Differential metabolic responses of periarticular ligaments and tendon to joint immobilization.

Parameters of collagen metabolic behavior were analyzed in the periarticular connective tissues, i.e., medial collateral ligament (MCL), anterior cruciate ligament (ACL), and patellar tendon (PT), of control and immobilized rabbit knees. Two periods of immobilization were studied: 9 and 12 wk. Collagen turnover and collagen cross-links were quantitatively assessed in the three tissues. The results showed that after 9 wk both synthesis and degradation were significantly increased in the MCL and ACL, whereas the PT showed lesser effects. After 12 wk all three tissues experienced significant losses of collagen mass, which resulted in tissue atrophy. The concentrations of the reducible collagen cross-links dihydroxylysinonorleucine and hydroxylysinonorleucine in the immobilized MCL and ACL were greater than their respective controls, indicating an increase in collagen synthesis, whereas concentrations of the nonreducible cross-link hydroxypyridinoline were observed to be decreased in these tissues. Of the reducible cross-links in the PT, only hydroxylysinonorleucine was found to be increased over control, whereas hydroxypyridinoline was slightly less concentrated. These results taken together have demonstrated that the ligamentous tissues are more susceptible to the effects of stress deprivation secondary to joint immobilization than the PT, and, in particular, the ACL of the three tissues studied appears to be most vulnerable.     

Collagen fibril D-period may change as a function of strain and location in ligament

The purpose of this study was to determine if the characteristic banding pattern (D-period) of collagen fibrils from rabbit medial collateral ligaments changes as a function of gross ligament strain and, if so, whether the changes are location dependent (insertion versus midsubstance). Femur–medial collateral ligament–tibia complexes were strained to 0, 8, or 12% and immediately chemically fixed in situ. Samples were taken from the medial collateral ligament midsubstance and bony insertions, and prepared for and observed under a transmission electron microscope. D-period length was measured and found to increase (albeit not significantly so, p=0.1) as a function of gross strain for samples obtained from the insertion sites but not for samples obtained from the ligament midsubstance. Results suggested that ligament strains are inhomogeneous at the ultrastructural level.     

A role for calcitonin gene-related peptide in rabbit knee joint ligament healing

Knee joint ligament healing has been shown to be improved when the torn ligament ends remain in contact, however, the rationale for these effects is unknown. The sensory neuropeptide calcitonin gene related peptide (CGRP) has potent trophic and vasodilatatory properties and as such is thought to be advantageous in wound repair. In ascertaining a role for CGRP in rabbit medial collateral ligament healing, the present study examined changes in CGRP-like immunoreactivity (CGRP-LI) and CGRP-mediated vasomotor responses in gap injured (non-contact), Z-plasty apposed (contact), and sham operated control medial collateral ligaments. At 6 weeks post-trauma, CGRP-LI decreased in the healing zone of gap injured and Z-plasty apposed medial collateral ligaments compared with controls, and non-contact ligament nerve fibres exhibited an abnormal morphology. Topical administration of CGRP (10(-13) to 10(-9) mol) caused a dose-dependent increase in ligament perfusion in each experimental group of knees. The CGRP-mediated vasodilatation associated with gap injured ligaments was not significantly different from controls (P = 0.06), whereas apposed medial collateral ligaments showed an augmented response to the peptide (P < 0.0005). These findings indicate that the beneficial effects of ligament interposition post-trauma may be related to an enhanced responsiveness to CGRP in conjunction with a more typical re-innervation profile. Conversely, the aberrant characteristics of CGRP-LI nerves occurring in gap injured tissue is suggestive of impaired CGRP release which may explain the poor functional recovery associated with these ligaments.     

Matrix metalloproteinase-13 expression in rabbit knee joint connective tissues: influence of maturation and response to injury.

