Neuropeptides regulate expression of matrix molecule, growth factor and inflammatory mediator mRNA in explants of normal and healing medial collateral ligament

Denervation degrades normal ligament properties and impairs ligament healing. This suggests that secreted neuromediators, such as neuropeptides, could be modulating cell metabolism in ligament and scar tissue. To test this hypothesis we investigated the effect of exogenous substance P (SP), neuropeptide Y (NPY) or calcitonin gene-related peptide (CGRP) on the mRNA levels for proteins associated with inflammation, angiogenesis, and matrix production in tissue-cultured specimens of normal and injured medial collateral ligament. SP and NPY induced increased mRNA levels for several inflammatory mediators in the 2-week post-injury specimens. All three neuropeptides induced decreases in mRNA levels for healing-associated growth factors and matrix molecules, including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and collagen types I and III. The results indicate that neuropeptides strongly influence the metabolic activity of cells in healing ligament, particularly at early time points after injury.     

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.     

Skeletal ligament healing using the recombinant human amelogenin protein

Injuries to ligaments are common, painful and debilitating, causing joint instability and impaired protective proprioception sensation around the joint. Healing of torn ligaments usually fails to take place, and surgical replacement or reconstruction is required. Previously, we showed that in vivo application of the recombinant human amelogenin protein (rHAM⁺) resulted in enhanced healing of the tooth‐supporting tissues. The aim of this study was to evaluate whether amelogenin might also enhance repair of skeletal ligaments. The rat knee medial collateral ligament (MCL) was chosen to prove the concept. Full thickness tear was created and various concentrations of rHAM⁺, dissolved in propylene glycol alginate (PGA) carrier, were applied to the transected MCL. 12 weeks after transection, the mechanical properties, structure and composition of transected ligaments treated with 0.5 μg/μl rHAM⁺ were similar to the normal un‐transected ligaments, and were much stronger, stiffer and organized than control ligaments, treated with PGA only. Furthermore, the proprioceptive free nerve endings, in the 0.5 μg/μl rHAM⁺ treated group, were parallel to the collagen fibres similar to their arrangement in normal ligament, while in the control ligaments the free nerve endings were entrapped in the scar tissue at different directions, not parallel to the axis of the force. Four days after transection, treatment with 0.5 μg/μl rHAM⁺ increased the amount of cells expressing mesenchymal stem cell markers at the injured site. In conclusion application of rHAM⁺ dose dependently induced mechanical, structural and sensory healing of torn skeletal ligament. Initially the process involved recruitment and proliferation of cells expressing mesenchymal stem cell markers.     

Hindlimb unloading alters ligament healing.

We investigated the hypothesis that hindlimb unloading inhibits healing in fibrous connective tissue such as ligament. Male rats were assigned to 3- and 7-wk treatment groups with three subgroups each: sham control, ambulatory healing, and hindlimb-suspended healing. Ambulatory and suspended animals underwent surgical rupture of their medial collateral ligaments, whereas sham surgeries were performed on control animals. After 3 or 7 wk, mechanical and/or morphological properties were measured in ligament, muscle, and bone. During mechanical testing, most suspended ligaments failed in the scar region, indicating the greatest impairment was to ligament and not to bone-ligament insertion. Ligament testing revealed significant reductions in maximum force, ultimate stress, elastic modulus, and low-load properties in suspended animals. In addition, femoral mineral density, femoral strength, gastrocnemius mass, and tibialis anterior mass were significantly reduced. Microscopy revealed abnormal scar formation and cell distribution in suspended ligaments with extracellular matrix discontinuities and voids between misaligned, but well-formed, collagen fiber bundles. Hence, stress levels from ambulation appear unnecessary for formation of fiber bundles yet required for collagen to form structurally competent continuous fibers. Results support our hypothesis that hindlimb unloading impairs healing of fibrous connective tissue. In addition, this study provides compelling morphological evidence explaining the altered structure-function relationship in load-deprived healing connective tissue.     

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.     

