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.

Origin of replacement cells for the anterior cruciate ligament autograft.

A rabbit model for anterior cruciate ligament (ACL) reconstruction using autogenous avascular patellar tendon (PT) was utilized to study the early events of graft incorporation. Histological observations demonstrated that autografts were centrally acellular with a peripheral rim of cells at 2 weeks, a central focal proliferation of cells at 3 weeks, and a cellular homogeneous distribution by 4-weeks postoperation. Graft necrosis followed by cellular proliferation suggested that a different population of cells other than the native PT fibroblasts may be inhabiting the graft. The extrinsic contribution of cells was studied by selective destruction of native PT cells with liquid nitrogen immersion prior to reconstruction of the ACL. The intrinsic contribution of cells was evaluated by sequestration of the PT graft in a semipermeable membrane before it was used to reconstruct the ACL. Histological analysis of tissue that was liquid N2 treated, used as an autograft, and then harvested 3-weeks postoperation revealed fibroblastic incorporation of the graft. In contrast, no cells were observed in semipermeable membrane sequestered autografts. These data suggest that autogenous ACL autografts of PT origin are repopulated by cells of external origin. In vitro control studies that were carried out in parallel demonstrated that PT fibroblasts could survive in tissue culture, but not in the synovial environment of the ACL. This suggests that fibroblasts from different sources have different, tissue-specific nutritional requirements.

Residual muscular swelling after repetitive eccentric contractions.

This investigation illustrates the morphological changes that take place following eccentric exercise and correlates those changes with intramuscular pressure readings. Eight healthy male subjects were asked to exercise their right lower leg anterior compartment eccentrically and their left concentrically. Four hundred submaximal contractions were performed in each exercise regimen over a 20-min period against a load corresponding to 15% of the individual's maximal dorsiflexion torque. Tissue fluid pressures were measured by the slit catheter technique before, during, and after exercise and 48 h later. Needle biopsies of both anterior tibialis muscles were also taken 48 h after completion of the exercise regimens. Overall morphology of the specimens revealed a greater cross-sectional fiber area (both type 1 and type 2) in the eccentrically exercised muscle as compared with the concentrically exercised muscle. Scant evidence of inflammation (only 1 of 8 of the "eccentric" muscle samples) and no fiber necrosis was observed. Fiber type proportions were equal on both sides and type 1 fiber biased (70%). Extremely large type 2 fibers were found in 4 of 8 subjects from the eccentric specimens. This incidence correlated significantly with the length of the time to return to resting pressure after eccentric exercise (r = 0.93, p less than 0.001). The percentage of water content was significantly higher in the eccentrically exercised muscle. Based on these findings, we conclude that muscle fiber swelling is a predominant feature following eccentric exercise and is directly associated with delayed muscle soreness.

New experimental procedures to evaluate the biomechanical properties of healing canine medial collateral ligaments.

Both clinical and animal studies have indicated that early mobilization and exercise may improve the healing of injured medial collateral ligaments (MCLs). To investigate these effects, transected canine MCLs were subjected to three different treatment regimens: (a) no surgical repair with 6 weeks mobilization; (b) surgical repair with 3 weeks immobilization followed by 3 weeks remobilization; and (c) surgical repair with 6 weeks immobilization. After sacrifice, knee laxity was measured in a newly designed varus-valgus (V-V) laxity device. Each knee was tested subsequently in tension to determine the structural properties of the femur-MCL-tibia complex (FMTC) and the mechanical properties of the healing MCL substance. It was found that the V-V laxity increased for all experimental knees, but that early mobilization enhanced joint stability. The structural properties of the FMTC and the mechanical properties of the MCL substance were also lower than the contralateral controls. Again, the early mobilization groups had better results.

Early histologic, metabolic, and vascular assessment of anterior cruciate ligament autografts.

A rabbit model for anterior cruciate ligament (ACL) reconstruction using autogenous patellar tendon was utilized to study the early events of autograft cellular dynamics. Biochemical, autoradiographic, histological, and vascular injection techniques demonstrated that the native autograft cell population rapidly necroses. This repopulation occurs without a vascular contribution; cells entering the autograft are reliant upon synovial fluid nutrition.