The hypothesis of the present work was that expression of matrix metalloproteinase-13 (MMP-13, collagenase-3) would be induced during conditions involving important matrix remodeling such as ligament maturation, scar healing and joint instability. Therefore, MMP-13 expression in the medial collateral ligament (MCL) during the variable situations of tissue maturation and healing was assessed. MMP-13 expression in three intra-articular connective tissues of the knee (i.e. articular cartilage, menisci and synovium) following the transection of the anterior cruciate ligament of the knee was evaluated at 3 and 8 weeks post-injury. MMP-13 mRNA (semi-quantitative RT-PCR) and protein (immunohistochemistry and Western blotting) were detected in all of the tissues studied. Significantly higher MCL mRNA levels for MMP-13 were detected during the early phases of tissue maturation (i.e. 29 days in utero and 2-month-old rabbits) compared to later phases (5- and 12-month-old rabbits). This pattern of expression was recapitulated following MCL injury, with very high levels of expression in scar tissue at 3 weeks post-injury and then a decline to levels not significantly different from control values by 14 weeks. Elevated mRNA levels correlated with increased protein levels for MMP-13 in both menisci and synovium following the transection of the anterior cruciate ligament and during medial collateral ligament healing. These results indicate that MMP-13 expression is regulated by a number of variables and that high levels of expression occur in situations when connective tissue remodeling is very active.     

Biomechanical studies of the rabbit patellar tendon after removal of its one-fourth or a half.

Effects of the overstressing induced by the harvest of grafts from the patellar tendon on the mechanical properties and morphometry of remaining tendon were studied using a rabbit model. The width of the patellar tendon was reduced by one-fourth or one-half equally removing the medial and lateral portions; by this surgery, the cross-sectional area was decreased by 25 or 50 percent from the original area. After all the rabbits were allowed unrestricted activities in cages for 3 to 12 weeks, their patellar tendons were harvested for mechanical and histological studies. The one-fourth removal induced no significant changes in the mechanical properties, but significantly increased the cross-sectional area. In the case of one-half removal, tensile strength and tangent modulus did not change in some tendons, although the cross-sectional area increased significantly. In the other central half tendons, mechanical strength decreased markedly, while the cross-sectional area increased; hypercellular areas and breakage of collagen bundles were observed in these tendons. These results indicate that the patellar tendon has an ability of functionally adapting to overstressing by changing the cross-sectional area, while keeping the mechanical properties unchanged, if the extent of overstressing is less than 30 percent.     

Plasminogen activators and plasminogen activator inhibitors in connective tissues and connective tissue cells: influence of the neuropeptide substance P on expression

Tissue segments isolated from ligament, epiligament, and synovial tissues from mature female New Zealand White Rabbits were demonstrated to consitutively secrete a plasminogen activator. Several tissues were also observed to constitutively secrete a plasminogen activator inhibitor which was detected in the form of a PA-PAI complex. Heterogeneity was observed in PA and PAI activity between the different connective tissues. Heterogeneity also existed between and within the medial collateral (MCL), lateral collateral (LCL), and the anterior cruciate (ACL) ligaments. In addition to the differences in constitutive expression of PA and PAI activity, differences in the responsiveness to the neuropeptide substance P (10^?5?10^?9 M) were also detected. This responsiveness to substance P was displayed by an increase in PA and PAI activity in the conditioned medium. The pattern of responsiveness reflected the degree of innervation of these tissues. That is, synovium and epiligament tissue were the most responsive tissues to substance P while the MCL, LCL and ACL were less responsive to the neuropeptide. Parallel results were obtained using cell culture with fibroblasts isolated from the above mentioned tissues. That is, the pattern of responsiveness was similar between cells and tissue segments. More specifically, cells isolated from both synovium and epiligament increased their both their PA (slightly) and PAI activity following exposure to substance P. This was demonstrated at both the protein and RNA level. Thus, cells within a tissue maintain their phenotype when removed from their three-dimensional matrix. These results are unique in demonstrating that normal ligament and synovial cells and tissue respond to substance P by altering the expression of PA and PAI activity. This investigation further supports the concept that innervation may be important in normal connective tissue function.     