Systemic administration of IGF-I enhances healing in collagenous extracellular matrices: evaluation of loaded and unloaded ligaments

Background  

Insulin-like growth factor-I (IGF-I) plays a crucial role in wound healing and tissue repair. We tested the hypotheses that systemic administration of IGF-I, or growth hormone (GH), or both (GH+IGF-I) would improve healing in collagenous connective tissue, such as ligament. These hypotheses were examined in rats that were allowed unrestricted activity after injury and in animals that were subjected to hindlimb disuse. Male rats were assigned to three groups: ambulatory sham-control, ambulatory-healing, and hindlimb unloaded-healing. Ambulatory and hindlimb unloaded animals underwent surgical disruption of their knee medial collateral ligaments (MCLs), while sham surgeries were performed on control animals. Healing animals subcutaneously received systemic doses of either saline, GH, IGF-I, or GH+IGF-I. After 3 weeks, mechanical properties, cell and matrix morphology, and biochemical composition were examined in control and healing ligaments.     

ACL/MCL transection affects knee ligament insertion distance of healing and intact ligaments during gait in the Ovine model

The objective of this study was to assess the impact of combined transection of the anterior cruciate and medial collateral ligaments on the intact and healing ligaments in the ovine stifle joint. In vivo 3D stifle joint kinematics were measured in eight sheep during treadmill walking (accuracy: 0.4±0.4 mm, 0.4±0.4°). Kinematics were measured with the joint intact and at 2, 4, 8, 12, 16 and 20 weeks after either surgical ligament transection (n=5) or sham surgery without transection (n=3). After sacrifice at 20 weeks, the 3D subject-specific bone and ligament geometry were digitized, and the 3D distances between insertions (DBI) of ligaments during the dynamic in vivo motion were calculated. Anterior cruciate ligament/medial collateral ligament (ACL/MCL) transection resulted in changes in the DBI of not only the transected ACL, but also the intact lateral collateral ligament (LCL) and posterior cruciate ligament (PCL), while the DBI of the transected MCL was not significantly changed. Increases in the maximal ACL DBI (2 week: +4.2 mm, 20 week: +5.7 mm) caused increases in the range of ACL DBI (2 week: 3.6 mm, 20 week: +3.8 mm) and the ACL apparent strain (2 week: +18.9%, 20 week: +24.0%). Decreases in the minimal PCL DBI (2 week: −3.2 mm, 20 week: −4.3 mm) resulted in increases in the range of PCL DBI (2 week: +2.7 mm, 20 week: +3.2 mm). Decreases in the maximal LCL DBI (2 week: −1.0 mm, 20 week: −2.0 mm) caused decreased LCL apparent strain (2 week: −3.4%, 20 week: −6.9%). Changes in the mechanical environment of these ligaments may play a significant role in the biological changes observed in these ligaments.     

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.     

Calcitonin gene-related peptide-like immunoreactivities in pheochromocytomas

Calcitonin gene-related peptide (CGRP) is reported to exist in high concentrations in plasma and tumor tissues of medullary thyroid carcinomas. CGRP-like immunoreactivity (LI) in tumor tissues of pheochromocytomas was investigated by radioimmunoassay. CGRP-LI in 9 pheochromocytomas ranged from 0.50 to 1240 ng/g wet tissue. Sephadex G-50 column chromatography revealed that most of CGRP-LI in tumor extracts was eluted in an identical position to synthetic human CGRP. Reverse-phase high pressure liquid chromatography revealed that CGRP-LI in tumor extracts was eluted in an identical position to synthetic human CGRP and in a more hydrophobic position. These results indicate that high concentrations of CGRP-LI also exist in tumor tissues of pheochromocytomas.     

