhage, inflammation, proliferation, and remodeling. At the cellular level, healing involves a cell's detachment from and attachment to the matrix adjacent to the wound area, migration, and proliferation. This study sought to investigate whether, during migration, the responses of the medial collateral and anterior cruciate ligament fibroblasts are intrinsically different under the same inflammatory conditions. Human medial collateral arid anterior cruciate ligament fibroblast cells were cultured, and in vitro wounds were simulated by streaking the cells with an inoculating loop, creating a cell-free area. The migration of the cells into this gap, thus filling the cell-free area, was observed. Two sets of experiments were conducted; one varied the wound width and the, other added the inflammatory factors tumor necrosis factor-a, complement C5a, and lipopolysaccharide the width of the wound increased, the rate of recovery decreased for both types of ligament cells (slope: anterior cruciate ligament, 0.13 hour/μm and medial collateral ligament, 0.10 hour/μm). Also, the three inflammatory factors used all inhibited the recovery rates of both ligaments to ones that were 1.4–2.3 times slower than controls. However, in both sets of experiments, the anterior cruciate ligament fibroblasts were more sensitive to inflammatory factors, and the medial collateral ligament fibroblasts had faster recovery rates (anterior cruciate ligament, 1.2–3.4 times slower than rates for medial collateral ligament fibroblasts, excluding those under lipopolysaccharide treatment). The results showed that medial collateral and anterior cruciate ligament fibroblasts responded differently under the same inflammatory conditions. This may suggest that these differences in intrinsic properties contribute to their different healing responses and abilities.     

Signal pathways and ligament cell adhesiveness

The influence of signal pathways involved in the adhesion of fibroblasts from the anterior cruciate and medial collateral ligaments to fibronectin was investigated. Specific emphasis was paid to the cyclic adenosine monophosphate and Ca²⁺/phospholipid pathways to determine the signaling mediated by integrin receptors during cell binding and spreading on a fibronectin-coated glass surface and to compare the roles of these two pathways in integrin-mediated adhesion in fibroblasts from the two ligaments. Individual cell adhesion strengths were determined using a micropipette-micromanipulation system after the cells were treated with signal pathway inhibiting agents. Adhesion in fibroblasts from the medial collateral ligament was significantly reduced by inhibiting agents for G_i protein, protein kinase A, protein kinase C, protein kinase G, phospholipase C, and calmodulin, which suggests a crucial role for cyclic adenosine monophosphate and Ca²⁺/phospholipid signaling in integrin-mediated adhesion of these fibroblasts. Adhesion in fibroblasts from the anterior cruciate ligament, however, was reduced only by a protein kinase C inhibiting agent and was increased by inhibiting agents for protein kinase A, protein kinase G, and calmodulin, which suggests only a partial role of Ca²⁺/phospholipid signaling in integrin-mediated adhesion of these fibroblasts. On the basis of additional parallel studies on the role of intracellular calcium in integrin-mediated adhesion, medial collateral ligament and anterior cruciate ligament fibroblast adhesion was calcium dependent throughout the 60 minute time course of adhesion experiments. Fibroblasts from the medial collateral ligament demonstrated a 2.2-fold increase in cytosolic free calcium upon binding to fibronectin, whereas fibroblasts from the anterior cruciate ligament demonstrated no significant increase in calcium. Overall, the study of the intrinsic differences between anterior cruciate ligament and medial collateral ligament fibroblasts in their signal pathways upon binding to fibronectin may reveal information important for further explaining the lack of functional healing response in the anterior cruciate ligament after injury.     

Integrin display increases in the wounded rabbit medial collateral ligament but not the wounded anterior cruciate ligament