Relationships between the size and spatial morphology of human masseter and medial pterygoid muscles, the craniofacial skeleton, and jaw biomechanics

The relationship between human craniofacial morphology and the biomechanical efficiency of bite force generation in widely varying muscular and skeletal types is unknown. To address this problem, we selected 22 subjects with different facial morphologies and used magnetic resonance imaging, cephalometric radiography, and data from dental casts to reconstruct their craniofacial tissues in three dimensions. Conventional cephalometric analyses were carried out, and the cross-sectional sizes of the masseter and medial pterygoid muscles were measured from reconstituted sections. The potential abilities of the muscles to generate bite forces at the molar teeth and mandibular condyles were calculated according to static equilibrium theory using muscle, first molar, and condylar moment arms. On average, the masseter muscle was about 66% larger in cross section than the medial pterygoid and was inclined more anteriorly relative to the functional occlusal plane. There was a significant positive correlation (P less than 0.01) between the cross-sectional areas of the masseter and medial pterygoid muscles (r = 0.75) and between the bizygomatic arch width and masseter cross-sectional area (r = 0.56) and medial pterygoid cross-sectional area (r = 0.69). The masseter muscle was always a more efficient producer of vertically oriented bite force than the medial pterygoid. Putative bite force from the medial pterygoid muscle alone correlated positively with mandibular length and inversely with upper face height. When muscle and tooth moment arms were considered together, a system efficient at producing force on the first molar was statistically associated with a face having a large intergonial width, small intercondylar width, narrow dental arch, forward maxilla, and forward mandible. There was no significant correlation between muscle cross-sectional areas and their respective putative bite forces. This suggests that there is no simple relationship between the tension-generating capacity of the muscles and their mechanical efficiency as described by their spatial arrangement. The study shows that in a modern human population so many combinations of biomechanically relevant variables are possible that subjects cannot easily be placed into ideal or nonideal categories for producing molar force. Our findings also confirm the impression that similar bite-force efficiencies can be found in subjects with disparate facial features.     

Structural properties of the medial collateral ligament complex of the human knee

The anatomy of the medial collateral ligament (MCL) complex consists of three identifiable passive restraining structures: the longitudinal fibres of the superficial medial collateral ligament (sMCL), the deep medial collateral ligament (dMCL), and the posteromedial capsule (PMC). The purpose of this study was to measure and compare the structural properties of these three individual structures. Eight human cadaveric knees (age 72-89 years, mean = 77 years, S.D. 5.3) were harvested and bone-ligament-bone tensile testing specimens prepared. After preconditioning, the specimens were extended to failure at 1000 mm/min in an Instron tensile testing machine. Ligament bundles failed either mid-substance or at their bony attachments. The ligament bundles had maximum loads of 534 N (sMCL), 194 N (dMCL), 425 N (PMC) and failed at 10.2, 7.1, and 12.0 mm mean extension, respectively. The maximum load and linear stiffness of the sMCL were significantly higher than those of the dMCL but not the PMC. The maximum load of the PMC was significantly higher than that of the dMCL; the linear stiffness of the PMC was higher than that of the dMCL but this did not reach statistical significance. The dMCL failed at a significantly lower extension than the other structures. The sMCL bundles that failed at their bony attachment were remounted using a freezing clamp fixture and again extended to failure, resulting in mid-substance failure at 884 N (74% higher). This study has shown that the PMC of the knee has comparable structural properties to the long superficial MCL and the short, deep MCL. In summary, the structural properties of the different component structures of the medial ligament complex indicate possible functional significance.     

Tensile properties, collagen content, and crosslinks in connective tissues of the immature knee joint

Background

The major connective tissues of the knee joint act in concert during locomotion to provide joint stability, smooth articulation, shock absorption, and distribution of mechanical stresses. These functions are largely conferred by the intrinsic material properties of the tissues, which are in turn determined by biochemical composition. A thorough understanding of the structure-function relationships of the connective tissues of the knee joint is needed to provide design parameters for efforts in tissue engineering.

Methodology/Principal Findings

The objective of this study was to perform a comprehensive characterization of the tensile properties, collagen content, and pyridinoline crosslink abundance of condylar cartilage, patellar cartilage, medial and lateral menisci, cranial and caudal cruciate ligaments (analogous to anterior and posterior cruciate ligaments in humans, respectively), medial and lateral collateral ligaments, and patellar ligament from immature bovine calves. Tensile stiffness and strength were greatest in the menisci and patellar ligament, and lowest in the hyaline cartilages and cruciate ligaments; these tensile results reflected trends in collagen content. Pyridinoline crosslinks were found in every tissue despite the relative immaturity of the joints, and significant differences were observed among tissues. Notably, for the cruciate ligaments and patellar ligament, crosslink density appeared more important in determining tensile stiffness than collagen content.