Overexpression of αCGRP promotes osteogenesis of periodontal ligament cells by regulation of YAP signaling

Human periodontal ligament cells (hPDLCs) are considered as an ideal cell type for periodontal tissue engineering as hPDLCs own mesenchymal stem cell-like properties. Additionally, it is suggested that α-calcitonin gene-related peptide (αCGRP) plays a pivotal role in the pathogenesis of periodontitis. However, the specific role of αCGRP on the regulation of alveolar bone regeneration which is essential for treatment of periodontitis remains unclear. In this study, lentiviral αCGRP expression vector was first transfected into hPDLCs. αCGRP expression and the osteogenesis-related gene (ALP, RUNX2, OCN, and BSP) expressions were detected. The results showed that expressions of osteogenic phenotypes were upregulated in αCGRP-transfected hPDLCs combined with an increased expression of Yes-associated protein (YAP), which is the key downstream effectors of Hippo pathway. Our observations suggest that αCGRP-mediated hPDLCs’ osteogenesis might relate with the activity of YAP signaling. These observations may reflect intrinsic functions of αCGRP in hPDLCs’ osteogenesis and its promising role in the treatment of bone deficiency in periodontal regeneration.     

The collateral ligaments of the knee joint in the cat and man. Morphological and functional study of the internal arrangement of fibers

The present investigation of fiber arrangement in the collateral ligaments of the knee was carried out in cats and man in various positions of flexion and extension, without compression load. In all knee joint positions, the fibers of the collateral ligaments are twisted except for the fibers in the meniscal part of the medial collateral ligament which have a parallel arrangement. Furthermore, most of the fibers in the collateral ligaments are taut in all positions of the knee joint in both cat and man. By means of planar models representing different fiber arrangements, the kinematic behavior of the collateral ligaments was analyzed. It appears that a crossed (twisted) arrangement of the fibers is most effective in rotatory movements, whereas a parallel orientation is most effective in translation. Our data further indicate that, in measuring the changes in lengths of ligaments during joint motion, one cannot neglect the internal arrangement of fibers and the geometry of the articular surfaces and menisci.     

Colocalization of protein kinase C beta-subtype and calcitonin gene-related peptide in rat spinal cord.

Colocalization of calcitonin gene-related peptide (CGRP) and protein kinase C beta-subtype (PKC-beta) like immunoreactivities (LI) and the subcellular localization of CGRP-LI were studied in the ventral horn of rat spinal cord. Ultrastructurally CGRP-LI was localized on the membranes of the Golgi-complexes, in multivesicular bodies and in vesicles adjacent to the Golgi-complex in motoneuron perikarya. The colocalization of PKC-beta and CGRP-LI was detected in most of the ventral horn motoneurons. However, few motoneurons were only PKC-beta-immunoreactive. These results suggest that PKC-beta may be involved in the regulation of CGRP release from motoric axon terminals.     

Neurogenic vasodilation and release of calcitonin gene-related peptide (CGRP) from perivascular nerves in the rat mesenteric artery

The effect of perivascular nerve stimulation (PNS) on the release of calcitonin gene-related peptide (CGRP) was examined by radioimmunoassay (RIA) in isolated, perfused rat mesenteric arteries. The released CGRP-like immunoreactivity (CGRP-LI) was identified to be CGRP itself and its oxidized form by combined analysis with RIA and high performance liquid chromatography. CGRP-LI was localized in the perivascular nerves of the large mesenteric artery and its branches. In the preparation precontracted by methoxamine, and perfused with a solution containing guanethidine, an adrenergic neuron blocker, PNS induced vasodilator responses and an increase of CGRP-LI in the perfusate in a frequency-dependent manner. Both the responses were attenuated by tetrodotoxin (10(-6) M), suggesting that they were neurogenic in origin. Removal of Ca2+ from the perfusing solution also abolished the PNS-induced release of CGRP-LI. These findings suggest that CGRP plays a transmitter role in the neurogenic vasodilation in the rat mesenteric vascular bed.     