The differential capacities of the anterior cruciate and medial collateral ligaments to heal may be related to differences in cellular function. This study tested the hypothesis that differential expression of integrins occurs in these ligaments after injury. The integrins are a family of cell surface receptors that mediate adhesion, migration, and other cellular functions critical to the healing of a wound. A similar complement and amount of the β₁ subfamily of integrins are known to be present on the unperturbed anterior cruciate and medial collateral ligaments in humans and rabbits. A partial laceration was surgically created in these two ligaments in 12 anesthetized New Zealand White rabbits. Immunohistochemistry was performed on sections from the ligaments at 1, 3, 7, and 10 days after injury, using monoclonal antibodies directed against the integrin subunits β₁, α₅, α₆, and α_v. Between 3 and 7 days, the wounded medial collateral ligament demonstrated a striking increase in staining for the β₁, α₅, and α_v subunits on the fibroblasts, within the repair site, and on capillary endothelium. Increased staining was most marked for the β₁ subunit and less marked for the α₅ and α_v subunits. The α₆ subunit stained exclusively vascular structures within the healing medial collateral ligament. In marked contrast, the anterior cruciate ligament, which does not mount an effective repair response, demonstrated no comparable alteration of integrin expression from baseline levels. This study demonstrates that increased expression of integrins occurs coincident with wound healing in the medial collateral ligament, whereas this expression remains at baseline levels in the nonhealing wounded anterior cruciate ligament. This observation suggests that a failure to alter expression of integrins subsequent to injury may play a role in the defective healing of the anterior cruciate ligament.     

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

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

Adhesiveness of human ligament fibroblasts to laminin

The adhesiveness of fibroblasts from the human anterior cruciate and medial collateral ligaments to the laminin molecule was studied, with particular emphasis on the intrinsic differences between fibroblasts from the two ligaments. Cellular adhesion strength, adhesion area, laminin concentration, and seeding time were examined. Cell adhesion to laminin anchored with poly-D-lysine to a cleaned cover glass was measured with a micropipette micromanipulation system after seeding. The adhesion strength of fibroblasts from the anterior cruciate ligament to laminin was greater than and significantly different from that of fibroblasts from the medial collateral ligament, depending on the laminin concentration. Fibroblasts from the anterior cruciate ligament also exhibited an increase in adhesion strength, dependent on laminin concentration of as much as 30 μg/ml, at which the laminin receptors were thought to be saturated. Fibroblasts from the medial collateral ligament did not show such an increase except at laminin concentrations of 5–10 μg/ml. There was no significant difference in adhesion area between fibroblasts from the two ligaments except after 45 minutes at a laminin concentration of 40 μg/ml. For both, the adhesion to laminin showed little correlation to seeding time during periods of as long as 60 minutes. Measurements of adhesion area also failed to show a significant correlation to seeding time for fibroblasts from either ligament at laminin concentrations of 20 and 40 μg/ml. Adhesion strength normalized by adhesion area had no correlation to seeding time. With all times taken into account, however, normalized adhesion strength for fibroblasts from the anterior cruciate ligament was approximately two times greater than and significantly different from that for fibroblasts from the medial collateral ligament at laminin concentrations of 20 and 40 μg/ml. Fibroblasts from the anterior cruciate ligament adhere more strongly to laminin than do those from the medial collateral ligament. This study may prove useful in the understanding of fibroblast adhesion to the basal lamina within the extracellular matrix and may provide insight into the natural healing processes of the anterior cruciate and medial collateral ligaments.     

Isolierte und kombinierte Kniebandverletzungen

Complex knee ligament injuries are characterized by simultaneous rupture of the anterior cruciate ligament (ACL) and/or the posterior cruciate ligament (PCL) and at least one collateral ligament. Isolated injury to the medial collateral ligament (MCL) and PCL have a high healing capacity and can be treated conservatively in many cases. Ruptures of the MCL can also be treated conservatively in complex injuries if the cruciate ligaments are reconstructed. Ruptures of the lateral structures usually need surgical reconstruction. Indications for acute surgical repair include meniscus dislocation, entrapment of collateral ligament portions in the joint, knee dislocation with severe knee instability, and displaced bony avulsions. The anatomy of the knee ligaments must be carefully respected in surgical reconstruction. Acute repair of collateral ligament injuries is possible only in the first 2 weeks after trauma. Acute arthroscopy is indicated only in combination with reconstructive surgery.     