Tensile properties of the medial collateral ligament as a function of age.

The biomechanical properties of the rabbit medial collateral ligament (MCL) as a function of maturation and age were investigated. Femur-MCL-tibia (F-M-T) preparations were obtained from rabbits of different age groups (open or closed epiphysis). Parallel increases in the animal body weight and ligament cross-sectional area were recorded with age. Cyclic and tensile failure tests were performed to obtain the structural properties of the F-M-T complex and the mechanical properties of the MCL substance. There were significant increases in the load at failure, energy-absorbing capability of the bone-ligament junction, and in the tensile strength of the ligament substance as a result of maturation and subsequent aging. Increases in the area of hysteresis obtained during cyclic loading-unloading were also documented. At the closing of the epiphysis, the mode of failure of the F-M-T structure progressed from tibial avulsion to failure in the midsubstance of the ligament. An asynchronous rate of maturation was observed between the structural properties of the bone-ligament complex and the mechanical properties of the ligament substance.

The phenomenon of "ligamentization": anterior cruciate ligament reconstruction with autogenous patellar tendon.

Reconstruction of the anterior cruciate ligament (ACL) with patellar tendon (PT) is a common procedure for the symptomatic ACL-deficient knee. Questions regarding graft incorporation, viability, and nutrition of the transplanted tissue are of concern. This relates to the graft's response to its new intrasynovial milieu and new physical forces. These factors were studied in a rabbit model of ACL reconstruction using PT and were evaluated with histological and biochemical parameters with respect to time. A histological and biochemical metamorphosis of the grafted PT occurred in this study. Autografts demonstrated a gradual assumption of the microscopic properties of normal ACL; by 30 weeks postoperatively, cell morphology was ligamentous in appearance. Normally, type III collagen is not observed in PT, however, a gradual increase in its concentration was seen in the grafts; by 30 weeks its concentration (10%) was the same as in normal ACL. Similarly, glycosaminoglycans content increased from its normally low level in PT to that found in native ACL. Collagen-reducible crosslink analysis demonstrated that grafted tissue changed from the normal PT pattern of low dihydroxylysinonorleucine (DHLNL) and high histidinohydroxymerodesmosine (HHMD) to the pattern seen in normal ACL (high DHLNL and low HHMD) by 30 weeks. These data suggest that when PT is placed in the anatomic and environmental milieu of the ACL, a "ligamentization" of the grafted tissue results; also the autograft initially depends on synovial fluid nutrition, as revascularization occurs after 6 weeks.

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 beta 1 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 beta 1, alpha 5, alpha 6, and alpha v. Between 3 and 7 days, the wounded medial collateral ligament demonstrated a striking increase in staining for the beta 1, alpha 5, and alpha v subunits on the fibroblasts, within the repair site, and on capillary endothelium. Increased staining was most marked for the beta 1 subunit and less marked for the alpha 5 and alpha v subunits. The alpha 6 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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 micrograms/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 micrograms/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 micrograms/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 micrograms/ml. Adhesion strength normalized by adhesion area had no correlation to seeding time.(ABSTRACT TRUNCATED AT 250 WORDS)

Immobilization of the knee joint alters the mechanical and ultrastructural properties of the rabbit anterior cruciate ligament.

The effects of immobilization of the knee joint on the mechanical and ultrastructural properties of the anterior cruciate ligament have not been well documented. Our goal was to determine these effects in a rabbit model and to assess the effect of knee flexion angle during immobilization. The knee joint was immobilized in either 170 degrees or 105 degrees of flexion, and new methodologies were utilized to determine the mechanical properties of the anterior cruciate ligament. In specimens from knees that had been immobilized, the cross-sectional area of the ligament was 74% of the control value. The stress-strain curve was altered slightly, and the strain at failure increased 32-40%. The modulus and stress at failure did not decrease significantly. There was no significant difference between the mechanical properties of the knees immobilized at 170 degrees and 105 degrees of flexion. Histological and ultrastructural evaluation demonstrated changes in the shape and intracellular make-up of the fibroblasts from the ligament after immobilization. This cellular response may account for the alterations in the mechanical properties of the anterior cruciate ligament.