Conclusions/Significance

To our knowledge, this study is the first to examine tensile properties, collagen content, and pyridinoline crosslink abundance in a direct head-to-head comparison among all of the major connective tissues of the knee. This is also the first study to report results for pyridinoline crosslink density that suggest its preferential role over collagen in determining tensile stiffness for certain tissues.     

A comparative evaluation of the mechanical properties of the rabbit medial collateral and anterior cruciate ligaments.

The biomechanical properties of the medial collateral and anterior cruciate ligaments from 30 New Zealand White rabbits were measured. Because of its complex geometry, the ACL was divided into two portions (medial and lateral) to provide uniform loading. This allowed an examination of the intra-ligamentous properties. A laser micrometer system was used to measure the cross-sectional area for tensile stress and a video dimension analyzer was used to measure the strain. The mechanical properties (stress-strain curves) of the MCL and ACL were different, with the modulus (determined between 4 and 7% strain) in the MCL (1120 +/- 153 MPa) more than twice that of either portion of the ACL (516 +/- 64 and 516 +/- 69 MPa for the medial and lateral portions, respectively). This higher modulus correlated with the more uniform and dense appearance of the collagen fibrils examined with scanning electron microscopy (SEM).     

Deficiency of macrophage migration inhibitory factor gene delays healing of the medial collateral ligament: a biomechanical and biological study

The role played by macrophage migration inhibitory factor (MIF) in the process of wound healing is controversial. Besides, there have been no reports that investigated the expression or the role of MIF in the repair process after ligament injury. In this study, we hypothesized that the deficiency in MIF gene might delay ligament healing in mice. The aim of this study was to clarify this hypothesis using MIF gene-deficient mice (MIFKO) and murine model of injury to the medial collateral ligament (MCL). Biomechanical testing showed that the levels of mechanical properties were significantly lower in MIFKO than in wild-type mice (WT) on day 28 after injury. Levels of matrix metalloproteinase (MMP)-2 and -13 mRNA in the healing tissue were significantly lower in MIFKO than in WT on day 28 and on day 7, respectively. Histologically, healing tissues in MIFKO exhibited prolonged hypertrophy, poor vascularity, and prolonged increase in cell number compared with those in WT. Taken together, it was suggested that MIFKO exhibited delayed healing of the MCL, which might be caused by lower mRNA expression of MMP-2 and -13.     

III度膝关节内侧副韧带损伤缝合锚重建术后异位骨化8例临床分析

目的:分析8例III度膝关节内侧副韧带损伤的患者行缝合锚重建术后异位骨化发生与损伤的关系。方法:回顾性收集8例Ⅲ度膝关节内侧副韧带损伤行缝合锚重建术后发生异位骨化的患者,对其临床一般资料、损伤程度及部位、膝关节活动度及异位骨化程度等进行分析。结果:8位中Ⅰ度异位骨化4例,膝关节活动度73.75°~176.25°,平均125°,Ⅱ°异位骨化4例,膝关节活动度78.75°~157.25°,平均117.4°。在发生内侧副韧带异位骨化的8名患者中,仅有1名为单纯内侧副韧带损伤导致,其余7名患者中5名合并前叉或前、后叉韧带损伤,1例伴有胫骨髁间棘的撕脱骨折,1例合并胫骨平台骨折,4例合并胫骨或股骨髁骨折。结论:膝关节内侧异位骨化是异位骨化的好发部位,其发生与膝关节多发韧带损伤有关。     

Effect of sulfated glycosaminoglycan digestion on the transverse permeability of medial collateral ligament