Synergistic effect of growth factors on cell outgrowth from explants of rabbit anterior cruciate and medial collateral ligaments

Cellular migration and proliferation are integral aspects of wound healing. An in vitro assay for cellular migration and proliferation using explants of rabbit anterior cruciate and medial collateral ligaments was developed previously. This study presents the effects of serum-free culture medium supplemented with basic fibroblast growth factor, bovine insulin, transforming growth factor-β1, and platelet-derived growth factor-B, added either individually or in combination, on cell outgrowth in explants of rabbit anterior cruciate and medial collateral ligaments. Outgrowth was assessed at 3 and 6 days by counting the number of cells surrounding the tissue explants. For explants of both ligaments, cell outgrowth was dependent on the presence of 10% fetal bovine serum or the combination of growth factors. Little outgrowth occurred in explants of either ligament in the presence of basic fibroblast growth factor, transforming growth factor-β1, or bovine insulin. Platelet-derived growth factor-B at concentrations of 20 and 100 ng/ml seemed to increase cell outgrowth from medial collateral ligament explants at 6 days. The outgrowth from the explants of both ligaments was much greater in the presence of all four growth factors than the sum of the outgrowth with the individual factors. This synergistic effect was as much as 10-fold and 20-fold for the anterior cruciate ligament explants at days 3 and 6, respectively, but no more than 3-fold for the medial collateral ligament explants at these times. Medial collateral ligament explants exhibited greater cell outgrowth than anterior cruciate ligament explants in 10% serum and in the presence of the four growth factors.     

Effect of growth factors on the proliferation of fibroblasts from the medial collateral and anterior cruciate ligaments

Growth factors have been shown to stimulate fibroblast division and thus may influence ligament healing. We analyzed the effects of individual growth factors on the proliferation of fibroblasts from the medial collateral and anterior cruciate ligaments of the rabbit in vitro in order to identify growth factors that might enhance proliferation of fibroblasts and to compare the responses of the fibroblasts from the two ligaments to these growth factors. Through measurement of the uptake of [³H]-thymidine into DNA, fibroblasts from these ligaments that had been treated with epidermal growth factor and basic fibroblast growth factor were found to proliferate nearly eight times more than control fibroblasts. Additionally, the fibroblasts of both ligaments proliferated at similar rates when exposed to platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, insulin-like growth factor-1, and interleukin-1-alpha. However, epidermal growth factor and transforming growth factor-beta caused the fibroblasts from the medial collateral ligament to proliferate at a rate 1.3–1.4 times greater than that of fibroblasts from the anterior cruciate ligament. The reverse was true with acidic fibroblast growth factor, which stimulated the fibroblasts from the anterior cruciate ligament to proliferate at a rate 1.3–1.6 times greater than that of fibroblasts from the medial collateral ligament. This study demonstrated that growth factors can stimulate cell division in ligaments and may be effective in enhancing ligament healing but that these differences were not great enough to explain fully the clinical differences observed between healing of the medial collateral and anterior cruciate ligaments.     

An irreducible knee dislocation: a case report

An 8-year-old man presented after sustaining an injury during a fall. A closed reduction attempt failed, and after several tests, an open reduction was performed. With posterolateral dislocation of the knee, there can be anterior cruciate ligament, posterior cruciate ligament, and medial collateral ligament disruption. At the 6-month (final) follow-up, the patient had no subjective pain or instability. With this type of injury, the approach can be conservative monitoring or repair of all of the ligaments. Because of the age and activity level of our patient, we opted for repair of the medial collateral ligament initially with the possibility of late anterior cruciate ligament and/or posterior cruciate ligament reconstruction.     

Rupture of superficial pes anserinus and partial rupture of patellar ligament as rare concomitant lesions of complex knee joint injuries

We present a rare case of combined knee joint lesions in a 25 year old patient. Besides the commonly reported injuries of the knee joint due to directly applied valgus force, forceful quadriceps muscle contraction, external rotation at flexed knee causing combined lesions such as rupture of the anterior cruciate ligament, rupture of the posterior cruciate ligament and rupture of the medial collateral ligament, a rare combination of the above mentioned lesions and a rupture of the lateral meniscus, an osteochondral fracture of the lateral femur condylus, a rupture of the medial patellofemoral retinacula as well as a complete rupture of the superficial pes anserinus and a partial rupture of the patellar ligament was encountered.     