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 Gi 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.

Measurement of mechanical properties of ligament substance from a bone-ligament-bone preparation.

This investigation presents a new approach in the measurement of the mechanical properties of the ligament substance from tensile testing of a bone-ligament-bone complex. Such basic information should be one of the necessary prerequisites in the evaluation of ligament repair as well as reconstruction by autogenous tissue grafts or artificial ligament implants. The use of a video system permits the determination of tensile strains of the mid-medial collateral ligaments from the canine, swine, and rabbit without mechanically interfering with the ligament deformation during testing. This methodology further eliminates the difficulties of measuring the initial length of the entire medial collateral ligament, as its insertions to bones are ambiguous and cover a large area. It was found that the strain of the ligament substance is consistently and considerably less than specific deformation of the bone-ligament-bone complex. These data suggest that the ligament-bone structure stretches nonuniformly with the highest deformation occurring near or at the ligament insertion sites to bone. Other interesting findings include the variation of tensile strains along the ligament substance for all animal species studied.

Delayed use of hyperbaric oxygen for treatment of a model anterior compartment syndrome.

This study examines the effect of delayed exposure to hyperbaric oxygen on muscle necrosis and edema development following compartment syndromes in the canine hindlimb. Compartment syndromes (100 mm Hg for 8 h) were generated in one anterolateral compartment of six anesthetized dogs. After a 2-h delay, three 1-h hyperbaric oxygen treatments (2 atm absolute pure oxygen) were given during the next 12 h. Two days later, technetium-99m stannous pyrophosphate (99mTc Sn-PYP) was injected intravenously; 3 h later, samples were obtained from the pressurized and contralateral control muscles, weighed for edema development, counted for 99mTC Sn-PYP uptake, and evaluated histologically. Hyperbaric oxygen treatments, even when delayed 2 h, reduced muscle necrosis and intramuscular edema to negligible levels (p less than 0.05) compared with untreated animals. In addition, muscle morphology remained essentially normal in all hyperbaric oxygen-treated animals. We conclude that even if hyperbaric oxygen treatments are delayed 2 h, edema and muscle necrosis are reduced significantly in a model compartment syndrome.

Flexor tendon repair.

Healing canine flexor tendons treated with either total immobilization, delayed protected mobilization, or early protected mobilization was studied by biomechanical, microangiographic, biochemical, and histologic techniques at intervals through 12 weeks. The healing characteristics of the early mobilization tendons showed higher tensile strengths and improved gliding function than the delayed mobilization and immobilization tendons. Protected passive motion brought about accelerated changes in peritendinous vessel density and configuration, as well as increased repair site total DNA content. While adhesions obliterated the space between the tendon surface and the tendon sheath of the immobilized repairs, the mobilized tendons demonstrated coverage of the repair site by cells from the epitenon by 10 days, and a smooth, gliding surface that was maintained free of adhesions through 42 days. A series of in vitro studies demonstrated the cellular processes involved in the repair: phagocytosis of cellular debris and collagenous fragments by cells from the epitenon, and collagen synthesis primarily by endotenon cells.

Synovial fluid nutrient delivery in the diathrial joint: an analysis of rabbit knee ligaments.

The role of synovial fluid in providing nutrition to rabbit knee ligaments and menisci was evaluated by intraarticular injection of a labeled collagen precursor, tritiated proline. Incorporation of this substrate as tritiated hydroxyproline was measured in collateral and cruciate ligaments and menisci. The injectate volume (0.35 ml) did not appreciably change the overall joint pressure as measured by a wick catheter; therefore, no alteration of synovial membrane diffusion characteristics resulted. The concentration of the injected proline (0.52 mg%) was well below that normally present in serum (2.65 mg%). Therefore, incorporation of this substrate was not driven by a concentration gradient and represented normal uptake of synovial fluid and physiological incorporation of label as measured by the presence of tritiated hydroxyproline. Autoradiography was performed on all ligaments and menisci, and demonstrated concentration of the isotope and its metabolite (tritiated proline and tritiated hydroxyproline, respectively) in and around fibroblasts. This study indicates that rabbit knee ligaments and menisci can derive nutrition from a synovial fluid source.