Dermatan and chondroitin sulfate glycosaminoglycans (GAGs) comprise over 90% of the GAG content in ligament. Studies of their mechanical contribution to soft tissues have reported conflicting results. Measuring the transient compressive response and biphasic material parameters of the tissue may elucidate the contributions of GAGs to the viscoelastic response to deformation. The hypotheses of the current study were that digestion of sulfated GAGs would decrease compressive stress and aggregate modulus while increasing the permeability of porcine medial collateral ligament (MCL). Confined compression stress relaxation experiments were carried out on porcine MCL and tissue treated with chondroitinase ABC (ChABC). Results were fit to a biphasic constitutive model to derive permeability and aggregate modulus. Bovine articular cartilage was used as a benchmark tissue to verify that the apparatus provided reliable results. GAG digestion removed up to 88% of sulfated GAGs from the ligament. Removal of sulfated GAGs increased the permeability of porcine MCL nearly 6-fold versus control tissues. Peak stress decreased significantly. Bovine articular cartilage exhibited the typical reduction of GAG content and resultant decreases in stress and modulus and increases in permeability with ChABC digestion. Given the relatively small amount of GAG in ligament (<1% of tissue dry weight) and the significant change in peak stress and permeability upon removal of GAGs, sulfated GAGs may play a significant role in maintaining the apposition of collagen fibrils in the transverse direction, thus supporting dynamic compressive loads experienced by the ligament during complex joint motion.     

Proteoglycan metabolism during repair of the ruptured medial collateral ligament in skeletally mature rabbits

The metabolism of the chondroitin/dermatan sulfate (CS/DS) proteoglycans (PGs) decorin and biglycan is markedly altered during short-term (3-6 weeks) and long-term (40 weeks-2 years) repair of surgically ruptured medial collateral ligaments from mature rabbits. A PG-rich extracellular matrix accumulates in injury gaps by 3 weeks postsurgery and extends into tissue regions containing the original ligaments, and elevated PG levels remain apparent up to 2 years postinjury. CS/DS PGs were prepared from such ligaments and identified after SDS-polyacrylamide gel electrophoresis by Alcian blue staining or immunoblotting. In normal ligaments, decorin is the most abundant proteoglycan (accounting for approximately 80% of the total); the remainder is biglycan and a large PG, possibly versican. In repairing ligaments, decorin is barely detected, but instead a large proteoglycan and abundant amounts of biglycan accumulate. Biglycan is present in two forms in repairing ligaments, and they can be separated on SDS-PAGE into 200- and 140-kDa forms. The slower migrating species is absent in normal ligaments and may represent a different glycoform (containing either a single or two short chondroitin/dermatan sulfate chains) of biglycan. Alteration in PG expression and posttranslational processing during medial collateral ligament repair are similar to those reported for repair and scar formation of other connective tissues. The accumulation of biglycan observed here may interfere with proper collagen network remodeling and may lead to persistent inflammatory and matrix turnover processes, thus preventing restoration of a long-term functional ligament tissue.     

The development and validation of a charge-coupled device laser reflectance system to measure the complex cross-sectional shape and area of soft tissues

Determination of the stresses in soft tissues such as ligaments and tendons under uniaxial tension require accurate measurement of their cross-sectional area. Of the many methods available, there are concerns regarding contact methods which exert external loads and deform the cross-sectional shape of soft tissues. Hence, the area measurements are affected. On the other hand, non-contact methods have difficulties in dealing with complex shapes, especially with concavities. To address these problems, a new measurement system using a charge-coupled device (CCD) laser displacement sensor has been developed and tested. This system measures the complete surface profile of the object by rotating the laser 360° around the soft tissue. Then, the cross-sectional shape is reconstructed and the cross-sectional area determined via Simpson's rule. The system's accuracy was first verified with objects of various cross-sectional shapes and areas (cylinder: 23.1, 76.5, 510.3 mm²; cuboid: 34.3, 163.8, 316.7 mm², and cylinder with concavities: 121.4 mm²). The CCD laser reflectance system's accuracy was within 2.0% for these objects. To test biological application, the goat Achilles tendon and the anteromedial bundle of the porcine anterior cruciate ligament specimens were measured and compared to values obtained using another accepted technique, the laser micrometer system. The areas obtained using the CCD laser reflectance system were 4.4% and 9.7% lower than those obtained with the laser micrometer system respectively. These differences could be mainly attributed to concavities. Thus, the CCD laser reflectance system is an improved method for measuring the cross-sectional shape and area of soft tissues since it can detect and account for concavities without physically contacting the specimen.     