The influence of ligament modelling strategies on the predictive capability of finite element models of the human knee joint

In finite element (FE) models knee ligaments can represented either by a group of one-dimensional springs, or by three-dimensional continuum elements based on segmentations. Continuum models closer approximate the anatomy, and facilitate ligament wrapping, while spring models are computationally less expensive. The mechanical properties of ligaments can be based on literature, or adjusted specifically for the subject. In the current study we investigated the effect of ligament modelling strategy on the predictive capability of FE models of the human knee joint. The effect of literature-based versus specimen-specific optimized material parameters was evaluated. Experiments were performed on three human cadaver knees, which were modelled in FE models with ligaments represented either using springs, or using continuum representations. In spring representation collateral ligaments were each modelled with three and cruciate ligaments with two single-element bundles. Stiffness parameters and pre-strains were optimized based on laxity tests for both approaches. Validation experiments were conducted to evaluate the outcomes of the FE models.Models (both spring and continuum) with subject-specific properties improved the predicted kinematics and contact outcome parameters. Models incorporating literature-based parameters, and particularly the spring models (with the representations implemented in this study), led to relatively high errors in kinematics and contact pressures. Using a continuum modelling approach resulted in more accurate contact outcome variables than the spring representation with two (cruciate ligaments) and three (collateral ligaments) single-element-bundle representations. However, when the prediction of joint kinematics is of main interest, spring ligament models provide a faster option with acceptable outcome.     

Calcitonin gene-related peptide expression at endplates of different fibre types in muscles in rat hind limbs

Calcitonin gene-related peptide (CGRP) occurs only in some motoneurons. In this study, the presence of CGRP in motor endplates in relation to muscle fibre types was examined in slow (soleus muscle) and fast [tibialis anterior (TA) and extensor digitorum longus (EDL)] leg muscles of the rat. CGRP was detected by use of immunohistochemical methods, and staining for the mitochondrial-bound enzyme NADH-TR was used for demonstration of fibre types. The fibres showing low NADH-TR activity were interpreted as representing IIB fibres. All such fibres located in the superficial portion of TA were innervated by endplates displaying CGRP-like immunoreactivity (LI), whereas in the deep portion of TA some of these fibres lacked CGRP-LI at their endplates. Thirty per cent of the IIB fibres in EDL showed CGRP-LI at the endplates. All fibres in TA and EDL displaying high NADH-TR activity and interpreted as type-IIA fibres, lacked CGRP-LI in their motor innervation. One third of the fibres with intermediate NADH-TR activity in TA exhibited CGRP-LI at their endplates, whereas in EDL only few such fibres displayed CGRP-LI in the endplate formation. These fibres are likely to belong to type-IIX or type-I motor units. CGRP-LI was very rarely detected at the endplates in the soleus muscle. These observations show that distinct differences exist between the slow muscle, soleus, and the fast muscles, TA and EDL, but that there are also differences between the different types of fibres in TA and EDL with respect to presence of CGRP-LI at the endplates. As CGRP-LI was frequently detected at endplates of IIB fibres, it is likely that CGRP has a particular role related to the differentiation and maintenance of these fibres.     

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.     

Ligament-bone interaction in a three-dimensional model of the knee

In mathematical knee-joint models, the ligaments are usually represented by straight-line elements, connecting the insertions of the femur and tibia. Such a model may not be valid if a ligament is bent in its course over bony-surfaces, particularly not if the resulting redirection of the ligament force has a considerable effect on the laxity or motion characteristics of the knee-joint model. In the present study, a model for wrapping of a ligament around bone was incorporated in a three-dimensional mathematical model of the human knee. The bony edge was described by a curved line on which the contact point of the line element representing a ligament bundle was located. Frictionless contact between the ligament bundle and the bone was assumed. This model was applied to the medial collateral ligament (MCL) interacting with the bony edge of the tibia. It was found that, in comparison with the original model without bony interactions, the bony edge redirected the ligament force of the MCL in such a way that it counterbalanced valgus moments on the tibia more effectively. The effect of the bony interaction with the MCL on the internal-external rotation laxity, however, was negligible.     