Injury of the anterior cruciate ligament: the role of collagenase in ligament degeneration.

Rapid degeneration of the anterior cruciate ligament (ACL) has been observed following acute ACL rupture. An understanding of this process might explain some of the poor clinical results of primary ACL repair. We created a surgical rabbit model of acute ACL injury and developed an in vitro assay for collagenase activity in the ACL and menisci. Microscopic evaluation revealed a rapidly degenerative process in injured ACLs, with loss of cellularity and matrix organization. This was associated with a significant increase in collagenase activity and a decrease in total collagen of the injured ACLs as compared with sham-operated controls. These findings confirm the observation that cut ACL ligament ends rapidly degenerate. This degenerative process might be partly due to a response of cells intrinsic to the ACL to injury. Left unchecked, this process may be detrimental to surgical attempts for primary ACL repair.

Use of patellar tendon autograft for anterior cruciate ligament reconstruction in the rabbit: a long-term histologic and biomechanical study.

To assess the degree of success of anterior cruciate ligament (ACL) replacement using the patellar tendon (PT) autograft, 29 New Zealand white rabbits underwent ACL reconstruction using a medial one-third PT autograft. The femur-ligament-tibia complexes were evaluated at 0, 6, 30, and 52 weeks postoperatively for gross and histologic appearances and tensile load to failure properties. Grossly, the autografts did not resemble the control ACLs. Histologically, the autografts progressed from being hypercellular with a random collagen fiber bundle organization to having a near normal cellularity with a more parallel collagen fiber bundle pattern. Anteroposterior knee laxity was more than two times that of the control knees 52 weeks after reconstruction. Biomechanically, the PT autografts plateaued at 30 weeks postoperatively. The ultimate load and stiffness were 15 +/- 5% and 24 +/- 6% of the control ACLs, respectively. At 52 weeks, the appearance of the PT autograft had some general histologic similarities as compared with the native ACL. However, these similarities did not extend to the functional properties of the autograft.

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

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

Characterization of arthroplasty tissue after 14 years post-cup arthroplasty: a morphological and biochemical assessment.

The cup arthroplasty has been reported to cause the formation of a fibrocartilaginous joint surface, which may result in a painless, functional joint. The joint surface of a 38-year-old man with a failed cup arthroplasty implanted for 14 years was examined histologically and biochemically. The joint surface tissue of this patient resembled fibrous connective tissue, with major types of collagen being Type I and Type III. No evidence of cartilaginous transformation in the healing scar was demonstrated, despite several years of successful functioning of the cup arthroplasty.

Flexor tendon repair in vitro: a comparative histologic study of the rabbit, chicken, dog, and monkey.

Flexor tendon healing in four different animal species was explored in a tissue culture system. Ninety percent transverse lacerations were made in 88 tendon segments obtained from rabbits, chickens, dogs, and monkeys. The tendons were removed from culture and studied by light and electron microscopy at intervals of 3, 6, 9, and 12 weeks. A characteristic sequence of repair including epitenon thickening, cellular differentiation, cell migration, and phagocytosis was seen in each of the repaired tendons. The endotenon cells of several animal tendons appeared to be synthesizing collagen. There was a consistent difference in the rate of healing between the four species. The rabbit tendons demonstrated nearly complete closure of the repair site by 12 weeks. A lesser response was seen in the chicken, followed by the dog and monkey. The differences in healing rate appeared to be due to the non-species-specific in vitro culture media. The in vitro flexor tendon culture system is particularly useful in studying the tendon repair responses of various species with the contributions of vascularity and synovial cells excluded.