Dehydration rates of meniscus and articular cartilage in vitro using a fast and accurate laser-based coordinate digitizing system

When used in in vitro studies, soft tissues such as the meniscus and articular cartilage are susceptible to dehydration and its effects, such as changes in size and shape as well as changes in structural and material properties. To quantify the effect of dehydration on the meniscus and articular cartilage, the first two objectives of this study were to (1) determine the percent change in meniscal dimensions over time due to dehydration, and (2) determine the percent change in articular cartilage thickness due to dehydration. To satisfy these two objectives, the third objective was to develop a new laser-based three-dimensional coordinate digitizing system (3-DCDS II) that can scan either the meniscus or articular cartilage surface within a time such that there is less than a 5% change in measurements due to dehydration. The new instrument was used to measure changes in meniscal and articular cartilage dimensions of six cadaveric specimens, which were exposed to air for 120 and 130 min, respectively. While there was no change in meniscal width, meniscal height decreased linearly by 4.5% per hour. Articular cartilage thickness decreased nonlinearly at a rate of 6% per hour after 10 min, and at a rate of 16% per hour after 130 min. The system bias and precision of the new instrument at 0 degrees slope of the surface being scanned were 0.0 and 2.6 microm, respectively, while at 45 degrees slope the bias and precision were 31.1 and 22.6 microm, respectively. The resolution ranged between 200 and 500 microm. Scanning an area of 60 x 80 mm (approximately the depth and width of a human tibial plateau) took 8 min and a complete scan of all five sides of a meniscus took 24 min. Thus, the 3-DCDS II can scan an entire meniscus with less than 2% change in dimensions due to dehydration and articular cartilage with less than 0.4% change. This study provides new information on the amount of time that meniscal tissue and articular cartilage can be exposed to air before marked changes in size and shape, and possibly biomechanical, structural and material properties, occur. The new 3-DCDS II designed for this study provides fast and accurate dimensional measurements of both soft and hard tissues.     

Ligament structure, physiology and function

Ligaments are specialized connective tissues with very interesting biomechanical properties. They have the ability to adapt to the complex functions that each are required to perform. While ligaments were once thought to be inert, they are in fact responsive to many local and systemic factors that influence their function within the organism. Injury to a ligament results in a drastic change in its structure and physiology and creates a situation where ligament function is restored by the formation of scar tissue that is biologically and biomechanically inferior to the tissue it replaces. This article will briefly review the basic structure, physiology and function of normal versus healing knee ligaments, referring specifically to what is known about two of the most extensively studied and clinically relevant knee ligaments, the anterior cruciate (ACL) and medial collateral (MCL) ligaments of the knee. Those readers wishing for more comprehensive sources of information on ligament biology and biomechanics are referred to many excellent reviews on these topics.     

A new method for determining cross-sectional shape and area of soft tissues

Assessment of the mechanical properties of soft tissues requires accurate measurement of the cross-sectional area. To date, techniques for determining cross-sectional areas of ligaments and tendons have been less than ideal due to the tissues' complex geometries and the fact that they deform easily under an applied external load. A new procedure has been developed for determining the cross-sectional area by means of an image reconstruction technique based on measurements from collimated laser beams. Using this procedure, the actual shape of the specimen cross-section can also be determined. The results are demonstrated to be highly accurate, and this methodology does not require mechanical contact with the specimen.     

Changes in the distribution of fibrillar collagens in the collateral and cruciate ligaments of the rabbit knee joint during fetal and postnatal development

Summary The collateral ligaments can be clearly distinguished in the 25-day fetal rabbit knee joint. Types I and V collagens are present in the extracellular matrix between the cells of the lateral and medial collateral ligaments and this distribution persists until the rabbit is skeletally mature. From 8 months onwards type III collagen is also present, particularly around the cells. Type I collagen mRNA is expressed by the cells from the 25-day fetal to 8-month-old adult ligament. The ligament sheath is composed of types III and V collagens. The cruciate ligaments are present between the femur and tibia in the 20-day fetus. The matrix is composed of types I and V collagens from the 25-day fetus until at 12- to 14-weeks postnatal, type III collagen appears in the pericellular regions together with type V. At 8 months and 2 years, the amount of type III collagen has increased. All the cells express the mRNA for type I collagen at 12- to 14-weeks, but only isolated cells express this mRNA at 8 months. Thus, both the collateral and cruciate ligaments undergo changes in their complement of collagens during postnatal development and ageing. The implications of these complex interactions of different types of collagen are discussed in relation to healing and the surgical replacement of torn ligaments by tendons.