Biomechanics of the rabbit knee and ankle: muscle, ligament, and joint contact force predictions

Mathematical models of small animals that predict in vivo forces acting on the lower extremities are critical for studies of musculoskeletal biomechanics and diseases. Rabbits are advantageous in this regard because they remodel their cortical bone similar to humans. Here, we enhance a recent mathematical model of the rabbit knee joint to include the loading behavior of individual muscles, ligaments, and joint contact at the knee and ankle during the stance phase of hopping. Geometric data from the hindlimbs of three adult New Zealand white rabbits, combined with previously reported intersegmental forces and moments, were used as inputs to the model. Muscle, ligament, and joint contact forces were computed using optimization techniques assuming that muscle endurance is maximized and ligament strain energy resists tibial shear force along an inclined plateau. Peak forces developed by the quadriceps and gastrocnemius muscle groups and by compressive knee contact were within the range of theoretical and in vivo predictions. Although a minimal force was carried by the anterior cruciate and medial collateral ligaments, force patterns in the posterior cruciate ligament were consistent with in vivo tibial displacement patterns during hopping in rabbits. Overall, our predictions compare favorably with theoretical estimates and in vivo measurements in rabbits, and enhance previous models by providing individual muscle, ligament, and joint contact information to predict in vivo forces acting on the lower extremities in rabbits.     

Temporal extracellular matrix adaptations in ligament during wound healing and hindlimb unloading

Previous data from spaceflight studies indicate that injured muscle and bone heal slowly and abnormally compared with ground controls, strongly suggesting that ligaments or tendons may not repair optimally as well. Thus the objective of this study was to investigate the biochemical and molecular gene expression of the collagen extracellular matrix in response to medial collateral ligament (MCL) injury repair in hindlimb unloaded (HLU) rodents. Male rats were assigned to 3- and 7-wk treatment groups with three subgroups each: sham control, ambulatory healing (Amb-healing), and HLU-healing groups. Amb- and HLU-healing animals underwent bilateral surgical transection of their MCLs, whereas control animals were subjected to sham surgeries. All surgeries were performed under isoflurane anesthesia. After 3 wk or 7 wk of HLU, rats were euthanized and MCLs were surgically isolated and prepared for molecular or biochemical analyses. Hydroxyproline concentration and hydroxylysylpyridinoline collagen cross-link contents were measured by HPLC and showed a substantial decrement in surgical groups. MCL tissue cellularity, quantified by DNA content, remained significantly elevated in all HLU-healing groups vs. Amb-healing groups. MCL gene expression of collagen type I, collagen type III, collagen type V, fibronectin, decorin, biglycan, lysyl oxidase, matrix metalloproteinase-2, and tissue inhibitor of matrix metalloproteinase-1, measured by real-time quantitative PCR, demonstrated differential expression in the HLU-healing groups compared with Amb-healing groups at both the 3- and 7-wk time points. Together, these data suggest that HLU affects dense fibrous connective tissue wound healing and confirms previous morphological and biomechanical data that HLU inhibits the ligament repair processes.     

Subfailure damage in ligament: a structural and cellular evaluation.

Subfailure damage in ligaments was evaluated macroscopically from a structural perspective (referring to the entire ligament as a structure) and microscopically from a cellular perspective. Freshly harvested rat medial collateral ligaments (MCLs) were used as a model in ex vivo experiments. Ligaments were preloaded with 0.1 N to establish a consistent point of reference for length (and strain) measurements. Ligament structural damage was characterized by nonrecoverable difference in tissue length after a subfailure stretch. The tissue's mechanical properties (via stress vs. strain curves measured from a preloaded state) after a single subfailure stretch were also evaluated (n = 6 pairs with a different stretch magnitude applied to each stretched ligament). Regions containing necrotic cells were used to characterize cellular damage after a single stretch. It should be noted that the number of damaged cells was not quantified and the difference between cellular area and area of fluorescence is not known. Structural and cellular damage were represented and compared as functions of subfailure MCL strains. Statistical analysis indicated that the onset of structural damage occurs at 5.14% strain (referenced from a preloaded length). Subfailure strains above the damage threshold changed the shape of the MCL stress-strain curve by elongating the toe region (i.e., increasing laxity) as well as decreasing the tangential modulus and ultimate stress. Cellular damage was induced at ligament strains significantly below the structural damage threshold. This cellular damage is likely to be part of the natural healing process in mildly sprained ligaments.