Effects of joint load on the stiffness and laxity of ligament-deficient knees. An in vitro study of the anterior cruciate and medial collateral ligaments

We measured the effects of serial section of the medial collateral ligament and anterior cruciate ligament and of the anterior cruciate ligament and medial collateral ligament on anterior-posterior force-versus-displacement and tibial torque-versus-rotation response curves for seven fresh frozen cadaver knees at zero and 20 degrees of flexion before and after application of as much as 925 newtons of compressive load on the tibiofemoral joint. Section of the anterior cruciate ligament always increased anterior laxity in an unloaded specimen; joint load reduced this increase by a greater amount at zero degrees than at 20 degrees of flexion. Joint load was more effective in limiting anterior laxity in anterior cruciate-deficient specimens at low levels of applied anterior force; at higher levels of applied force, the effects of joint congruency were overcome and ligament restraints came into play. Section of the medial collateral ligament increased anterior laxity in an unloaded knee only for specimens in which the anterior cruciate ligament had been previously sectioned; joint load eliminated this increase at full extension but did not do so at 20 degrees of flexion. The medial collateral ligament was the more important of the two ligaments in controlling torsional laxity. Secondary section of either ligament (the other ligament having been sectioned first) produced a greater increase in laxity than did primary section of that ligament in an intact knee. Increases in torsional laxity due to primary section of either ligament were unaffected by the application of joint load. Joint load reduced increases in laxity that were due to secondary section of the medial collateral ligament.     

Maturation of composite ligament

A variety of synthetic fibrillar ligaments are being developed and clinically investigated. The tissue response to the variety of polymeric fibrils is similar. The soft-tissue response to carbon fiber braid substituting for lateral collateral ligament in three human knees and for the anterior cruciate ligament in one was examined 12-42 months after surgery. An advanced stage of maturity of the composite structure was particularly evident by dense bundles of collagen in one of the collateral ligaments and in the substitute for the anterior cruciate ligament. Associated with the newly formed collagen the carbon fibril initially formed unique carbon collagen of composite structural units. Eventually, subsidence of the fibroblast response was accompanied by an increase in density and width of the collagen fibers and by a loss of the configuration of the carbon-collagen units. Even the mature collagenous structure was not comparable to the natural ligament.     

Effect of growth factors on matrix synthesis by ligament fibroblasts

Although it has been reported that several growth factors modulate soft-tissue healing, the specific effects of growth factors on protein synthesis during ligament healing have not been widely investigated. In this study, we examined the effects of basic and acidic fibroblast growth factors, transforming growth factor β1, and epidermal growth factor on collagen and noncollagenous protein synthesis by cultured fibroblasts from medial collateral ligament and anterior cruciate ligament in vitro. Uptake of tritiated proline was used to measure synthesis of collagen and noncollagenous protein, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the type of collagens synthesized. Our data showed that transforming growth factor β1 increased both collagen and noncollagenous protein synthesis by medial collateral and anterior cruciate ligament fibroblasts on a dose-dependent basis. Collagen synthesis by cultured fibroblasts from the medial collateral and anterior cruciate ligaments was increased by treatment with transforming growth factor β1 by as much as approximately 1.5 times that of untreated controls. Although the response to transforming growth factor β1 by anterior cruciate ligament fibroblasts was equal to that by medial collateral ligament fibroblasts, the amounts of matrix proteins synthesized by anterior cruciate ligament fibroblasts were approximately half of that by medial collateral ligament fibroblasts. The increase was mostly in type-I collagen. Treatment of anterior cruciate ligament fibroblasts with epidermal growth factor increased collagen synthesis by approximately 25% but had little effect on medial collateral ligament fibroblasts. Neither basic nor acidic fibroblast growth factor increased either collagen or noncollagenous protein synthesis. These findings suggest that topical application of transforming growth factor β1, alone or in combination with epidermal growth factor, may have the potential to strengthen the ligament by increasing matrix synthesis during its remodeling and healing processes.     

Increased expression of the β1, α5 and αv 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 β₁, α₅, α₆, and α_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 β₁, α₅, and α_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 β₁ subunit protein in both ligaments from knees that were stress deprived for 9 and 12 weeks, as compared with the control ligaments. These studies establish that stress deprivation in this animal model is accompanied by increased expression of integrin cell-surface receptors containing the β₁, α₅, and α_v subunits on anterior cruciate and medial collateral ligament fibroblasts, with a time course similar to that previously reported for indices of extracellular matrix remodeling induced by stress deprivation. This outcome suggests an important role for these integrins in the remodeling of stress-deprived ligaments.